Ib qho Distinctive Distribution Of Hypoxia-inducible Factor-1 nyob rau hauv Cultured Renal Tubular Cells Nrog Hypoperfusion Simulated Los Ntawm Cov Ntawv Pov Thawj

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Tomoko Honda¹| Yosuke Hirakawa¹ | Kiichi Mizukami²| Toshitada Yoshihara²| Tetsuhiro Tanaka¹| Seiji Tobita²| Masaomi Nagaku

Abstract

Kev mob hypoxia nyob rau hauv lub raum tubulointerstitium plays lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm cov mob ntev.raumkab mob(CKD). Yog li ntawd nws yog ib qho tseem ceeb los soj ntsuam tubular hypoxia thiab kev ua ntawm hypoxia-inducible factor (HIF)-1 nyob rau hauv teb rau hypoxia. Rarefaction ntawm peritubular capillary ua rau hypoperfusion hauv CKD; Txawm li cas los xij, qhov cuam tshuam ntawm hypoperfusion ntawm HIFs tsis tshua tau tshawb xyuas. Peb induced hypoperfusion tshwm sim los ntawm coverlip tso rau hauv tib neegraum-2 cov hlwb thiab pom cov pa oxygen gradient hauv qab daim npog. Immunocytochemistry ntawm HIF-1 tau pom ib qho kev tsim ntawm lub khob noom cookie rau ntawm ntug ntawm thaj chaw pimonidazole-zoo, uas peb hu ua "HIF-ring". Cov pa oxygen nro ntawm HIF-ntiv nplhaib tau kwv yees li ntawm 4 mmHg thiab 20 mmHg. Cov txiaj ntsig no tsis sib haum nrog cov kev tshawb fawb yav dhau los uas qhia HIF-1 tsub zuj zuj hauv cov tshuaj anoxic nrog homogeneous oxygen nro. Peb tau soj ntsuam ntxiv qhov muaj pH gradient nyob rau hauv daim npog npog, nrog rau kev hloov ntawm HIF lub nplhaib vim kev hloov pauv hauv pH ntawm kab lis kev cai nruab nrab, qhia tias lub nplhaib HIF tau tsim los ntawm kev tawm tsam ntawm HIF-1 cuam tshuam rau pH tsawg. Qhov kev tshawb fawb no pom tau hais tias HIF-1 kev ua kom zoo li lub cev lub cev hauv cov kab lis kev cai nrog hypoperfusion.

KEYWORDShypoperfusion, hypoxia, hypoxia-inducible factor, oxygen gradient, pH

Cistanche tshuaj ntsuabtiv thaiv kab mob raum, nyem qhov no kom tau txais cov qauv

1|Taw qhia

Qhov tshwm sim ntawm chronicraumkab mob(CKD) tab tom nce thoob plaws ntiaj teb raws li lub hnub nyoog hauv zej zog (Tonelli & Riella, 2014). Kev loj hlob ntawm CKD yog qhov hloov tsis tau thaum lub raum kev puas tsuaj nce mus txog qib siab thiab thaum kawg ua rau mob raum kawg (ESRD), tsis hais tus kab mob hauv qab. Qhov kev tawm tuaj no qhia txog qhov muaj nyob ntawm "qhov kawg ntawm txoj hauv kev." Raws li yav dhau los kev tshuaj ntsuam xyuas pathological, kev poob rau hauv lub raum kev ua haujlwm muaj kev sib raug zoo nrog tubulointerstitial kev puas tsuaj ntau dua li kev puas tsuaj glomerular. Muaj ntau cov ntaub ntawv qhia pom tias lub raum fibrosis induces chronic hypoxia nyob rau hauv tubulointerstitium, thiab tubulointerstitial hypoxia aggravates CKD thiab ua rau ESRD. Cov pov thawj no qhia tau hais tias tubulointerstitial hypoxia plays ib feem ntawm txoj hauv kev kawg ntawm CKD (Mimura & Nangaku, 2010; Nangaku, 2006). CovraumNws yog qhov cuam tshuam rau hypoxia vim nws qhov kev thov siab rau oxygen, thiab muaj cov pa oxygen shunt ntawm cov hlab ntsha thiab cov hlab ntsha (Nangaku, 2006; Welch et al., 2001; Zhang et al., 2014). Yog li ntawd, peb txiav txim siab tias nws yog ib qho tseem ceeb los soj ntsuam hypoxia thiab cov lus teb rau hypoxia hauv lub raum tubulointerstitium.

Thawj cov lus teb rau cov kab mob hypoxia hauv cov kab mob muaj sia tshwm sim los ntawm txoj hauv kev hypoxia-inducible (HIF) (Hypoxia, 2011; Semenza & Wang, 1992; Zhou et al., 2003). HIF muaj - thiab -subunits. Txawm hais tias -subunit yog constitutively active, lub -subunit degraded nyob rau hauv lub xub ntiag ntawm oxygen. Nyob rau hauv cov xwm txheej normoxic, HIF- yog hydroxylated los ntawm prolyl hydroxylase domain (Ph.D.), ua rau nws pom tau los ntawm von Hippel-Lindau qog suppressor (VHL). Qhov kev lees paub no ua rau ubiquitination thiab degradation ntawm hydroxylated HIF- hauv cov proteasome. Nyob rau hauv cov xwm txheej hypoxic, HIF- accumulates hauv cytosol, vim tias tsis yog-hydroxylated HIF- dim degradation. Accumulated HIF- yog translocated los ntawm cytosol mus rau lub nucleus, qhov twg nws dimerizes nrog HIF-, thiab ua raws li ib tug transcriptional yam, txhawb kev qhia ntawm downstream noob. Ntawm peb lub isoforms ntawm HIF-subunits-HIF-1 , HIF-2 , thiab HIF-3 -lub raum tubular epithelial hlwb paub qhia HIF-1 (Tanaka li al ., 2016). Cov kev tshawb fawb yav dhau los tau pom tias muaj kev cuam tshuam los yog cheeb tsam ntawm HIF nyob rau hauvraumnrog CKD (Goldfarb et al., 2006; Yu et al., 2012), thiab HIF no tau qhib rau hauv qhov txo qis oxygen hauv CKD milieu. Qhov tsis zoo rau hypoxia hauv CKD tuaj yeem tshwm sim los ntawm qhov muaj qhov cuam tshuam ntawm HIF txoj hauv kev (Asai li al., 2016; Tanaka li al., 2013; Thangarajah li al., 2009). Kev tiv thaiv ntawm HIF ua kom muaj kev tshaj tawm hauv ntau tus tsiaj qauv, suav nrog streptozotocin-induced diabetic nas thiab 5/6th nephrectomy nas (Deng li al., 2010; Nordquist li al., 2015). Ph.D. Cov tshuaj inhibitors tsis ntev los no tau nyiam cov kev kho tshiab rau lub raum ntshav ntshav hauv cov neeg mob CKD (Akizawa li al., 2019; Chen li al., 2017, 2019; Coyne et al., 2017; Miyata et al., 2011; Pergola et al. ., 2016; Provenzano et al., 2016). Txawm li cas los xij, cov ntsiab lus ntawm kev loj hlob ntawm lub raum hypoxia, thiab txoj hauv kev uas HIF accumulation tshwm sim hauv lub raum.raumnrog CKD, nyob twj ywm tsis meej. Oxygen-dependent hydroxylation ntawm HIF- tshwm sim hauv cytosol, thiab yog li ntawd nws yog ib qho tseem ceeb rau kev ntsuas intracellular thiab extracellular oxygen nro. Muaj ntau txoj hauv kev siv los ntsuas cov pa oxygen, suav nrog kev siv cov microelectrodes lossis ntshav oxygen qib-dependent magnetic resonance imaging (BOLD-MRI). Peb siv phosphorescence lifetime imaging microscopy (PLIM) rau kev ntsuas qhov ntsuas ntawm cov pa oxygen hauv lub cev (Hirakawa li al., 2017; Yoshihara li al., 2015). BTPDM1 yog phosphorescent dye raws li iridium (III) complex BTP, (bp)2Ir(acc) (bp=benzoate-pyridine, aac=acetylacetone) nrog ib pawg cationic dimethylamino, uas yog passively. faib nyob rau hauv intracellular lysosomes. Nws tau raug tsim los ua ib qho phospho- tsis ntev los no soj ntsuam los ntsuas cov pa oxygen hauv lub cev (Yoshihara li al., 2015). PLIM nrog BTPDM1 tau ua kom tau txais cov duab siab daws teeb meem ntawm ib feem ntawm cov pa oxygen hauv lub raum tubular hlwb ntawm lub raum ntawm cov nas ib txwm muaj, muab cov ntaub ntawv qhia tias muaj cov pa oxygen gradient, txawm tias nyob hauv ob lub raum (Hirakawa li al., 2018). ).

Muaj cov pa oxygen gradients nyob rau hauv lub cev, nrog raulub raum, txawm tias nyob rau hauv ib txwm muaj (Hirakawa li al., 2018; Kietzmann, 2017; Zhdanov et al., 2015), thiab cov gradients no tuaj yeem xav tau hauv CKD. Hypoperfusion yog tshwm sim los ntawm qhov tsis tshua muaj tshwm sim ntawm microvasculature hauv CKD, nyob rau hauv qhov kev raug mob glomerular ua rau txo qis hauv peritubular capillary ntshav txaus, ua rau muaj kev cuam tshuam ntawm interstitial fibrosis. Interstitial fibrosis impairs lub diffusion thiab muab cov pa oxygen mus rau tubular hlwb, thiab ua rau cov tsis tshua muaj microvasculature, ntxiv aggravating tubulointerstitial hypoxia (Mimura & Nangaku, 2010; Nangaku, 2006). Cov teebmeem lom ntawm hypoperfusion, txawm li cas los xij, tseem tsis paub meej, vim tias feem ntau cov kev tshawb fawb tau tshawb xyuas qhov cuam tshuam ntawm hypoxia thiab HIF-1 ntawm cov kab lis kev cai nyob rau hauv cov xwm txheej ntawm homogenous perfusion thiab oxygen tension (Rexius-Hall li al., 2017). Cov kev tshawb fawb yav dhau los tau pom tias cov kab lis kev cai monolayered npog nrog daim npog npog muab ib qho qauv zoo ntawm hypoperfusion induced los ntawm kev cuam tshuam rau diffusion hauv kab lis kev cai nruab nrab. Tus qauv hypoperfusion yog txuam nrog cov pa oxygen gradient vim tias daim npog npog tiv thaiv oxygen diffusion los ntawm sab saum toj ntawm nruab nrab mus rau cov hlwb (Pitts & Toombs, 2004; Takahashi & Sato, 2010; Yoshihara et al., 2015). Nyob rau hauv ib lub monolayer ntawm kab lis kev cai npog nrog ib daim npog npog, intracellular oxygen nro poob nrog kev ncua deb ntawm ntug ntawm cov npog npog vim tias tsis muaj oxygen diffusion mus rau hauv cov kab lis kev cai. Raws li qhov tshwm sim, cov pa oxygen gradient yog tsim los ntawm ntug mus rau qhov chaw ntawm cov ntaub npog, thiab cov pa oxygen intracellular yuav poob mus rau ib qho anoxic ntau yam nyob rau hauv nruab nrab ntawm daim npog (Takahashi & Sato, 2010; Yoshihara li al., 2015) . Hauv kev tshawb fawb tam sim no, peb tau tsom mus rau HIF ua kom muaj zog thiab tshawb xyuas seb puas muaj cov pa oxygen-yooj yim hloov pauv hauv HIF-kev qhia nyob rau hauv lub xub ntiag ntawm hypoperfusion nrog oxygen gradient. Txij li thaum hypoperfusion nrog cov pa oxygen gradient muaj nyob rau hauv lub raum tubules hauv vivo, kev soj ntsuam ntawm cov pa oxygen ywj pheej ntawm HIF- kev qhia hauv cov qauv npog tuaj yeem muab kev nkag siab rau hauv cov txheej txheem hauv qab tsis txaus ntawm HIF-sau hauv CKD.

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2|MATERIAL S THIAB METHODS

2.1|Cell kab lis kev cai

Cultivated hlwb raug incubated nyob rau hauv ib tug humidified incubator nrog 5 feem pua ​​CO2. Hypoxic incubation tau ua nyob rau hauv tus kheej CO2 / multi-gas incubator APM-30D (Astec). Anoxia raug ntxias nrog lub hnab anoxia, AnaeroPack, thiab Anaerobic cultivation sets #A-13 (Mitsubishi Gas Chemical).

HK-2 hlwb (Homo sapiens, humanraum, txiv neej, CRL- 2190, ATCC, RRID: CVCL_0302), ib qho immortalized proximal tubule epithelial cell kab los ntawm ib txwm neeg laus lub raum, tau coj los ntawm Dulbecco's modified Eagle's nruab nrab / nutrient sib tov F{{2} } Ham (DMEM/F12) (D8062, Sigma Aldrich) muaj 10 feem pua ​​​​fetal bovine serum (FBS) (F7524, Sigma Aldrich), thiab tshuaj penicillin-streptomycin (15070063, Thermo Fisher Scientific) hauv ib lub tais 10 cm Rau txoj kev, HK-2 hlwb raug dissociated nrog trypsin (204-16935, Wako Pure Chemical Industries, Ltd.) thiab centrifuged ntawm 300 g rau 5 min.

HeLa qog nqaij hlav qog noj ntshav thiab tib neeg embryonicraumhlwb 293 (HEK293) tau coj mus kuaj hauv DMEM nrog qis (1000 mg / L) qabzib (D6046, Sigma Aldrich) muaj 5 feem pua ​​FBS hauv 10 cm kab lis kev cai tais. Cov hlwb no tau dhau los ua HK-2 hlwb.

Renal proximal tubule epithelial cells (RPTECs) (CC- 2553, Cambrex) tau khaws cia siv RenaLifeTM Comp cov khoom siv (LRC-LL0025, Lonza Ltd.). Rau txoj kev, RPTECs tau dissociated siv trypsin, neutralized nrog Trypsin Neutralizing Solution (CC-5002, Lonza Ltd.), thiab centrifuged ntawm 200 g rau 5 min.

2.2|Tsim kom muaj tus qauv hypoperfusion los ntawm qhov chaw npog npog

Round-shaped coverlips ntawm 15 mm inch (C015001, Matsunami) tau ntxuav siv ultrasound thiab khaws cia hauv 99.5 feem pua ​​​​ethanol ua ntej siv. Ob txoj hauv kev tau ua haujlwm, raws li kev soj ntsuam ntawm cov hlwb sab nraum daim npog.

Monolayered kab lis kev cai tau muab tso rau ntawm daim npog npog thiab hauv qab ntawm lub tais (Daim duab S1a, b), lossis lwm txoj kev (Daim duab S1c), raws li tau piav qhia hauv qab no. Ob qho tib si los yog lwm txoj hauv kev tau raug xaiv los tsim peb cov qauv npog rau kev yees duab nyob, txawm tias pom cov pa oxygen lossis PH imaging. Rau immunocytochemistry (ICC), lwm txoj kev raug xaiv, vim hais tias thaum siv cov txheej txheem ib txwm siv, feem ntau ntawm cov hlwb feem ntau detached los ntawm qab ntawm lub tais thaum lub sij hawm npog npog tshem tawm rau cell fixation (Daim duab S2a).

2.2.1|Txoj kev cai

Hnub 1, cov hlwb tau muab tso rau hauv qab ntawm 27 hli iav-raws li tais (3910-035, Iwaki) ntawm qhov sib xyaw ntawm 100 feem pua ​​* (kwv yees li 5.0 × 105 / phaj). Lawv tau coj mus kuaj hauv DMEM / F12 uas muaj 10 feem pua ​​FBS yam tsis muaj tshuaj tua kab mob thaum hmo ntuj. Hnub 2, daim ntawv npog tau muab tso rau ntawm cov kab lis kev cai rau lub sijhawm qhia (Daim duab S1b).

2.2.2|Lwm txoj kev

Cov noob tau cog rau ntawm daim npog npog hauv 35 mm-kev coj noj coj ua tais ntawm 100 feem pua ​​​​ntawm cov khoom sib xyaw (kwv yees li 5.0 × 105 / phaj) Hnub.

1. Lawv tau coj mus kuaj hauv DMEM/F12 uas muaj 10 feem pua ​​FBS yam tsis muaj tshuaj tua kab mob ib hmos. Hnub 2, lub npog-daim npog tau thim tsis tau, txhawm rau txuas rau saum npoo ntawm nws cov hlwb mus rau hauv qab ntawm cov khoom tshiab 27 lub sijhawm (daim duab S1C).

Peb kuj tau tsim cov qauv npog uas siv cov ntawv npog npog nrog lub cheeb ntawm 10 hli (CS01005, Matsunami) (Daim duab S2b).

2.3|Nyob oxygen duab nrog BTPDM1

Cultivated hlwb tau npaj rau hnub 1, raws li tau piav saum toj no. Hnub 2, cov hlwb tau yaug ob zaug nrog Hanks 'Balanced Salt tov (HBSS) (H8264, Sigma Aldrich) thiab incubated nrog 500 nM BTPDM1, iridium-based cationic lipophilic dye siv los ua ib qho intracellular phosphorescent sojntsuam (Yoshihara, et al. 2015), hauv DMEM/F12 yam tsis muaj phenol liab (21041-025, Thermo Fisher Scientific) rau 30 min. Tom qab cov hlwb tau ntxuav ob zaug nrog HBSS, lawv tau muab qhwv rau ntawm daim npog npog thiab hauv qab ntawm lub tais, raws li tau piav qhia saum toj no. Kev siv phosphorescence los ntawm BTPDM1 nyob rau hauv cov kab lis kev cai uas npog nrog cov npog npog tau pom siv lub tshuab lim dej kom zoo thiab tso tawm, thiab BTPDM1 phosphorescence (Hirakawa li al., 2015) tau kuaj pom siv lub fluorescence microscope, BZ-X710 (Keyence Corporation). Cov duab tau los ntawm lub tshuab hluav taws xob inverted fluorescent tau hloov kho rau qhov ci thiab qhov sib txawv siv BZ-X tsom xam software.

2.4|Immunocytochemistry

Cov cell ntawm daim npog npog tau raug coj los siv nrog 200 µM pimonidazole HCl (HP3-100, Hypoxyprobe, Inc.) hauv DMEM / F12 yam tsis muaj phenol liab rau 1 h, tom qab ntawd cov ntaub npog tau tig thiab txuas rau lub hau npog iav tais, xws li piav yav tas los. Cov hlwb tom qab ntawd tau coj mus kuaj hauv DMEM / F12 yam tsis muaj phenol liab rau lub sijhawm teev tseg. Thaum tsis siv pimonidazole counterstaining, pretreatment nrog pimonidazole raug tshem tawm.

Tom qab ua tiav ntawm lub sijhawm kab lis kev cai, txhua daim npog tau raug sau, thiab cov hlwb tau kho tam sim nrog methanol / acetone (1: 1) ntawm cov dej khov, qhov chaw lawv nyob rau 30 feeb. Tom qab ntxuav ob zaug nrog Dulbecco's phosphate-buffered saline (PBS) (D5652, Sigma Aldrich), cell membrane tau permeabilized rau 30 min, incubated nrog 5 feem pua ​​​​bovine serum albumin (BSA) (A3059, Sigma Aldrich) rau 30 feeb, thiab nrog cov protein tsis pub dawb (X0909, DAKO) rau 10 feeb. Cov hlwb tau stained nrog thawj cov tshuaj tiv thaiv kab mob thiab tom qab ntawd nrog lub thib ob, fluorescent antibody. Cov npe ntawm thawj cov tshuaj tiv thaiv yog muab rau hauv Table S1. FITC-swine polyclonal anti-luv immunoglobulin (F0205, 1:20 dilution, DAKO) tau siv los ua thawj cov tshuaj tiv thaiv yog tias tus tswv tsev yog luav. Cov tshuaj fluorescent antibody Alexa Fluor 594 streptavidin (S11227, 1:500 dilution, Thermo Fisher Scientific) tau siv los ua thawj cov tshuaj tiv thaiv yog tus tswv tsev yog nas, ua raws li biotinylated anti-nas IgG (H plus L) (BA{{23}). }, 1: 1000 dilution, Vector Laboratories), raws li cov tshuaj tiv thaiv thib ob. Nuclear staining tau ua tiav siv bisBenzimide H 33342 trihydrochloride (B2261, Sigma Aldrich) rau txhua tus qauv.

Cov teeb liab fluorescent tau pom los ntawm kev siv lub tshuab hluav taws xob inverted fluorescent, BZ-X710 (Keyence Corporation) nrog cov ntxaij lim dej hauv qab no: Texas Liab nrog lub zog excitation wavelength (Ex) ntawm 560/40 nm, emission wavelength (Em) ntawm 630/75 nm, GFP ( Piv txwv li: 470/40 nm, Em: 525/50 nm), thiab DAPI (Ex: 360/40 nm, Em: 460/50 nm). Cov duab tau hloov kho rau qhov ci thiab qhov sib piv siv BZ-X tsom xam software.

2.5|ICC ntawm HIF ntawm HK-2 hlwb kho nrog cobalt chloride

Lwm txoj hauv kev tau hloov kho los tsim cov qauv npog ntawm HK-2 hlwb kho nrog cobalt chloride. HK-2 cov hlwb tau cog rau ntawm ib nrab-qhov ntom ntom (2.5 × 105 / phaj) ntawm daim npog npog rau Hnub 1 thiab kho nrog 300 µM cobalt chloride hexahydrate (C 8661, Sigma Aldrich) rau 16 teev ntawm Hnub 2. Hnub 3, cov ntaub ntawv npog tau hloov pauv los txuas cov xov tooj ntawm tes mus rau hauv qab ntawm 27 hli iav-raws li phaj tshiab rau 3 h, thiab ICC ntawm HIF tau ua tiav.

2.6|Western blotting

Txhawm rau tshawb xyuas HIF{{0}} tsub zuj zuj ntawm cov pa sib txawv ntawm cov pa sib txawv thiab ntawm qib pH sib txawv, 1.0 × 106 HK-2 hlwb ib 10 cm kab lis kev cai tais tau incubated nyob rau hauv normoxia, 2 feem pua ​​oxygen, 1 feem pua ​​oxygen, los yog anoxia, rau 5 h, los yog nyob rau hauv DMEM / F12 ntawm pH 7.4, pH 6.0, los yog pH 5.0 rau 5 h.

Cov hlwb no tau lysed hauv RIPA tsis muaj 50 mM Tris-buffer (pH 8.0), 150 mM NaCl, 0.5 w/v feem pua ​​sodium deoxycholate, 0.1 w/v feem pua ​​SDS, thiab 1.0 w/v feem pua ​​NP40.

Rau western blotting, SDS qauv tsis muaj {{0}}}.35 M Tris-HCl (pH 6.8), 10 feem pua ​​SDS, 36 feem pua ​​glycerol, 0.012% bromophenol xiav, thiab 0.1 M dithiothreitol (DTT) tau ntxiv rau cov proteins. Cov proteins uas muaj SDS cov qauv tsis tau raug tshem tawm los ntawm qhov kub ntawm 95 degree rau 5 min.

Cov proteins tau sib cais los ntawm electrophoresis ntawm 10 feem pua ​​SDS polyacrylamide gels. Cov proteins tau raug xa mus rau AmershamTM HybondTM PVDF membrane (10600023, GE Healthcare) hauv kev hloov pauv tsis zoo (48 mM Tris-base buffer, 39 mM glycine, 0.04% SDS, thiab 20 v / v feem pua ​​​​methanol) siv Trans-Blot®. TurboTM Transfer System (Bio-Rad). Cov daim nyias nyias tau muab tso rau hauv chav sov nrog cov tshuaj tiv thaiv thawj zaug, tshuaj tiv thaiv HIF1 los tiv thaiv lub cev (NB100-134, 1:500 dilution, Novus Biologicals, RRID: AB_350071), thiab tshuaj tiv thaiv actin antibody (A2066). , 1:2000 dilution, Sigma Aldrich, RRID: AB_476693), thiab tom qab ntawd hauv cov tshuaj tiv thaiv kab mob thib ob, polyclonal tshis tiv thaiv luav immunoglobulin / HRP (P0448, 1: 10000 dilution, DAKO, RRID: AB{{26 }}). PierceTM ECL Plus Western Blotting Substrate (32132, ThermoFisher Scientific) tau siv los kuaj xyuas. Chemiluminescence tau soj ntsuam siv Luminoimage Analyzer ImageQuantLAS4000mini (GE Healthcare). Reproducibility tau lees paub los ntawm kev ua yam tsawg kawg peb qhov kev sim ywj pheej. Qhov kev siv ntawm cov bands tau suav nrog siv National Institutes of Health ImageJ software (Schneider li al., 2012).

2.7|Luciferase tus neeg sau xov xwm soj ntsuam

Kev ntsuam xyuas tus neeg sau xov xwm luciferase tau ua los ntsuas HIF1 tsub zuj zuj hauv homogenous hypoxic kab lis kev cai incubation. Yav dhau los peb tau tsim ib lub cim npe luciferase uas tau tsav los ntawm lub ntsiab lus hypoxia-responsive (HRE) thiab tsim Hypoxia-Responsive Reporter Vector (Transgene Construction). Qhov no tau tsim los ntawm tandem luam ntawm HRE los ntawm nas vascular endothelial kev loj hlob yam noob noob sub-cloned rau hauv 5' cheeb tsam ntawm am CMV-promoter-luciferase transcription unit (pre-Luc) (Chiang et al., 2011; Tanaka et ib., 2004).

HK{{0}} cov hlwb ntawm qhov concentration ntawm 1.0 × 105 ib qhov dej tau npaj rau hauv 12-cov kab lis kev cai zoo (150628, Thermo Fisher Scientific). Cov hlwb raug co-transfected nrog 500 ng ntawm pGL3-Basic HRE-luciferase vectors thiab 30 ng ntawm pRL-SV40 Renilla luciferase tswj vectors (Promega), siv 2 µl FuGENE® HD Transfection Reagent (E2311, Promega) ib. zoo. HK-2 cells co-transfected nrog 500 ng ofpGL3-Basic firefly luciferase vectors thiab 30 ng ofpRL-SV40 Renilla luciferase tswj vectors (Promega) tau siv los ua kev tswj tsis zoo.

Cov kab mob kis tau raug tsim nyob rau hauv normoxia, 2% hypoxia, 1% hypoxia, lossis ib lub hnab anoxia, rau 5 teev. Tom qab ntawd cov hlwb tau sau tseg siv 100 µl ntawm passive protein lysis tsis, rau kev ntsuam xyuas dual luciferase. Ib qho LB9507 luminometer (EG thiab Berthold) tau siv rau kev ntsuas. Txhawm rau kho kom zoo rau kev hloov pauv, tus nqi txheeb ze ntawm firefly luciferase lub teeb chav tsev tau muab faib los ntawm Renilla luciferase.

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2.8|Analysis ntawm apoptosis

Cov kab mob qog noj ntshav tau npog nrog daim npog npog rau 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, thiab 24 h, thiab tom qab ntawd sau nrog trypsin. Cells kho nrog 3 feem pua ​​​​ntawm hydrogen peroxide (081- 04215, Wako Pure Chemical Industries, Ltd.) rau 30 feeb tau npaj ua kev tswj hwm apoptotic. Kev soj ntsuam ntau ntawm apoptosis tau ua tiav siv Muse Annexin V thiab Dead Cell Cov Khoom Siv (MCH100105, Millipore) hauv Muse™ Cell Analyzer (Millipore), raws li cov chaw tsim khoom cov lus qhia.

2.9|Quantitative real-time PCR (qRT-PCR)

Tag nrho RNA rho tawm thiab cDNA synthesis tau ua raws li cov chaw tsim khoom cov lus qhia rau RNAiso Plus (9109, Takara) thiab PrimeScript™ RT Master Mix (RR036B, Perfect Real Time) (Takara) raws. PCR Real-time tau ua tiav siv THUNDERBIRD SYBR qPCR Mix (QPS- 201, Toyobo) hauv CFX Txuas Real-Time PCR Detection System (Bio-Rad). Cov ntawv sau tau zoo li qub mus rau qib ntawm -actin mRNA qhia. qRT-PCR tau ua nyob rau hauv triplicate siv cov noob tshwj xeeb primers. HIF-1 tau nthuav dav siv rau pem hauv ntej, 5′-CCATTAGAAAGCAGTTCCGC-3′ thiab thim rov qab, 5′-TGGGTAGGAAGATGGAGATGC-3′ primers. -actin tau nthuav dav siv rau pem hauv ntej, 5′-TCCCCCAACTTGA GATGTATGAAG-3′, thiab thim rov qab 5′-AACTGGTCTCAAG TCAGTGTACAGG-3′ primers.

2.10|Nws Kev hloov pauv nrog siRNA

Txhawm rau tshawb xyuas RNAi-induced HIF-1 knockdown hauv HK-2 hlwb, 5.0 × 104 HK-2 hlwb ntawm ib qhov dej tau npaj rau hauv 6 lub qhov dej. Ob hom RNAi—HIF-1 siRNA (siHIF-1 #1 [HSS104774, Thermo Fisher Scientific] thiab siHIF-1 #2 [HSS104775, ThermoFisher Scientific])—tau siv nrog Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Raws li kev tswj tsis zoo, Stealth RNAi™ siRNA Negative Control Med GC Duplex #3 (12935-113) tau siv; 1.5 µl ntawm txhua siRNA thiab 5 µl RNAiMAX tau sib xyaw. siRNA- cov kab mob kis tau raug tsim nyob rau hauv cov xwm txheej normoxic lossis hypoxic (1 feem pua ​​O2) rau 48 teev thiab RNA raug rho tawm. Kev ua tau zoo ntawm-HIF-1 knockdown tau tshuaj xyuas siv qRT-PCR.

2.11|Nyob daim duab ntawm cov teebmeem ntawm PH gradient

Peb pom qhov pH gradient intracellular ntawm cov hlwb nyob hauv qab daim npog. Cells tau kho nrog pHrodo Green AM Intracellular pH Qhov Ntsuas (p35373, ThermoFisher Scientific) raws li cov chaw tsim khoom cov lus qhia, ua ntej siv cov qauv npog. Ib qho inverted fluorescence microscope, BZ-X710 (Keyence Corporation) nrog GFP lim tau siv los saib xyuas lub sijhawm hloov pauv ntawm fluorescence siv gradient hauv qab daim npog.

2.12|Quantitative tsom xam ntawm HIF nplhaib

2.12.1|Qhov chaw ntawm HIF lub nplhaib

Peb ntsuas qhov kev ncua deb ntawm HIF rings thiab pimonidazole-zoo thaj chaw los ntawm cov npoo npog siv ImageJ, raws li hauv qab no. Cov cheeb tsam ntawm lub voj voog sab nrauv thiab sab hauv ntawm HIF lub nplhaib thiab pimonidazole-zoo lub voj voog raug txiav txim, thiab lub vojvoog ntawm txhua lub voj voog raug xam. Txhua tus nqi raug rho tawm ntawm 7.5 hli, uas yog lub vojvoog ntawm 15 hli npog, txhawm rau suav nws qhov deb ntawm ntug npog. Peb qhov kev sim ywj pheej ntawm ICC ntawm HIF hauv txhua qhov xwm txheej tau ua.

2.12.2|Lub ntsiab lus ntawm HIF lub nplhaib

Peb siv cov duab fluorescent tau los ntawm cov qauv npog npog hauv qab normoxia thiab nruab nrab pH. Peb txhais qhov chaw ntawm lub nplhaib HIF thiab nws sab nraud thiab sab hauv li 2.5–3.0 nplai (0.22–0.45 mm), 0.5 –1. Peb ntsuas tsib qhov chaw ntawm HIF-1 cov cim rau ib qho qauv thiab suav qhov nruab nrab. Peb qhov kev sim ywj pheej ntawm ICC ntawm HIF tau ua.

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2.13|Kev ntsuas cov pa oxygen los ntawm PLIM duab

2.13.1|Kev tsim kho cov kab calibration los ntsuas cov pa oxygen

Kev ntsuas qhov nkhaus ntawm HK-2 hlwb tau tsim, raws li txoj hauv kev tau piav qhia hauv peb tsab ntawv tshaj tawm dhau los (Yoshihara li al., 2015). HK-2 hlwb loaded nrog 500 nM BTPDM1 rau 30 min tau coj mus rau hauv lub pa oxygen-concentration-changeable multi-gas incubator nruab nrog lub inverted fluorescent microscope uas txuas nrog lub neej ntsuas qhov system. Ib txoj kab calibration raws li kev soj ntsuam Stern-Volmer tau tsim los siv phosphorescence lifetime (PL) ntawm HK-2 hlwb nyob rau hauv ntau qhov sib txawv oxygen tensions siv

Kev sib npaug (1)

qhov twg τp sawv cev rau PL hauv pO2, τ0 sawv cev rau PL hauv deoxygenation, kq sawv cev tus nqi quenching tas li, thiab pO2 sawv cev rau ib nrab siab ntawm oxygen. Siv cov kab calibration no, cov pa oxygen siab tuaj yeem xam los ntawm PL.

2.13.2|PLIM duab ntawm tus qauv npog

HK-2 cell loaded nrog 500 nM BTPDM1 rau 30 min tau npaj. Cov qauv npog npog tau tsim thiab coj mus rau hauv O2 concentration-changeable multi-gas incubator nruab nrog lub tshuab hluav taws xob inverted fluorescent, txuas nrog lub neej ntsuas qhov system.

Phosphorescence lifetime imaging microscopy (PLIM) dluab raug kaw siv ib qho inverted fluorescence micro-scope nruab nrog ib tug confocal scanning system (Hirakawa li al., 2018). PLIM cov duab tau txais tom qab 30 min incubation hauv 21 feem pua ​​O2 lossis 4 feem pua ​​O2. Plaub PLIM cov duab ib qho piv txwv, los ntawm sab sauv, hauv qab, sab laug, thiab sab xis nyob ze ntawm ntug npog, tau coj los txiav txim siab qhov nruab nrab PL, suav nrog kev hloov pauv hauv PLs hauv daim npog.

2.13.3|Kev txheeb xyuas lub nplhaib HIF hauv daim duab PLIM

Pimonidazole tau zoo hauv qab 10 mmHg ntawm oxygen siab. PL sib npaug ntawm 10 mmHg ntawm cov pa oxygen siab yog 4034.6 ns, raws li txoj kab calibration, yog li cov kab sab nrauv ntawm lub voj voog pimonidazole hauv daim duab PLIM tuaj yeem txheeb xyuas. Tom qab ntawd, cov chaw ntawm sab nrauv thiab sab hauv HIF rings hauv PLIM duab tuaj yeem pom los ntawm kev siv qhov ntsuas ntsuas qhov deb. Peb ntsuas qhov nruab nrab PLs sib npaug rau HIF lub nplhaib hauv 21 feem pua ​​O2 thiab 4 feem pua ​​O2.

2.13.4|Kev ntsuas cov pa oxygen ntawm lub nplhaib HIF

Peb xam qhov ntau ntawm oxygen pressures ntawm HIF lub nplhaib los ntawm PLs, siv cov kab calibration. PLIM cov duab raug tshuaj xyuas siv SPCImage 5.0 (Becker & Hickl GmbH).

2.14|Kev txheeb cais

Dunnett qhov kev sim tau siv los sib piv cov kev sim thiab tswj pawg, thiab qhov tseem ceeb < .05="" raug="" txiav="" txim="" siab="" los="" qhia="" qhov="" sib="" txawv="" tseem="" ceeb.="" cov="" tub="" ntxhais="" kawm="" qhov="" kev="" xeem="" t-test="" tau="" siv="" los="" sib="" piv="" peb="" lossis="" ntau="" pawg,="" thiab="" bonferroni="" hloov="" p-tus="" nqi="" raug="" siv.="" txhua="" qhov="" kev="" txheeb="" cais="" tau="" ua="" tiav="" siv="" jmp="" pro="" ver13.2.1="" (sas).="" cov="" txiaj="" ntsig="" tau="" suav="" tias="" yog="" muab="" los="" ntawm="" cov="" pej="" xeem="" ib="" txwm="" faib,="" thiab="" tau="" pom="" tias="" yog="" qhov="" txhais="" tau="" tias="" ±="" tus="" qauv="" sib="" txawv="">

dab tsi yog cistanche

3|TSEEM CEEB

3.1|Oxygen gradient tsim nyob rau hauv tus qauv hypoperfusion

Peb tau lees paub tias muaj cov pa oxygen gradient nyob rau hauv cov qauv hypoperfusion induced los ntawm coverslip qhov chaw los ntawm ncaj qha soj ntsuam oxygen tension, siv phosphorescence. Phosphorescence siv qhov ntsuas tuaj yeem kwv yees qhov sib piv ntawm cov pa oxygen siab (Hirakawa li al., 2015; Yoshihara li al., 2015). Peb tau soj ntsuam phosphorescence siv ntawm cov qauv npog ntawm HK-2 hlwb kho nrog BTPDM1. Cov duab ntawm phosphorescence siv tau pom tias hypoxia nyob rau hauv feem ntau ntawm cov cheeb tsam hauv cov ntaub npog, tab sis tsis muaj hypoxia nyob ze ntawm ntug, ib qho kev soj ntsuam uas qhia tias muaj cov pa oxygen gradient nyob ib ncig ntawm ntug ntawm cov ntaub npog hauv HK-2 cov cell npog nrog ib tug daim ntawv npog (Daim duab 1a). Txij li qhov kev siv ntawm phosphorescence nyob ntawm qhov concentration ntawm qhov kev sojntsuam, thiab nyob rau lub sijhawm ex-citation, kev ntsuas phosphorescence lifetime (PL) yog qhov zoo rau kev soj ntsuam ntau ntawm oxygen siab (Yoshihara li al., 2015). Yog li, rau qhov ntsuas kom muaj nuj nqis ntawm cov pa oxygen nruj ntawm cov kab lis kev cai nyob ib puag ncig ntawm ntug ntawm daim npog npog, peb tau txais PLIM cov duab ntawm HK-2 cov cell npog nrog cov npog npog rau 30 min (Daim duab 1b). PL txuas ntxiv raws li qhov kev ncua deb ntawm cov npoo npog tau nce. Peb tau lees paub tias cov pa oxygen nro, suav nrog cov kab calibration (Daim duab S3), txo qis raws li qhov kev ncua deb ntawm cov npoo npog tau nce, muab pov thawj rau lub neej ntawm cov pa oxygen nyob ib ncig ntawm ntug ntawm cov ntaub npog (Daim duab 1c). Kev soj ntsuam ntawm lub cev ntawm lub cev ntawm cov phosphorescence siv tau qhia tias cov pa oxygen gradient pib tsim 10 min tom qab muab cov ntaub npog npog.

(Daim duab 1d).

3.2|Tshwj xeeb HIF-1 kev faib tawm hauv cov qauv hypoperfusion, "HIF nplhaib"

Peb tau tshuaj xyuas HIF-1 kev faib tawm hauv cov qauv hypoperfusion nrog cov pa oxygen gradient. Lub ICC ntawm HIF-1 nyob rau hauv HK-2 hlwb tau pom ib qho kev txhim kho zoo li lub khob noom cookie rau ntawm ntug ntawm thaj chaw pimonidazole-zoo, uas peb hu ua "HIF nplhaib" (Daim duab 2a). Qhov kev siv ntawm HIF-1 teeb liab tau siab dua ntawm HIF lub nplhaib dua li ntawm nws qhov chaw sab hauv lossis sab nrauv (Daim duab 2b). Qhov tshwm sim no tau lees paub tias siv ICC nrog lwm HIF-1 cov tshuaj tiv thaiv kab mob (Daim duab S4a) lossis lwm cov kab cell tubular (Daim duab S4b, c). Peb kuj tau ua ICC tsom xam ntawm HIF-1 hauv lwm hom kab lis kev cai, xws li HeLa hlwb qog noj ntshav. Txawm hais tias qhov tsis muaj zog adhesion ntawm cov cell mus rau ib daim npog npog tau ua rau kev soj ntsuam ntxaws ntawm HIF-1 kev faib tawm nyuaj, peb tau pom qhov ua kom zoo li lub khob noom cookie ntawm HIF-1 hauv HeLa hlwb (Daim duab S4d).

Lub nplhaib HIF thiab pimonidazole-zoo thaj chaw pib tsim ob peb teev tom qab muab cov ntaub npog npog, thiab lub nplhaib HIF tau tshwm sim zoo li qub los ntawm 3 h (Daim duab 2c). HIF-1 knockdown ua rau ploj ntawm lub teeb liab HIF-1 , suav nrog lub nplhaib HIF (Daim duab 2d, Daim duab S4e), qhia tias lub nplhaib HIF yog nyob ntawm HIF-1 .

Txhawm rau tshawb xyuas qhov ua tau tias HIF lub nplhaib tsim yog nyob ntawm cov pa oxygen, peb tau tshawb xyuas qhov kev faib tawm ntawm HIF-1 hauv cov qauv npog hauv qab incubation nrog cov pa sib txawv. Peb tso ib lub tais rau hauv cov chav tsev hypoxic nrog cov pa sib txawv ntawm cov pa oxygen tam sim ntawd tom qab ua cov qauv npog. Thaum lub sij hawm hypoxic incubation, peb pom tias ob lub nplhaib HIF thiab pimonidazole-zoo cheeb tsam tshwm sim nthuav tawm sab nraud (Daim duab 3a). Peb ntsuas qhov kev ncua deb ntawm txhua lub nplhaib HIF thiab thaj chaw pimonidazole-zoo los ntawm cov npoo npog hauv qab normoxia, 4 feem pua ​​O2, thiab 1 feem pua ​​O2 incubation. Ob lub nplhaib HIF thiab thaj chaw pimonidazole-zoo tau nthuav tawm sab nraud raws li cov pa oxygen nyob ib puag ncig txo qis (Daim duab 3b). Cov txiaj ntsig no tau qhia tias lub nplhaib HIF yog nyob ntawm HIF-1 thiab cov pa oxygen.

Txhawm rau kom paub meej tias cov khoom sib xyaw ua ke yog ib qho tshwm sim tshwj xeeb ntawm HIF, peb tau ua ICC nrog cov noob tu vaj tse hauv cov qauv npog. GAPDH thiab actin cov cim tau khaws cia thoob plaws thaj tsam ntawm daim npog npog thiab sib piv ntawm thaj tsam ntawm HIF lub nplhaib thiab lwm thaj chaw (Daim duab S5a, b).

3.3|Kev ntsuas qhov ntau ntawm oxygen siab ntawm HIF lub nplhaib

Peb tau lees paub lub nplhaib zoo li kev txhim kho ntawm HIF-1 tsub zuj zuj hauv cov kab lis kev cai uas npog nrog cov npog npog, ib qho tshwm sim nyob ntawm cov pa oxygen. Txhawm rau txiav txim siab qhov sib txawv ntawm cov pa oxygen, peb tau txheeb xyuas cov pa oxygen siab ntawm HIF lub nplhaib siv cov txheej txheem phosphorescence lub neej. Peb tau txais PLIM cov duab ntawm cov qauv npog tom qab 30 min incubation hauv 21 feem pua ​​O2 lossis 4 feem pua ​​O2 (Daim duab 4a). Lub nplhaib HIF tsim rau ntawm ntug ntawm lub voj voog pimonidazole hauv ICC ntawm HIF (Daim duab 3b). Peb tau txheeb xyuas qhov chaw nyob rau hauv daim duab PLIM uas yog sib npaug rau thaj tsam pimonidazole-zoo nyob rau hauv ICC ntawm HIF thiab tom qab ntawd tau txheeb xyuas qhov chaw sib raug rau lub nplhaib HIF (Daim duab 4b), siv ntau qhov kev soj ntsuam ntawm cov ntaub ntawv nyob deb ntawm lub nplhaib HIF thiab cov pimonidazole-positive area (Daim duab 3b). Peb kuj ntsuas qhov ntau ntawm PLs ntawm lub nplhaib HIF thiab tom qab ntawd siv cov kab calibration (Daim duab S3) los xam cov pa oxygen siab ntawm lub nplhaib HIF hauv 21 feem pua ​​O2 (4.20 [3.46–4.97] ~ 35.9 [28.5–44.9] mmHg). , thiab 4 feem pua ​​O2 (2.19 [0.21–4.32] ~ 20.4 [17.1–24.1] mmHg) (Daim duab 4c).

3.4|HIF-1 tsub zuj zuj nyob rau hauv homogeneous oxygen nro

Thaum lub zog tsis muaj zog ntawm HIF-1 sab nraum lub nplhaib HIF hauv cov qauv npog tuaj yeem raug ntaus nqi los ntawm hypoxia tsis txaus, cov neeg nyob hauv lub nplhaib, qhov twg HIF-1 yuav tsum tau ua kom muaj zog ntau dua los ntawm cov pa qis dua, yuav tsum yuav tswj tau los ntawm qhov tshwm sim tsis muaj oxygen. Txhawm rau tshuaj xyuas qhov tshwm sim uas ua rau muaj qhov tsis txaus siab tshaj plaws ntawm HIF-1 hauv thaj chaw anoxic tshwm sim tsuas yog hauv cov qauv hypoperfusion, peb tau tshuaj xyuas seb puas muaj kev sib raug zoo ntawm cov pa tension thiab HIF-1 tsub zuj zuj hauv incubation nrog homogeneous oxygen nro. Western blot tsom xam ntawm cell lysates nyob rau hauv sib txawv homogeneous oxygen tensions pom qhov nce hauv HIF-1 tsub zuj zuj thaum lub sij hawm anoxic incubation (Daim duab 5a). HRE-luciferase reporter assay kuj tau qhia tias HIF-1 tsub zuj zuj zuj zus nrog qis oxygen tension nyob rau hauv cov xwm txheej ntawm homogeneous oxygen tension (Daim duab 5b). Cov txiaj ntsig no tau qhia tias qhov sib sau ntawm HIF-1 yog oxygen tension-dependent, nrog rau qhov siab tshaj plaws tau pom nyob rau hauv ntau yam anoxic. Cov yam ntxwv ntawm HIF-1 yuav tsum sib txawv ntawm tus qauv hypoperfusion thiab tus qauv nrog homogenous oxygen nro. Peb kwv yees tias qhov tshwm sim tshwj xeeb ntawm HIF-1 tsub zuj zuj hauv cov qauv hypoperfusion no tuaj yeem txiav txim siab los ntawm lwm yam uas tsis yog cov pa oxygen.

3.5|HIF nplhaib tsim yog ywj siab ntawm PhD

Peb tau tshuaj xyuas seb HIF-1 degradation, lub peev xwm ntawm HIF-nplhaib tsim, puas tau kho hauv lub nplhaib HIF. Lub tswv yim no zoo li tsis muaj tseeb, vim tias hydroxylation los ntawm Ph.D., tus nqi-txheej txheem ntawm HIF degradation, yuav tsum tau oxygen raws li substrate. Cobalt chloride yog dav siv los ua tshuaj HIF stabilizer. Peb tsim cov qauv npog ntawm HK-2 hlwb kho nrog cobalt chloride thiab ua ICC tsom xam ntawm HIF. HIF-1 teeb liab tsis tau nce ntxiv hauv HIF lub nplhaib, uas tsis meej vim tias qhov nce ntxiv ntawm HIF-1 sab nraum lub nplhaib (Daim duab 6c). Lub ICC ntawm HK-2 hlwb nrog knockdown ntawm PHD2, uas ntseeg tau tias yog thawj HIF prolyl hydroxylase hauv kev sim kab lis kev cai ntawm tes (Strowitzki li al., 2019), ua tau zoo sib xws (Daim duab S6). Cov txiaj ntsig no tau qhia tias PHD-VHL axis ntawm HIF degradation tsis cuam tshuam rau HIF lub nplhaib tsim.

3.6|Kev cuam tshuam ntawm pH ntawm HIF-1 tsub zuj zuj

Ib daim ntawv tshaj tawm yav dhau los tau piav qhia txog kev siv cov qauv npog ntawm lub plawv myocytes uas muaj pH poob rau hauv cov ntaub npog (Pitts & Toombs, 2004). Peb tau tshawb xyuas pH hauv qab daim npog thiab nws qhov cuam tshuam ntawm HIF lub nplhaib tsim. Peb kho HK-2 hlwb nrog qhov ntsuas pH intracellular, qhov kev siv fluorescence uas tso cai rau kev ntsuas pH hauv cov hlwb. Cov duab nyob tau pom tias pH txo qis dua feem ntau ntawm thaj chaw sab hauv, tab sis tsis nyob ze ntawm ntug ntawm daim npog npog, qhia tias muaj pH gradient nyob ib ncig ntawm ntug hauv cov qauv npog (Daim duab 6a). Kev txheeb xyuas ntau ntawm kev sib raug zoo ntawm fluorescence siv thiab kev ncua deb ntawm lub npog npog tau pom tias yav dhau los tau nce siab vim qhov kev ncua deb tau nce, kev soj ntsuam uas txhawb nqa lub neej ntawm pH thiab oxygen gradients nyob ib ncig ntawm cov npoo npog (Daim duab 6b). Western blot tsom xam ntawm cell lysates nyob rau hauv homogeneous oxygen concentrations pom tias incubation nrog pH 5.0 nruab nrab suppressed HIF-1 accumulation nyob rau hauv hypoxic tej yam kev mob (Daim duab S7a–d). Peb kuj tau lees paub tias HIF-1 tsub zuj zuj nyob rau hauv hypoxia nyob rau hauv txawv pH tej yam kev mob txo qis dua pH 6.0 (Daim duab S8a–c).

Ib daim ntawv tshaj tawm hais txog qhov tseem ceeb ntawm cov kua qaub me me, ntawm pH nyob ib ncig ntawm 6.0. Raws li txoj kev tshawb no, reoxygenation tom qab hypoxia acidified cov xov xwm, uas ua rau nucleolar sequestration ntawm VHL. Nyob rau hauv lem, HIF degradation raug tiv thaiv nyob rau hauv C2C12 myotubes, PC12 neurons, thiab 786-0 raum mob qog noj ntshav (Mekhail li al., 2004). Hauv peb txoj kev tshawb fawb, tsis muaj kev hloov pauv hauv VHL kev faib tawm hauv tubular hlwb ntawm thaj tsam nrog HIF-1 tsub zuj zuj thiab cov uas nws tau raug txwv, hauv cov qauv npog nrog pH qhov tseem ceeb ntawm 5.0 mus rau 7.4 (Daim duab S9a–c). Tubular hlwb raug nthuav tawm rau ntau yam ntawm pH (Burke li al., 1999; Pavuluri li al., 2019; Raghunand et al., 2003), yog li peb tau tshawb xyuas qhov hloov pauv ntawm HIF lub nplhaib hauv xov xwm ntawm pH sib txawv. tus nqi ntawm 5.0 thiab 8.5. Lub nplhaib HIF tau pom meej meej hauv xov xwm nrog pH 8.5 thiab 7.4 (Daim duab 6c). Lub nplhaib HIF kuj muaj nyob ntawm pH 6.5 tab sis tsis pom (Daim duab 6c). HIF-1 teeb liab tau txo qis hauv tag nrho cheeb tsam ntawm daim npog npog ntawm pH 5.0. (Daim duab 6c). Peb tau ua qhov kev soj ntsuam ntau ntxiv ntawm qhov kev sib raug zoo ntawm lub nplhaib HIF thiab ntug ntawm thaj chaw pimonidazole-zoo (Daim duab 7a) thiab pom tias txoj haujlwm ntawm lub nplhaib sib txawv nyob ntawm pH. Lub voj voog sab hauv zoo li nthuav tawm sab nraud ntawm pH 6.5, piv nrog pH 7.4, thaum lub voj voog sab nrauv poob qis rau sab hauv ntawm pH 8.5, raws li ntug ntawm thaj chaw pimonidazole-zoo (Daim duab 7b, c). Peb kuj tau lees paub tias cov kev ua ub no ntawm tsev tu noob tau khaws cia hauv cov qauv npog hauv qab acidic incubation (Daim duab S10). Yog li, pH zoo li ua lub luag haujlwm tseem ceeb hauv cov txheej txheem hauv qab HIF lub nplhaib tsim.

4|Kev sib tham

Hauv txoj kev tshawb no, peb tau txheeb xyuas qhov sib txawv ntawm HIF-1 nyob rau hauv lub raum tubular hlwb, siv tus qauv ntawm hypoperfusion induced los ntawm coverslip tso. Peb pom tias kev tsim thiab kev saib xyuas ntawm HIF lub nplhaib tau tswj hwm los ntawm cov pa oxygen siab thiab pH, ob qho tib si muaj nyob rau hauv ib qho gradient nyob rau hauv cov npoo npog ntawm cov qauv npog. Raws li cov txiaj ntsig no, peb npaj cov txheej txheem ua tau rau HIF lub nplhaib tsim, koom nrog HIF-1 accumulating nrog txo cov pa tension, thiab cov tsub zuj zuj raug suppressed vim pH tsawg nyob rau hauv ib tug tej yam deb ntawm ib tug coverlip ntug (Daim duab 8 ).

Hypoperfusion yog induced los ntawm rarefaction ntawm microvasculature hauv CKD (Mimura & Nangaku, 2{{20}}}10; Nangaku, 2006). Hauv peb txoj haujlwm dhau los, peb tau siv hauv vivo imaging los ua kom pom qhov pom ntawm oxygen gradient nyob rau hauv lub raum tubular hlwb ntawm lub raum nas ib txwm (Hirakawa li al., 2018). Oxygen nro kuj tseem xav tias yuav muaj ntau yam hauv tubular hlwb hauv CKD. Txawm li cas los xij, nws tseem tsis tau paub meej tias qhov muaj nyob ntawm hypoperfusion nrog oxygen gradient cuam tshuam rau kev tiv thaiv kev tiv thaiv hypoxia, HIF. Hauv txoj kev tshawb no, peb tau tshawb xyuas HIF kev faib tawm hauv cov kab mob sib kis ntawm cov tubules nyob rau hauv cov qauv hypoperfusion thiab pom tias cov pa oxygen gradient nyob rau hauv cov kab mob raum tubular hlwb tau ua tiav ua qauv siv ib puag ncig iav npog. Cov ntaub ntawv pov thawj los ntawm kev sim siv homogeneous oxygen nro qhia tau tias HIF-1 , uas nws hydroxylation thiab tom qab degradation feem ntau yog nyob ntawm cov pa oxygen, khaws cia. Qhov ntawd yog tus nqi ntawm HIF-1 nce ntxiv thaum cov pa oxygen txo qis. Hauv peb cov qauv npog, txawm li cas los xij, HIF-1 tau raug tshem tawm hauv thaj chaw anoxic thiab muaj qhov sib txuam siab tshaj plaws ntawm qhov deb ntawm qhov npog npog. Qhov no lub khob noom cookie zoo li HIF tsub kom tsis tau pom ua ntej. Peb tau pom tias HIF lub nplhaib for-motion tuaj yeem pom tsis hais txog ntawm cov pa oxygen ntawm qhov chaw tsim khoom, tab sis nws qhov chaw nyob yog nyob ntawm lub zog oxygen. Qhov ntau ntawm oxygen nro ntawm lub nplhaib HIF tau ntsuas ntawm kwv yees li 4-20 mmHg siv PLIM nrog BTPDM1. Feem ntau cov kev tshawb fawb yav dhau los, uas tau tsom mus rau cov kab lis kev cai hauv ib qho chaw uas muaj cov pa sib xyaw ua ke, pom tias HIF{12}} cov protein ntau ntxiv hauv cov kab mob hypoxic lossis anoxic (Ameri li al., 2002; Carrera li al. , 2014). Yog li, hauv peb cov qauv, HIF lub nplhaib tsim yuav tsum tau cuam tshuam los ntawm ib qho tseem ceeb uas tsis yog cov pa oxygen; nws yog ywj pheej ntawm Ph.D., tus nqi-txheej txheej txheem ntawm HIF degradation. Ib qho kev ua tiav hauv kev nthuav dav ntawm cov txheej txheem ntawm HIF lub nplhaib tsim yog peb qhov kev tshawb pom ntawm lub luag haujlwm ntawm pH gradient, nrog rau cov pa oxygen gradient, hauv cov qauv npog-duab, uas txwv kev nkag mus ntawm cov xov xwm zoo ib yam li hauv vivo hypoperfusion. qauv. Hauv cov qauv hypoperfusion no, pH intracellular txo qis vim qhov kev ncua deb ntawm lub hau npog-slip edge nce, thiab pH gradient tau pom nyob ib ncig ntawm cov npoo npog. Peb tau pom tias HIF-1 tsub zuj zuj ntawm pH qis, nyob ib ncig ntawm pH 5.0, piv nrog rau kev tsim tawm ntawm qhov nruab nrab pH nyob rau hauv homogeneous oxygen nro. Lub npoo sab nraud ntawm lub nplhaib HIF nthuav tawm sab nraud ntawm pH 6.5, thiab sab hauv ntawm lub nplhaib HIF poob rau sab hauv ntawm pH 8.5, raws li ntug ntawm thaj chaw pimonidazole-zoo. Lub nplhaib HIF tau pom nyob rau hauv cov kua qaub, thiab HIF{26}} teeb liab ploj mus ntawm pH 5.0. Yog li, peb tau piav qhia qhov tseem ceeb ntawm pH rau HIF lub nplhaib tsim hauv cov qauv npog. Peb tau ua pov thawj tias lub nplhaib HIF tau tsim los ntawm kev tawm tsam ntawm HIF-1 tsub zuj zuj los ntawm pH qis hauv lub nplhaib.

Ntau qhov kev tshawb fawb yav dhau los siv txoj kev npog npog tau siv cov qog nqaij hlav cancer. Raws li ib tsab ntawv ceeb toom siv tib neeg hepatoma cell kab Hep3B, uas qhia txog cov pa oxygen-dependent redshift ntawm ntsuab fluorescent protein (AcGFP1), nyob imaging ntawm cov hlwb uas npog nrog ib daim npog daim duab qhia ib tug redshift raws li qhov deb ntawm ib tug coverlip ntug nce (Takahashi & Sato, 2010). Lub emission spectrum ntawm hepatoma hlwb hloov los ntawm lub wavelength ntawm ntsuab fluorescent protein (GFP) fluorescence rau cov liab raws li cov pa tensions txo. Hauv lwm txoj kev tshawb fawb, cov pa oxygen ntawm SCC-7 hlwb nrog ib puag ncig 15-mm npog npog tau ntsuas siv cov txheej txheem phosphorescence lub neej. Cov pa oxygen yog 6.9 mmHg thiab 166 mmHg, suav los ntawm PL, uas yog 3.89 µs thiab 893 µs, 0–2 mm sab hauv, thiab 0–1 mm sab nraud ntawm cov npoo npog, ntsig txog (Yoshihara li al., 2015). Txoj kev npog npog kuj tau siv nrog cov hlab plawv myocytes, ib qho system uas tau nyiam cov xim raws li kev cog lus hauv vivo ischemia-reperfusion qauv. Kelly thiab al qhia tias myocytes npog nrog ib daim npog npog tau pom cov kev hloov pauv ntawm lub sijhawm, nrog rau kev hloov pauv hauv mitochondrial membrane muaj peev xwm thiab plasma membrane dynamics, thaum kawg ua rau myocyte tuag. Lawv kuj tau pom tias cov pH ntawm lub cev ntawm cov myocytes npog nrog cov npog npog poob sai sai mus rau kwv yees li pH 4 nyob rau hauv nruab nrab ntawm daim npog npog (Pitts & Toombs, 2004). Tus qauv npog-slip, inhibiting oxygen los yog khoom noj khoom haus diffusion rau cov kab mob hauv cov kab lis kev cai hauv qab daim npog, tuaj yeem ua raws li tus qauv hauv vivo ntawm ischemic, ib txwm, thiab marginal zones hauv nruab nrab, sab nraud, thiab ntug ntawm daim npog npog feem. Ntau qhov kev tshawb fawb tau siv cov qauv no ua tus qauv ischemia-reperfusion ntawm myocytes hauv vitro (Chun et al., 2015; Pitts et al., 2008; Solhjoo & O'Rourke, 2015; Wang et al., 2012). Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, peb txoj kev tshawb fawb yog thawj zaug siv cov txheej txheem npog rau tubular cells. Hauv peb lub cev, qhov kev ncua deb ntawm cov npoo npog mus rau thaj tsam sib npaug rau 10 mmHg ntawm oxygen tension, qhov twg pimonidazole yuav tsum tig zoo, yog 1.22 hli, thiab cov pa tensions poob qis li xoom nyob rau hauv qhov kev ncua deb ntawm 2 hli los ntawm cov npog npog. . Cov txiaj ntsig no tuaj yeem ua tau raws li cov ntaub ntawv dhau los uas siv nyob ib puag ncig 15 hli npog, ua kom pom qhov muaj pa oxygen tsawg kawg li ntawm 2 hli ntawm ntug (Akiyama li al., 2018; Yoshihara li al., 2015). Peb tau tshuaj xyuas tus nqi ntawm apoptosis ntawm cov hlwb hauv cov ntawv npog (Daim duab S11) thiab pom tias GAPDH thiab actin tau khaws cia txawm tias nyob hauv lub nplhaib HIF, qhia tias lub nplhaib HIF tsis yog tshwm sim los ntawm cell apoptosis lossis tuag hauv HIF lub nplhaib.

Ntau qhov kev tshawb fawb tau ntsuas cov pa oxygen hauv lub raum siv ntau txoj hauv kev. Qee qhov piv txwv ntawm kev ntsuas cov pa oxygen ntawm lub raum ib txwm muaj raws li hauv qab no: 50 mmHg thiab 30 mmHg hauv cortex thiab medulla siv microelectrodes; thiab 49 mmHg thiab 41 mmHg nyob rau hauv S1 thiab S2 ntu ntawm tubular hlwb ntawm cortex siv PLIM (Hirakawa li al., 2017, 2018; Zhang et al., 2014). Cov pa oxygen ntawm lub raum mob yuav qis dua. Raws li tsab ntawv tshaj tawm dhau los siv cov pa microelectrodes, cov nas mob ntshav qab zib tau qis dua lub raum parenchymal oxygen nro: 36 mmHg thiab 11 mmHg hauv lub cortex thiab medulla raws li (Heyman li al., 2013; Palm et al., 2003). Txij li cov pa microelectrodes ntsuas qhov nruab nrab parenchymal oxygen nro, qhov dav dav yuav tsum nyob rau hauv cov mob raum. Lub intracellular oxygen nro txo ​​mus rau xoom mmHg nyob rau hauv ib tug qauv ntawm lub raum ischemic uas nws lateral lub raum hlab ntsha thiab cov hlab ntsha raug clamped (Hirakawa li al., 2015). Xav txog cov kev tshawb pom no, qhov ntau ntawm oxygen nro ntawm lub nplhaib HIF, kwv yees li 4-20 mmHg, zoo li plausible hauv vivo.

Peb tau tshawb pom ntxiv tias pH cuam tshuam rau HIF lub nplhaib tsim nrog rau cov pa oxygen. Txij li thaum tubular epithelial hlwb nyob rau hauv lub raum tau txuas mus rau cov kua tso zis, pH ntawm tubular hlwb yuav tsum raug cuam tshuam los ntawm cov zis. Xav txog ntau yam pH hauv cov zis, peb txiav txim siab los kawm cov hlwb incubated ntawm pH ntau ntawm 5.0–8.5. Muaj kev tshawb fawb txog qhov ntau ntawm pH hauv lub raum ib txwm. Ib txoj kev tshawb nrhiav pom tias pH ntawm lub cortex yog 7.39 ± 0. }}}.09, raws li ntsuas siv microelectrodes (Burke li al., 1999). Lwm txoj kev tshawb fawb, siv MRI-raws li pH duab, qhia pH qhov tseem ceeb ntawm 7.3 ± 0.13 thiab 7.0 ± 0.29 raws li (Raghunand li al., 2003). Nws tau tshaj tawm tias lub raum pH txo qis los ntawm 6.5 txog 6.32, thiab qis dua li 5.83 hauv qhov mob hnyav, hauv nas qauv ntawm CKD nrog acidosis (Pavuluri li al., 2019). Cov kev soj ntsuam no tau muab pov thawj ntawm pH poob thiab hloov pauv hauv CKD. Txawm li cas los xij, qhov cuam tshuam ntawm pH ntawm HIF-1 tshwm sim los ntawm kev mob hypoxia hauv CKD tsis tau kuaj pom zoo. Hauv txoj kev tshawb fawb tam sim no, kev hloov pauv ntawm HIF lub nplhaib tau pom ntawm pH 6.5 thiab pH 7.4, pH ntau yam uas yog plausible hauv CKD. Qhov txiaj ntsig no txhawb qhov kev xav tias qhov qis me me hauv pH cuam tshuam rau HIF-1 tsub zuj zuj hauv CKD. Raws li cov ntaub ntawv pov thawj ntawm pH poob qis dua 6.0 hauv qhov mob hnyav ntawm CKD, nws tseem yuav tsum tau soj ntsuam HIF-1 lub cev lub cev ntawm pH qis. Txawm hais tias muaj cov ntaub ntawv tshaj tawm tias ib puag ncig acidic, txawm tias nyob hauv normoxia, stabilizes HIF-1, feem ntau ntawm cov ntawv ceeb toom no tau tshawb xyuas cov mob me me acidity, tshaj pH 6.0 (Filatova li al., 2016; Mekhail li al., 2004) . Hauv kev ua haujlwm no, peb tau pom kev txwv ntawm HIF-1 tsub zuj zuj hauv tubular hlwb ntawm pH 5.0, thiab attenuation ntawm HIF-1 cov cim hauv cov qauv npog npog ntawm pH 5.0. Yog li, lub raum pH txo qis hauv CKD hauv vivo tuaj yeem cuam tshuam nrog kev ua haujlwm tsis txaus HIF, nrog rau cov tshuaj uremic toxins hauv lub raum hypoxic, ua rau CKD nce ntxiv (Tanaka li al., 2013).

Peb txoj kev tshawb fawb muaj ntau yam kev txwv. Ua ntej, hauv thaj tsam ntawm hypoperfusion, kab lis kev cai xov xwm yuav tsum tsis muaj cov as-ham, ntxiv rau qhov muaj oxygen tsis txaus thiab txo pH. Txawm li cas los xij, nws tsis yooj yim rau delineate cov teebmeem ntawm pH, oxygen tension, thiab lwm yam vim li cas los ntawm hypoperfusion. Hauv lub cev, ischemia, pathological hypoperfusion ntawm cov ntshav tshwm sim los ntawm kev sib xyaw ua ke ntawm cov pa oxygen thiab cov as-ham uas tsis tshua muaj, tshwm sim hauv cov kabmob thiab cov hlwb. Nws yog ib qho tseem ceeb kom nkag siab qhov sib txawv ntawm hypoxia sensing thiab ischemia sensing. Txawm hais tias muaj teeb meem hauv kev nthuav tawm cov txiaj ntsig lom neeg ntawm txhua yam, peb tus qauv hypoperfusion yog lub cev plausible, ua raws li cov xwm txheej hauv vivo. Qhov thib ob, ua tus qauv npog yuav tsum muaj kev txawj ntse thiab kev xyaum, txij li feem ntau ntawm cov hlwb qee zaus tau raug tshem tawm thaum tshem tawm cov npog npog, tshwj xeeb tshaj yog thaum siv cov txheej txheem ib txwm siv (Daim duab S2a). Qhov teeb meem no nyob ntawm cov kab ntawm tes siv. Piv txwv li, nws nyuaj rau peb siv cov qauv npog rau HEK 293 lub hlwb vim tias lawv qhov adhesion rau daim npog npog yog qhov tsis muaj zog. Thib peb, nws yog qhov tsim nyog kom txo qis kev puas tsuaj rau cov kab lis kev cai hauv qab daim npog. Tus naj npawb ntawm cov hlwb puas tau nce ntxiv raws li lub sijhawm uas lawv tau them ntxiv, raws li qhia los ntawm kev txheeb xyuas ntawm apoptosis. Peb tau txiav txim siab tias 3 h yog qhov tsim nyog rau kev kwv yees lub nplhaib HIF thaum nws zoo li zoo li qub, thiab kwv yees li 10 feem pua ​​​​ntawm cov hlwb ua apoptotic (Daim duab S11). Qhov kev txwv thib plaub yog qhov nyuaj ntawm kev txuas cov kab lis kev cai rau lub npog ntsej muag hauv txhua qhov qauv (Daim duab S2b, c). Qhov sib txawv ntawm kev sib txuas ntawm ib txwm siv thiab lwm txoj hauv kev los ua tus qauv npog-duab kuj tuaj yeem cuam tshuam txoj kev kawm. Peb ntsuas qhov sib npaug ntawm cov pa oxygen sib npaug rau HIF lub nplhaib los ntawm cov txheej txheem phosphorescence lub neej, uas peb tau tsim cov qauv npog siv ib txwm siv. Txij li thaum lub nplhaib HIF thaum lub sij hawm immunocytochemistry tau pom los ntawm kev siv lwm txoj kev, qhov tseeb oxygen nro yuav txawv. Peb txo qis qhov yuam kev no los ntawm kev siv cov pov thawj tias pimonidazole tau zoo ntawm 10 mmHg lossis tsawg dua ntawm cov pa oxygen. Qhov thib tsib txwv yog tias pH intracellular tsis tas yuav zoo ib yam li pH ntawm kab lis kev cai nruab nrab. Ntau cov ntawv tshaj tawm yav dhau los tau pom tias pH intracellular zoo ib yam nrog nruab nrab pH rau qee qhov hauv cov kab lis kev cai (Michl li al., 2019), peb tuaj yeem ntsuas tsuas yog qhov sib txawv hauv pH intracellular los ntawm kev hloov pH nruab nrab. Hauv vivo, kev sib raug zoo ntawm pH ntawm thaj chaw intracellular thiab extracellular, xws li cov zis los yog cov kua hauv lub cev, yog qhov nyuaj dua li hauv cov kab lis kev cai. Yog li, kev tshawb fawb ntxiv txog txoj hauv kev uas pH hloov pauv tswj kev sib sau ntawm HIF-1 protein nyob rau hauv vivo yuav tsim nyog rau yav tom ntej.

Cistanche txiaj ntsig: txhim kho raummuaj nuj nqi

Lwm qhov kev txwv yog tias pH yuav tsum muaj kev cuam tshuam rau hypoxia marker, pimonidazole. Peb piv qhov kev ncua deb ntawm ntug ntawm pimonidazole-zoo cheeb tsam los ntawm qhov chaw ntawm ib daim ntaub npog nyob rau hauv txawv pH tej yam kev mob nyob rau hauv peb cov qauv npog. Qhov ntug ntawm thaj chaw pimonidazole-zoo yog piv ntawm pH 6.5, 7.4, thiab 8.5 (Daim duab S12a, b). Ib txoj kev tshawb fawb yav dhau los kuj tau qhia txog kev saib xyuas ntawm pimonidazole khi ntawm pH qhov sib txawv (Kleiter li al., 2006). Raws li cov ntaub ntawv pov thawj no, peb tau xaus lus tias pimonidazole yog tus cim hypoxia tsim nyog rau peb cov kev sim npog npog. Qhov kawg txwv yog tias muab ntau qhov kev sib zog ntawm cov pa oxygen ntawm kwv yees li 4 mmHg thiab 20 mmHg (0.52 feem pua ​​O2 txog 2.6 feem pua ​​O2) ntawm HIF lub nplhaib, Pt(II)- thiab Pd(II)-porphyrins, uas tau siv dav hauv kev lom neeg. oxygen probes, muaj qhov zoo rau kev ntsuas cov pa oxygen tsawg heev vim tias lawv lub neej ntev dua phosphorescence piv nrog cov siv Ir (III) complexes (Yoshihara li al., 2017). Txawm li cas los xij, feem ntau, kev ntsuas cov pa oxygen ntau raws li phosphorescence, suav nrog Stern-Volmer equation, muaj kev ua tau zoo dua ntawm qhov qis O2 (Papkovsky & Zhdanov, 2016). Ob peb cov kev tshawb fawb yav dhau los tau qhia txog Ir(III) complex-raws li ntsuas ntawm oxygen tensions tsawg li ntawm 10 mmHg (Akiyama li al., 2018; Yoshihara li al., 2015). Yog li ntawd, peb ntseeg hais tias qhov ntau ntawm oxygen nro ntawm lub nplhaib HIF yuav tsum yog qhov tseeb.

5|Cov lus xaus

Hauv cov ntsiab lus, peb pom tias kev nkag siab lub luag haujlwm ntawm ob qho tib si oxygen thiab pH yog qhov tseem ceeb rau kev nkag siab txog HIF-1 lub cev lub cev hauv CKD, thiab tuaj yeem tau txais los ntawm kev tshawb xyuas tubular hlwb nrog hypoperfusion. Cov qauv npog npog, nrog rau nws cov kev txwv ntawm kev nkag mus ntawm kev tshaj tawm, yog ib qho qauv zoo ntawm hypoperfusion hauv vivo, tshwj xeeb tshaj yog nyob rau hauv cov tubules hauv CKD, vim qhov tsis tshua muaj ntawm peritubular capillaries yog ib qho tseem ceeb ntawm CKD (Mimura & Nangaku, 2010; Nangaku, 2006). Tus qauv hypoperfusion no tuaj yeem ua raws li cov pa oxygen thiab pH gradients hauv vivo, xws li qog nqaij hlav thiab ischemic lesions. Tus qauv muab ib txoj hauv kev zoo rau kev elucidation ntawm cov txheej txheem lom neeg.

Kev tsis sib haum xeeb ntawm kev txaus siab

Txhua tus kws sau ntawv tsis muaj teeb meem ntawm kev txaus siab los tshaj tawm.

AUTHORS 'CONTRIBUTIONS

Txhua tus kws sau ntawv tau pab tsim lub tswv yim tag nrho. TH tau ua qhov kev sim tag nrho. TH thiab YH txheeb xyuas cov txiaj ntsig thiab ua cov duab. TH sau cov ntawv sau qub. KM, TY, thiab ST tau ua qhov kev sim siv cov txheej txheem phosphorescence lub neej thiab kho ib feem ntawm cov ntawv sau. YH, TT, thiab MN kho tag nrho cov ntawv sau. Txhua tus kws sau ntawv tau nyeem thiab pom zoo cov ntawv sau kawg.

CEEB TOOM

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