Kev nthuav qhia Tus Qauv Ntawm -Tubulin, Inversin Thiab Nws Lub Hom Phiaj Dishevelled -1 Thiab Morphology ntawm Thawj Cilia hauv Tib Neeg Lub raum Kev Loj Hlob Thiab Kab Mob
Mar 10, 2022
Abstract:Lub spatiotemporal qhia ntawm -tubulin, inversion, thiab disheveled-1 (DVL-1) cov proteins txuam nrog Wnt-signaling pathway, thiab thawj cilia morphology tau soj ntsuam hauv kev tsimlub raum(14th – 38th lub lis piam txoj kev loj hlob), noj qab nyob zoo postnatal (1.5- thiab 7-xyoos), thiab pathologically hloov tib neeglub raum,suav nrog multicystic dysplasticlub raum(MCDK), focal segmental glomerulosclerosis (FSGS) thiab nephrotic syndrome ntawm Finnish type (CNF). Kev tsom xam tau ua los ntawm ob chav immunofluorescence, electron microscopy, semiquantitative thiab statistical method. Cytoplasmic co-expression ntawm -tubulin, inversion, thiab DVL-1 tau pom nyob rau hauv proximal convoluted tubules (pct), distal convoluted tubules (dct), thiab glomeruli (g) ntawm cov ntaub so ntswg soj ntsuam. Thaumraumtxoj kev loj hlob, tag nrho cov lus qhia ntawm -tubulin, inversion, thiab DVL-1tso tseg, thaum lub sij hawm postnatal me ntsis nce. Qhov siab tshaj plaws ntawm -tubulin thiab inversion characterized dct thiab g, thaum siab DVL-1 characterized pct. -tubulin, inversion, thiab DVL-1 cov qauv qhia hauv MCDK, FSGS, thiab CNFlub raumho txawv ntawm kev noj qab haus huv tswj. Muab piv rau noj qab nyob zoolub raum, pathologically hloovlub raummuaj dysmorphic thawj cilia. Kev qhia txawv txav ntawm -tubulin, inversion, thiab DVL-1 thaumraumkev loj hlob tuaj yeem qhia tau tias kev hloov pauv ntawm canonical thiab noncanonical Wnt-signaling yog qhov tseem ceeb rau qhov qubraummorphogenesis. Nyob rau hauv sib piv, lawv cuam tshuam qhia nyob rau hauv pathologicallub raumTej zaum yuav cuam tshuam nrog qhov txawv txav ntawm thawj cilia, ua rau mob ntevkab mob raum.
Ntsiab lus:tib neeg lub raum kev loj hlob; - tubulin; inversin; DVL-1; MCDK; FSGS; CNF; lub cev, lub cev
CISTANCHE yuav txhim kho rau lub raum/ raum mob
Taw qhia
Kev loj hlob ntawm qhov tseeb lossis metanephriclub raumpib thaum lub lim tiam thib tsib lub cev xeeb tub (GW), uas tom qab ntawd sib txawv tsis tu ncua los tsim lub raum mus tas li [1,2]. Cov kev taw qhia kev sib cuam tshuam ntawm metanephric mesenchyme thiab ureteric bud ua kom zooraumkev loj hlob thaum lub sij hawm nephrogenesis. Luv luv, lub ureteric bud induces mesenchymal-rau-epithelial hloov (MET) nyob rau hauv metanephric mesenchyme, uas condensates thiab cov ntaub ntawv.lub raumvesicles, ua raws li comma-shaped thiab S-shaped lub cev, thiab thaum kawg ua rau glomeruli tsim [3]. Nyob rau hauv rov qab, lub mesenchyme induces ntxiv branching ntawm lub ureteric bud. Lub raum metanephric dhau los ua cov khoom ua haujlwm ua haujlwm ntawm lub lim tiam 11 ntawm tib neeg txoj kev loj hlob. Txawm li cas los xij, nephrogenesis tau ua tiav nyob rau hauv 34th mus rau 36th lub lim tiam ntawm kev loj hlob ntawm fetus, thaum muaj ntau yam kev tshwm sim tiav lawm, tab sis cov txheej txheem sib txawv ntawm ob lub raum txuas ntxiv mus rau lub sijhawm postnatal [4,5]. Kev cuam tshuam ntawm cov kev cuam tshuam tsis zoo no ua rau muaj ntau yam txawv txav ntawm lub raum thiab cov zis (CAKUT), suav nrog dysplasia, polycystic raum kab mob, multicystic dysplastic.mob raum(MCDK), yog li ua rau mob ntevmob raum(CKD) [6].
Hauv txoj kev tshawb no, peb tau tsom mus rau thawj cilia zoo li thiab Wnt-signaling txoj hauv kev, uas ua lub luag haujlwm tseem ceeb thaum lub sij hawm ib txwm muaj nephrogenesis thiabraumkho cov txheej txheem, tom qab mob hnyav lossis mob ntevmob raum[7]. Lub hav zoov ntawm thawj cilia thaum lub sij hawm nephrogenesis thiab ntau ntawm kev loj hlobraumCov teeb meem tshwm sim nyob rau hauv cov neeg mob uas muaj tus kab mob ciliary taw qhia tias qhov tseem ceeb cilia muaj nuj nqi yog qhov tsim nyog rau qhov qubraumorganogenesis [8,9]. Thawj cilium yog microtubule-raws li organelle tseem ceeb rau cov ntaub so ntswg homeostasis, qhov twg -tubulin yog lub hauv paus tivthaiv [10]. Kev poob ntawm -tubulin acetylation hauv cov hlwb tsis txawj tuag ua rau kev hloov pauv ntawm epithelial-to-mesenchymal (EMT), yog li qhia tias acetylated -tubulin tseem ceeb hauv kev ruaj khov ntawm microtubules [11]. Thaum lub sij hawm tib neeg txoj kev loj hlob, thawj cilia-mediated Wnt txoj kev ua rau cov cell proliferation, sib txawv thiab cov ntaub so ntswg morphogenesis [12]. Tus naj npawb tseem ceeb ntawm cov menyuam yaus uas muaj kev cuam tshuam thawj cilia muaj nuj nqi thiab cuam tshuam Wnt-signaling txoj hauv kev tsim CKD, qhov chaw yug me nyuam.lub raumkev tsis sib haum xeeb yog lub luag haujlwm rau yuav luag ib nrab ntawm cov xwm txheej [13]. Feem ntau congenital cystic kab mob nyob rau hauv cov me nyuam yog multicystic dysplasticmob raum(MCDK) [14]. Ntau lub hlwv uas tsis sib txuas lus [15] thiab tsis muaj qhov qublub raumparenchyma yog cov yam ntxwv ntawm kev tshawb pom microscopic rau MCDK. Focal segmental glomerulosclerosis (FSGS) yog ib qho ntawm cov kab mob glomerular feem ntau uas coj mus rau theem kawg.mob raum[16]. Thaum pib, FSGS yog tus yam ntxwv ntawm kev ua focal, tsuas yog ib feem ntawm cov glomeruli thiab segmental, nrog rau kev hloov pauv hauv ib ntu ntawm lub voj voog glomerular [17]. Congenital nephrotic syndrome ntawm Finnish hom (CNF) yog ib qho tsis tshua muaj autosomal recessively inherited.mob raumsawv cev los ntawm kev tshwm sim ua ntej yug me nyuam ntawm kev poob protein ntau [18], cuam tshuam nrog cystogenesis ntawm proximal tubules [19]. Ntau hom kab mob thawj glomerular tsim cov proteinuria los ntawm kev puas tsuaj glomerular filtration barrier [20]. Ntau qhov kev tshawb fawb pom tau hais tias kev raug mob thiab kev ua haujlwm tsis zoo ntawm cov podocytes muaj lub luag haujlwm tseem ceeb hauv cov kab mob ntawm CKD proteinuria [21]. Txoj kev tshawb no los ntawm Vukojevic et al. qhia tau hais tias muaj ntau ntxiv ntawm ciliated thiab tsis zoo sib txawv podocytes hauv CNF [22]. Cov kev tshawb fawb yav dhau los tau qhia tias Wnt / -catenin signaling txoj hauv kev muaj lub luag haujlwm tseem ceeb hauv kev ua haujlwm ntawm podocyte tsis ua haujlwm nrog cov proteinuria [23]. Muaj ob txoj hauv kev Wnt-signaling, ib qho hu ua canonical -catenin-dependent thiab lwm yam noncanonical, -catenin ywj siab. Thaum nasraumtxoj kev loj hlob, canonical Wnt-signaling yog ua haujlwm ntawm cov lus qhia ureteric bud thiab hauv S-zoo li lub cev [24]. Tsis tas li ntawd, Wnt ntawm canonical signaling muaj feem xyuam rau kev tswj xyuas MET thaum lub sij hawm nephrogenesis [25]. Namely, khi ntawm cov protein complex uas muaj Dvl rau daim nyias nyias receptor yog yuav tsum tau rau Wnt txoj kev ua kom, thaum disabling cov ntsiab lus tseem ceeb ntawm txoj kev no ua rau lub perinatal tuag thaum ntxov vim tsis muaj nephrogenic cheeb tsam nyob rau hauv lubraum[25].
Ib qho protein tseem ceeb raws li kev hloov pauv molecular ntawm canonical thiab noncanonical Wnt signaling txoj hauv kev tau pom tias yog inversion [26]. Txawm hais tias kev sib cuam tshuam ntawm ntau yam proteins nrog inversion tau raug kuaj pom, nws txoj haujlwm tseeb tseem tsis tau qhia meej meej. Hauvlub raumepithelial hlwb, inversion nyob rau hauv thawj cilia, ua raws li ib tug ciliary protein [27]. Tsis tas li ntawd, inversion tsim ib qho chaw ruaj khov nrog tubulin hauv kab lis kev cai ntawmlub raumcell, thiab lawv colocalize hauv vivo [27,28]. Inversion zoo li ua lub luag haujlwm tseem ceeb hauv kev ua haujlwm ntxov ntawm pronephric system thiab sab laug-txoj cai symmetry kev txiav txim siab thaum lub sij hawm kev loj hlob [29,30]. Ib qho xwm txheej tseem ceeb rau ob qho tib si canonical thiab noncanonical Wnt-signaling txoj hauv kev nrhiav neeg ua haujlwm ntawm cytoplasmic Dvl mus rau lub cell membrane. Txij li cov kev tshawb pom yav dhau los tau pom tias kev hloov pauv thiab Dvl colocalize nyob rau hauv lub raum epithelial hlwb, nws tuaj yeem kwv yees tias kev hloov pauv kuj tseem muaj lub luag haujlwm hauv kev teeb tsa tsis yog. Dysregulation ntawm Wnt-signaling mediated los ntawm inversion ua rau aberrant proliferation hauv tubules [31]. Kev ntsuam xyuas caj ces tau nthuav tawm DVL-1 kev hloov pauv hauv cov neeg mob uas muaj tus mob autosomal-dominant Robinow syndrome, nthuav tawm cov kab mob sib txawv hauv qee cov neeg mob cuam tshuam nrog urogenital thiablub raumkev tsis sib haum xeeb [32]
CISTANCHE yuav txhim kho lub raum / raum mob ntshav qab zib
Cov qauv kev loj hlob ntawm -tubulin, inversion thiab DVL-1 tau raug tshawb xyuas hauv cov qauv tsiaj sib txawv. Yog li, lawv cov lus qhia hauv pronephros ntawm Xenopus laevis tau raug lees paub los ntawm kev siv intravital microscopy [31], thaum lawv tau pom nyob rau hauv cov kab ntawm tes muab los ntawm murine proximal tubular hlwb los ntawm kev siv confocal microscopy thiab loj spectrometry [33]. Tsis tas li ntawd, lub teeb microscopy thiab ob chav immunofluorescence qhia lawv muaj nyob rau hauv cov naslub raum[34]. Cov kev tshawb fawb ntawm inversion knockout nas qhia kom loj, diffused cysts nyob rau hauv lublub raummedulla thiab cortex txuam nrog situs visceral inversion [35]. Hauv tib neeg, kev hloov pauv ntawm kev hloov pauv tau nthuav tawm nrog cov menyuam mos nephronophthisis uas ua rau kev txiav cev xeeb tub [36]. Txawm tias muaj ntau yam kev tshawb fawb ntawmlub raum-tubulin, inversion thiab DVL-1 kev qhia qauv thiab kev ua haujlwm, rau qhov zoo tshaj plaws ntawm peb txoj kev paub, ib qho qauv qhia ntawm cov proteins nyob rau hauv tib neeg fetal thiab postnatal tib neeg cov ntaub so ntswg tseem tsis tau tshawb nrhiav thiab coj mus rau kev sib raug zoo. Tsis tas li ntawd, tseem tsis tau muaj pov thawj qhia txog kev qhia txog kev hloov pauv thiab DVL-1 hauv kev txhim kho tib neeg thaum ntxov. Yog li, qhov kev tshawb fawb no tau tsom mus rau kev txheeb xyuas cov qauv ntawm lub cev thiab lub cev ntawm -tubulin, inversion thiab DVL-1 thaum lub sij hawm fetal thiab postnatal theem ntawm cov tib neeg noj qab nyob zoo.lub raum, zoo li hauvraumcov ntaub so ntswg ntawm MCDK, FSGS thiab CNFlub raumtxhawm rau tsim kom muaj qhov tsis tuaj yeem txuas ntawm cov chaw no. Namely, peb npaj siab hais tias cuam tshuam cov qauv ntawm -tubulin, inversion thiab DVL-1 cov proteins tej zaum yuav yog cov txheej txheem cystogenesis thiab ib qho kev ua haujlwm txawv txav ntawm thawj cilia ua rau mob ntev.kab mob raum.
Cov txiaj ntsig
Kev tsom xam ntawm kev loj hlob thiab postnatalraumcov ntaub so ntswg tau ua los ntawm cov qauv sib txawv ntawm cov qauvraum:Cov tubules sib thooj (pct), cov tubules distal convoluted (dct) thiab glomeruli (g). Thaum lub sij hawm ntsuam xyuas kev loj hlob thiab nyob rau hauv postnatalraumcov ntaub so ntswg, -tubulin, inversion thiab DVL-1 tau pom cov qauv qhia zoo tab sis nrog qhov sib txawv ntawm kev siv, kev faib tawm thiab ntau. Analysis ntawm pathologicalcov ntaub so ntswgntawm MCDK, FSGS thiab CNF tau ua tiav ntawm tag nrho cov duab suav ntawm cov hlwb zoo piv rau kev noj qab haus huv raum cov ntaub so ntswg.
2.1. Lub teeb Microscopy, Electron Microscopy thiab Immunohistochemistry (-Tubulin) ntawm Kev Noj Qab Haus Huv thiab Pathologically Hloov Postnatal Tib Neeg Raum Cov Kab Mob
2.1.1. Noj qab nyob zoo Postnatal raum teeb
microscopy ntawm postnatalraumcov ntaub so ntswg qhia tau hais tias zooraumcov qauv, nrog qhov sib txawv ntawm medulla thiab cortex thiab tsim ntawm pct, dct, thiab g hauv thaj tsam cortical. Immunohistochemical staining ntawm -tubulin qhia pom muaj ib qho tseem ceeb cilium nyob rau hauv nruab nrab ntawm lub apical cell nto ntawm txhua lub tubular cell thiab nyob rau hauv 0.018 feem pua ± 0.00014 feem pua SD nyob rau saum npoo ntawm glomerular hlwb. Electron microscopy qhia pom tias tsuas yog ib qho tseem ceeb cilium tshwm sim los ntawm lub cev basal ntawm lub apical cell nto ntawm tubule (Daim duab 1a).
2.1.2. CNF
Feem ntau ntawm cov kev soj ntsuam g yog me me thiab lobulated (80 feem pua), thaum cov haiv neeg tsawg yog qhov loj me lossis hypertrophic (20 feem pua, n=100). Segmental lossis thoob ntiaj teb sclerosis ntawm g yog pom, thaum pct thiab dct yog ib nrab dilated thiab coated nrog atrophic epithelium. Ultrastructurally, poob ntawm microvilli thiab txo qis hauv cell qhov siab nrog multifragmentation ntawm nuclei yog pom. Hauv podocytes, cov txheej txheem ko taw yog diffusely ploj. Tubular thawj cilia yog qhov tsis sib xws, tshwj xeeb tshaj yog nyob rau hauv cov tubules 'cysts, qhia txog kev hloov ntawm qhov ntev thiab cytoplasmic txoj hauj lwm (Daim duab 1b).
2.1.3. MCDK
Nyob rau hauv lub raum cov ntaub so ntswg, sporadic immature thiab mature g tuaj yeem pom.Lub raumcov ntaub so ntswg puv nrog ntau oval cysts. Columnar epithelial hlwb npog cov hlwv thiab ncig los ntawm cov ntaub so ntswg xoob. Immunohistochemical staining rau -tubulin qhia ntau qhov ntev thiab tsis sib haum xeeb thawj cilia nyob rau saum npoo ntawm cov hlwb epithelial (Daim duab 1c).
2.1.4. FSGS
Nyob rau hauv 90 feem pua ntawm g, qhov dav segmental sclerosis pom (n=100), txuam nrog interstitial fibrosis thiab cov cim qhia ntawm tubular puas. Qhov txo qis hauv qhov siab ntawm cov hlwb epithelial nrog kev poob ntawm microvilli yog pom nyob rau hauv electron microscope, thaum lub sij hawm endothelial hlwb thiab mesangial cheeb tsam muaj ib txwm ultrastructure. Hauv electron microscope, qhov ntev heev thiab dislocated (decentralized) cilium yog pom nyob rau saum npoo ntawm lub distal tubular cell (Daim duab 1e). Podocytes qhia txog kev ploj ntawm ko taw nrog cov ntaub so ntswg dav microvillus. Immunohistochemical staining rau ib-tubulin qhia ntau qhov ntev thiab tsis sib haum xeeb thawj cilia nyob rau saum npoo ntawm cov hlwb epithelial (Daim duab 1c).
2.2. Immunohistochemical Staining rau 心Tubulin, Inversion thiab DVL-1- thiab Kev Tshawb Fawb Txog Kev Txhim Kho thiab Kev Noj Qab Haus Huv Tom Qab Tib Neeg Raum
Nyob rau hauv lub 14th, 15th thiab 16th gestational lub lis piam, lubraumcov ntaub so ntswg nthuav tawm nrog cov theem sib txawv ntawm kev tsim nephron: los ntawm cov khob metanephric,lub raumvesicles theem kom txib S-zoo li nephrons, yog li tsim nephrogenic cheeb tsam nyob rau hauv lub cortex sab nrauv (Daim duab 2). Kev sib txawv pct, dct thiab g tuaj yeem pom nyob rau hauv thaj chaw cortical ze dua rauraummedulla. Thaum lub sijhawm 22nd GW, qhov chaw ntawm medulla thiab cortex tau ua kom pom tseeb, thaum nyob rau hauv 38th GW,raumcov qauv zoo li txawv heev, muaj cov ntaub ntawv paub tab ntawm pct, dct thiab g
2.2.1. ib- Tubulin
Thaum lub sij hawm txoj kev loj hlob, a-tubulin yog ib qho kev nthuav qhia hauv txhua qhov kev loj hlobraumcov qauv, feem ntau pom pom daim ntawv ntawm thawj cilia ntawm qhov chaw ntawm parietal epithelial hlwb ntawm Bowman's capsule, g, pct thiab dct. Hauvraumcov ntaub so ntswg, 82-97 feem pua ntawm cov hlwb pct qhia a-tubulin, thaum nyob rau hauv dct qhia poob los ntawm 99 feem pua rau 72 feem pua hauv 38th GW (saib daim duab 3, Table 1). Ntawm cov theem kev loj hlob sib txawv, qhov muaj zog tshaj plaws ntawm a-tubulin pom nyob rau hauv g ntawm 16th GWlub raum, muaj 93 feem pua ntawm cov hlwb zoo. lithe 14th, 15th thiab 22nd GW (saib daim duab 3a, b, Table 1), nyob ib ncig ntawm 70 feem pua ntawm cov g hlwb qhia a-tubulin, thaum qhov tsawg tshaj plaws qhia tau pom nyob rau hauv 38th GW (p <0.01) nrog="" 34="" feem="" pua="" ntawm="" cov="" hlwb="" zoo="" (daim="" duab="" 3c).="" hauv="">raumcov ntaub so ntswg (1.5- xyoo thiab 7- xyoolub raum), qhov muaj zog tshaj tiv thaiv kab mob rau a-tubulin yog pom ntawm apical cell nto ntawm pct thiab dct (Daim duab 3d-f). Lub a-tubulin qhia kuj tseem muaj nyob rau hauv perinuclear cytoplasm ntawm dct thiab parietal epithelial hlwb ntawm Bowman's capsule. a-tubulin stains tag nrholub raumcov qauv nrog qhov nruab nrab ntawm 50 feem pua ntawm cov hlwb zoo hauv pct, 94 feem pua hauv dct thiab 85 feem pua hauv g (Daim duab 3f). Dct stains txawv ntawm pct (p <0.0001). tag="" nrho="" cov="" txheej="" txheem="" tshawb="" fawb="" stain="" zoo="" rau="" a-tubulin,="" nrog="" 77="" feem="" pua="" ntawm="" cov="" hlwb="" zoo="" hauv="" pct,="" 72="" feem="" pua="" hauv="" dct="" thiab="" 58="" feem="" pua="" hauv="" g="" (daim="">
Daim duab 3. Immunofluorescence staining ntawm -tubulin thiab DAPI nyob rau hauv lub tsim thiab postnatal tib neeg lub raum cov ntaub so ntswg (a–e). Lub raum ntawm 14th, 15th / 16th, 38th GW (a–c);lub raumntawm 1.5- thiab 7- menyuam yaus hnub nyoog (d,e). Qhov zoo staining ntawm -tubulin ( xub) yog pom nyob rau hauv txhua tus qauv hauv lub cortex los ntawm kev loj hlob thiab postnatal theem (a-e), proximal convoluted tubules (pct), distal convoluted tubules (dct) thiab glomeruli (g). Cov ntsiab lus ntawm thawj cilia hauv pct (d) thiab dct (a–c, e) tau pom tias muaj ntau dua magnification insets (dawb lub thawv). Kev nthuav dav × 40, nplai bar 100 µm. Kev faib tawm ntawm -Tubulin zoo hlwb rau ib tus qauv thoob plaws hauv ntau theem ntawm kev loj hlob thiab nyob rau hauv lub postnatal lub sij hawm yog qhia nyob rau hauv daim duab (f). Graph (g) (tag nrho cov protein-zoo hlwb hauv cov qauv pom) qhia cov protein qhia ntsig txog lub sijhawm kev loj hlob (Linear regression) thiab kev sib cuam tshuam ntawm -Tubulin rau DVL-1 los ntawm kev loj hlob thiab kev loj hlob (Ob-Way ANOVA ua raws los ntawm SIDAK's posthoc test). Cov ntaub ntawv yog qhia raws li txhais tau tias ± SD.
Kev faib tawm ntawm -tubulin-zoo hlwb rau ib tus qauv thoob plaws hauv ntau theem ntawm kev loj hlob thiab hauv lub sijhawm postnatal yog qhia hauv daim duab (f). Graph (g) (tag nrho cov protein-zoo hlwb hauv cov qauv pom) qhia cov protein qhia ntsig txog lub sijhawm kev loj hlob (Linear regression) thiab kev sib cuam tshuam ntawm -tubulin rau DVL-1 los ntawm kev loj hlob thiab kev loj hlob (ob txoj kev ANOVA ua raws los ntawm Sidak's post hoc test). Cov ntaub ntawv yog qhia raws li txhais tau tias ± SD.
2.2.2. Muab ob npaug rau Immunoflfluorescence Staining rau Inversion thiab DVL-1 thiab DAPI Nuclear Stain hauv Kev Tsim Kho thiab Kev Noj Qab Haus Huv Postnatal Raum Inversion Qhia hauv Kev Tsim Kho thiab Postnatal Raum Cov Ntaub Ntawv
Nyob rau hauv lub 14th, 15th thiab 16th GW, inversion qhia muaj zog granular qhia nyob rau hauv g, thiab me me granular qhia nyob rau hauv lub cytoplasm ntawm pct thiab dct (Daim duab 4a, b), thaum nyob rau hauv lub 22nd thiab 38th GW (saib daim duab 4c), lub zog. Kev qhia zoo yog pom nyob rau hauv g (Table 1). Nyob nruab nrab ntawm 14th thiab 22nd GW, nyob ib ncig ntawm 50 feem pua ntawm cov hlwb nthuav tawm version. Nyob rau hauv lub 16th GW, pct hlwb qhia inversion nyob rau hauv 73 feem pua ntawm cov hlwb, thaum txo mus rau 29 feem pua ntawm cov hlwb (p <0.05) nyob="" rau="" hauv="" lub="" 38th="" gw="" (saib="" daim="" duab="" 4f).="" kev="" qhia="" zoo="" ntawm="" kev="" hloov="" pauv="" muaj="" nyob="" hauv="" kwv="" yees="" li="" 80-90="" feem="" pua="" ntawm="" dct="" thiab="" g="" hlwb="" hauv="">raumcov ntaub so ntswg ntawm 14th thiab 16th GW, nrog qhov qhia qis tshaj plaws hauv dct ntawm 38th GW (p < 0.05,="" p="">< 0.0001,="" feem)="" qhov="" twg="" 66="" feem="" pua="" ntawm="" cov="" hlwb="" tau="" zoo.="" hauv="">lub raum, pct stains muaj zog ntawm apical cell membrane, thaum dct stains feem ntau ntawm cov basal cell membrane thiab hauv perinuclear cytoplasm (Daim duab 4d, e). Nyob rau theem ntawd, peb pom kev qhia txog kev hloov pauv hauv txhua qhov kev tshawb nrhiavlub raumcov qauv, suav nrog pct, dct thiab g, nrog lub ntsiab lus ntawm cov hlwb zoo ntawm 92 feem pua rau pct, 88 feem pua rau dct thiab 90 feem pua rau g (Daim duab 4f). Peb tsis tau soj ntsuam qhov sib txawv tseem ceeb hauv lub teeb liab lub zog ntawm inversion qhia ntawm cov qauv. Qhov siab tshaj plaws ntawm inversion yog pom nyob rau hauv g, nrog ib tug txhais tau tias ntawm 94 feem pua ntawm cov hlwb zoo (Daim duab 4f). Qhov sib txawv tseem ceeb yog pom tias muaj qhov sib txawv ntawm g (qhov twg 94 feem pua ntawm cov hlwb stain zoo) rau pct, qhov twg 58 feem pua ntawm cov hlwb stain zoo (p < 0.01)="" thiab="" dct,="" qhov="" twg="" 49="" feem="" pua="" ntawm="" cov="" hlwb="" yog="" zoo="" (p=""><0.0001, daim="" duab="">
DVL-1 Kev nthuav qhia hauv Kev Loj Hlob thiab Postnatal raum Hniav
Kev qhia me me rau kev muaj zog ntawm DVL{{0}} yog pom nyob rau hauv cytoplasm ntawm pct, dct thiab g nyob rau hauv lub sij hawm fetal (saib daim duab 4a–c, Table 1). Hauv pct, 76–86 feem pua ntawm cov hlwb qhia DVL-1 hauv 14th–22nd GW, thaum nyob hauv 38th GW, muaj 57 feem pua ntawm cov hlwb zoo (Daim duab 4g). Hauv dct ntawm 14th thiab 15th GW, 68–70 feem pua ntawm cov hlwb qhia DVL-1, hauv 16th thiab 22nd GW, tus lej ntawm cov hlwb zoo poob rau 42–5{{41} } feem pua, thaum nyob rau hauv 38th GW qhia (p <0.001), yog="" pom="" nyob="" rau="" hauv="" 19="" feem="" pua="" ntawm="" dct="" hlwb.="" kev="" nthuav="" qhia="" ntawm="" dvl-1="" nce="" hauv="" g="" thoob="" plaws="" hauv="" lub="" cev="" xeeb="" tub:="" hauv="" 14th="" gw,="" nws="" yog="" 6="" feem="" pua,="" thaum="" nyob="" rau="" hauv="" 15th,="" 16th="" thiab="" 22nd="" gw,="" nws="" nce="" mus="" rau="" 10="" feem="" pua="" (p=""><0.01). hauv="" 38="" gw,="" 14="" feem="" pua="" ntawm="" g="" hlwb="" qhia="" dvl-1="" (daim="" duab="" 4="" g).="" hauv="" lub="" sijhawm="" postnatal,="" dvl-1="" immunoreactivity="" yog="" tawg="" mus="" rau="" hauv="" cytoplasm="" (table="" 1)="" ntawm="" ob="" qho="" tib="" si="" pct="" thiab="" dct="" (daim="" duab="" 4d,="" e).="" cov="" teeb="" liab="" pom="" muaj="" nyob="" rau="" hauv="" tag="" nrho="" cov="" kev="" soj="" ntsuam="" cov="" qauv="" tab="" sis="" feem="" ntau="" nyob="" rau="" hauv="" lub="" perinuclear="" cytoplasm.="" qhov="" kev="" siv="" ntawm="" dvl-1="" qhia="" tau="" ntau="" dua="" hauv="" pct="" piv="" rau="" dct="" thiab="" g="" (p=""><0.01). 39–45="" feem="" pua="" ntawm="" cov="" hlwb="" hauv="" pct="" thiab="" dct="" qhia="" dvl-1,="" thaum="" nyob="" rau="" hauv="" g,="" 21="" feem="" pua="" ntawm="" cov="" hlwb="" yog="" qhov="" zoo="" (daim="" duab="" 4="" g).="" peb="" tsis="" pom="" qhov="" sib="" txawv="" tseem="" ceeb="" ntawm="" feem="" pua="" ntawm="" cov="" hlwb="" immunoreactive="" hauv="" qhov="" sib="">lub raumcov qauv. Qhov feem pua ntawm DVL-1 immunoreactive hlwb yog 34 feem pua hauv pct, 36 feem pua hauv dct thiab 27 feem pua hauv g, raws li (Daim duab 4g).
Daim duab 4. Ob chav immunofluorescence staining ntawm inversion (ntsuab), DVL-1 (liab) thiab DAPI (xiav) nyob rau hauv cov ntaub so ntswg loj hlob thiab postnatal raum (14th–38th GW, 1.5- thiab {{6 }} xyoo laus cov ntaub so ntswg). Qhov zoo staining (xub xub) yog qhia nyob rau hauv txhua tus qauv thoob plaws txhua theem ntawm kev loj hlob (a–c) thiab lub sij hawm postnatal (d,e). Kev sib koom ua ke microphones nrog rau cov qauv kev txaus siab hauv cortex: proximal convoluted tubules (pct), distal convoluted tubules (dct) thiab glomeruli (g). Colocalization ntawm inversion/DVL-1 (lub taub hau) yog qhia nyob rau hauv merged microphotographs. Kev tswj tsis zoo stainings yog qhia raws li insets ntawm inversion thiab DVL-1 (a). Erythrocytes tuaj yeem pom zoo li cov hlwb uas muaj zog nyob ze dct. Cov ntsiab lus tau pom raws li cov khoom loj dua. Kev nthuav dav × 40, nplai bar 100 µm. Dynamic zoo cell tis ntawm inversion thiab DVL-1 nyob rau hauv lub raum qauv (pct, dct, g) thoob plaws hauv kev loj hlob thiab postnatal theem yog qhia nyob rau hauv graphs (f,g). Graph (h) qhia cov protein qhia (tag nrho cov naj npawb ntawm cov hlwb zoo hauv cov qauv) ntsig txog lub sij hawm loj hlob (Linear regression) thiab kev sib cuam tshuam ntawm inversion rau DVL-1 los ntawm kev loj hlob thiab kev loj hlob (ob txoj kev ANOVA ua raws li Sidak's post hoc test ). Cov ntaub ntawv yog qhia raws li txhais tau tias ± SD.
Kev nthuav qhia ntawm kev hloov pauv thiab DVL-1 yog pom nyob rau hauv cytoplasm ntawm lub raum g, dct thiab pct thaum lub sij hawm kev loj hlob (saib daim duab 4a–c, merging). Hauv lub sijhawm tom qab yug menyuam, kev sib koom ua ke ntawm inversion thiab DVL-1 characterizes txawv cellular compartments ntawm tubular cells nyob rau hauv pct thiab dct, thaum co-expression nyob rau hauv g yog tus cwj pwm los ntawm cov muaj zog prevalence ntawm inversion qhia (saib daim duab 4d, ua ke. ). Hauv 7-xyoo lub raum tom qab yug me nyuam, co-expression ntawm inversion thiab DVL-1 yog pom nyob rau hauv tubular hlwb ntawm dct thiab pct, thiab nyob rau hauv g qhov inversion qhia me ntsis prevails nyob rau hauv kev sib piv rau DVL-1 (Daim duab 4e, sib koom ua ke).
2.2.3. Qhov sib txawv ntawm Kev Tshaj Tawm ntawm -Tubulin, Inversion thiab DVL-1 nruab nrab ntawm Kev Txhim Kho Raum Sib Txawv thiab Lub raum Postnatal
Tus naj npawb ntawm -tubulin-zoo hlwb nyob rau hauv pct ntawm lub raum cov ntaub so ntswg nyob rau hauv cov ntaub so ntswg ntawm lub raum yog cov ntaub ntawv ntau dua nyob rau hauv kev sib piv rau cov me nyuam hnub nyoog muaj hnub nyoog kaum xyoo cov ntaub so ntswg (13th (p < 0).="" 0{="" {11}}1),="" 15="" (p="">< 0).="" dct="" ntawm="" cov="" me="" nyuam="" hauv="" plab="" thaum="" ntxov="" (13th,="" 15th,="" 16th="" thiab="" 22nd="" gw)="" muaj="" cov="" hlwb="" zoo="" dua="" piv="" rau="" 38th="" gw="" thiab="" 1.="" thaum="" cov="" theem="" sib="" txawv="" ntawm="" kev="" loj="" hlob="" tau="" muab="" piv,="" peb="" pom="" cov="" theem="" qis="" ntawm="" kev="" hloov="" pauv="" hauv="" pct="" ntawm="" 13th="" (p=""><0.0001), 15th="" (p=""><0.00001), 22nd="" (p=""><0.001) thiab="" 38th="" (p=""><0.0001) gw="" piv="" rau="">lub raumcov ntaub so ntswg los ntawm 1.5- menyuam yaus hnub nyoog. Nyob rau hauv dct, statisticically tseem ceeb tshaj qhia ntawm inversion tau pom muab piv 16th GW rau 38th GW raum ntaub so ntswg (p < 0.0001).="" tsis="" tas="" li="" ntawd,="" kev="" qhia="" qis="" ntawm="" kev="" hloov="" pauv="" hauv="" dct="" tau="" pom="" nyob="" rau="" hauv="" 7-="" cov="" menyuam="" yaus="" lub="" raum="" cov="" ntaub="" so="" ntswg;="" muab="" piv="" rau="" 13th,="" 15th="" (p=""><0.001, ob="" qho="" tib="" si),="" 16th="" thiab="" 1.5-cov="" ntaub="" so="" ntswg="" raum="" rau="" dct="" ntawm="" 7-="" xyoo-laus="" cov="" ntaub="" so="" ntswg="" (p="">< 0.00001,="" raws="" li,="" daim="" duab="" 4f).="" cov="" teeb="" liab="" pom="" ntawm="" dvl-1="" qhia="" los="" ntawm="" ntau="" theem="" qhia="" tias="" pct="" ntawm="" 13th="" (p=""><0.01), 15th,="" 16th="">< 0.001,="" respectively)="" and="" 22nd="" (p="" <="" 0.0001)="" gw="" had="" higher="" expression="" of="" immunoreactive="" cells="" compared="" to="" the="" pct="" of="" 7-year-old="" children="" kidney="" tissue.="" dvl-1="" immune-expression="" in="" dct="" of="" the="" kidney="" from="" 13th="" and="" 15th="" gw="" was="" significantly="" higher="" than="" in="" the="" 38th="" gw="" tissue="" (p="" <="" 0.0001,="" respectively,="" figure="">
2.2.4. Kev sib raug zoo ntawm Kev Tshaj Tawm ntawm -Tubulin thiab Inversion rau DVL-1 hauv Kev Tsim Kho thiab Postnatal raum
-Tubulin thiab inversion qhia tau muab piv rau DVL-1 qhia thoob plaws hauv cov theem kev loj hlob thiab hauv cov ntaub so ntswg tom qab yug menyuam. -tubulin muaj cov ntaub ntawv qhia ntau dua hauv txhua theem pom, piv rau qhov qhia ntawm DVL-1 (p < 0.001,="" daim="" duab="" 3g).="" los="" ntawm="" tag="" nrho="" cov="" theem="" pom,="" inversion="" muaj="" ib="" qho="" kev="" qhia="" ntau="" dua="" thaum="" piv="" rau="" dvl-1="" (p="">< 0.0001,="" daim="" duab="" 4="">
2.2.5. Kev sib piv ntawm Immunohistochemical Staining rau -Tubulin, version thiab DVL-1- thiab Statistical Analysis of Pathologically Changeed Kidney Tissue (MCDK, CNF, FSGS) -Tubulin Qhov txawv ntawm -tubulin staining hauv MCDK, FSGS thiab CNF yog qhov tseem ceeb hauv kev txheeb cais. kom noj qab nyob zoo tom qab lub raum cov ntaub so ntswg ua ib pawg tswj hwm (p < 0.0001,="" raws="" li).="" dysplastic="" raum="" cov="" ntaub="" so="" ntswg="" muaj="" kev="" qhia="" ntau="" dua="" piv="" rau="" cov="" pab="" pawg="" tswj,="" thaum="" fsgs="" thiab="" cnf="" pom="" tau="" hais="" tias="" qis="" dua="" ntawm="" -tubulin="" thaum="" piv="" rau="" kev="" noj="" qab="" haus="" huv="" (daim="" duab="">
Kev hloov pauv
Inversion yog qhia nyob rau hauv lub cytoplasm ntawm disorganized epithelial hlwb thiab tubules ntawm MCDK (Daim duab 5a). Hauv CNF, inversion qhia characterizes g thiab dct, thaum nws pom tsawg intensively hauv pct cysts (Daim duab 5b). Hauv FSGS, kev hloov pauv tau hais tawm hauv g thiab dct tab sis tsis tshua muaj zog hauv pct cysts (Daim duab 5c). Spatial qhia ntawm inversion tau pom qhov muaj txiaj ntsig zoo dua nyob rau hauv cov ntaub so ntswg noj qab haus huv (Daim duab 5g) thaum piv rau MCDK, FSGS thiab CNF (p < 0.0001,="" raws="" li="">
Daim duab 5. Ob chav immunofluorescence staining ntawm inversion (ntsuab), DVL-1 (liab) thiab DAPI (xiav) hauv cov ntaub so ntswg raum hauv MCDK (a), CNF (b) thiab FSGS (c): tubules (t) , glomeruli (g), pct cyst (c), qhov siab ntawm pct epithelial hlwb (*). Cov qauv thiab cell co-localization ntawm inversion thiab DVL-1 ( xub xub) yog qhia nyob rau hauv cov merged seem nrog cov ntsiab lus nyob rau hauv ntau dua magnification insets. Kev tswj tsis zoo stainings yog qhia raws li insets ntawm inversion thiab DVL-1 (a). Magnifification × 40, nplai bar 10{{2{29}}}} µm. Kev sib raug zoo ntawm -tubulin (d) thiab inversion (e) rau DVL-1 qhia hauv MCDK, FSGS thiab CNF (ob txoj kev ANOVA ua raws li SIDAK's post hoc test). Qhov sib txawv ntawm epithelial cell qhov siab (f) ntawm FSGS, CNF thiab MCDK pct piv rau kev tswj kev noj qab haus huv (ib txoj kev ANOVA ua raws li Tukey's post hoc test, n=50). Qhov sib txawv ntawm tag nrho cov cell zoo feem pua ntawm -tubulin, inversion thiab DVL-1 hauv MCDK, CNF thiab FSGS thaum piv rau kev tswj kev noj qab haus huv (g–i), ib txoj kev ANOVA ua raws li Tukey's post hoc test). Cov ntaub ntawv yog qhia raws li txhais tau tias ± SD. Qhov sib txawv tseem ceeb yog qhia los ntawm p-tus nqi (* p < 0.05,="" **="" p="">< 0.01,="" ****="" p="">< 0.0001).="" dvl-1="" dvl-1="" tsuas="" yog="" qhia="" me="" me="" rau="" hauv="" cov="" tubules="" tsis="" sib="" haum="" xeeb="" ntawm="" mcdk,="" thaum="" nyob="" hauv="" cnf,="" nws="" cov="" lus="" qhia="" yog="" dct="" thiab="" g="" (daim="" duab="" 5a,b).="" hauv="" fsgs,="" dvl-1="" tau="" pom="" tias="" muaj="" qhov="" ua="" rau="" me="" me="" hauv="" g,="" dct="" thiab="" pct="" (daim="" duab="" 5c).="" lub="" raum="" noj="" qab="" nyob="" zoo="" stained="" rau="" dvl-1="" tau="" pom="" qhov="" ntau="" dua="" ntawm="" staining="" (daim="" duab="" 5="" h)="" sib="" piv="" rau="" mcdk="" thiab="" fsgs="" (p=""><0.0001, ob="" qho="" tib="" si),="" thaum="" tsis="" muaj="" qhov="" sib="" txawv="" tseem="" ceeb="" rau="">
2.2.6. Kev sib raug zoo ntawm kev nthuav qhia ntawm -Tubulin thiab Inversion rau DVL-1 hauv Pathologically Changeed Kidney Tissue (MCDK, CNF, FSGS)Hauv MCDK, FSGS thiab CNF, -tubulin stained ntau dua DVL-1 (p < 0.0001,="" raws="" li,="" daim="" duab="" 5d).="" inversion="" stained="" ntau="" dua="" thaum="" piv="" rau="" dvl-1="" hauv="" mdck="" (p=""><0.0001) thiab="" hauv="" fsgs="" (p=""><0.01, daim="" duab="">
2.2.7. Qhov sib txawv hauv Epithelial Cell Qhov siab ntawm Proximal Convoluted Tubules ntawm Kev Tswj Xyuas Kev Noj Qab Haus Huv thiab Pathologically Hloov Raum Hniav (MCDK, CNF, FSGS)Qhov siab ntawm pct epithelial hlwb (n {{0}}} ib pab pawg)) tau muab piv rau ntawm tubule hlwb hauv cov ntaub so ntswg noj qab nyob zoo thiab cov ntaub so ntswg hloov raum (Daim duab 5b,c,f). Qhov nruab nrab ntawm lub cell qhov siab hauv HC yog 12.91 µm ± 1.847 µm thiab tau siab dua thaum piv rau CNF thiab FSGS (p < 0.0001,="" raws="" li="" ).="" qhov="" siab="" ntawm="" tes="" hauv="" cnf="" pct="" yog="" 9.011="" µm="" ±="" 1.453="" µm,="" thaum="" nyob="" rau="" hauv="" fsgs="" yog="" 8.114="" µm="" ±="" 0.9248="" µm.="" pct="" epithelial="" hlwb="" ntawm="" mcdk="" tsis="" pom="" qhov="" hloov="" pauv="" loj="" hauv="" qhov="" siab="" (12.17="" µm="" ±="" 1.476="" µm)="" thaum="" piv="" rau="">
2.2.8 ib. Qhov sib txawv hauv Primary Cilia Length ntawm Kev Tswj Xyuas Kev Noj Qab Haus Huv thiab Pathologically Hloov Raum Hniav (MCDK, CNF, FSGS)
Thawj cilium ntev (n{0}} ib pab pawg) tau muab piv ntawm HC thiab cov ntaub so ntswg hloov pauv raum (Daim duab 6c). Cov ntaub so ntswg noj qab nyob zoo thiab MCDK tau stained nrog -tubulin kom paub meej qhov tshwj xeeb ntawm -tubulin cilium staining (Daim duab 6a,b). Hauv cov ntaub so ntswg noj qab nyob zoo, thawj cilium yog 5.065 µm ± 1.229 µm ntev, thaum nyob hauv MCDK, CNF thiab FSGS tau ntev dua (p<0.0005, respectively).="" primary="" cilium="" length="" in="" mcdk="" was="" 9.908="" µm="" ±="" 2.434="" µm,="" in="" cnf="" 13.65="" µm="" ±="" 3.218="" µm,="" while="" in="" fsgs="" was="" significantly="" longer,="" 18.29="" µm="" ±="" 4.717="" µm,="" when="" compared="" to="" hc=""><0.0001) and="" other="" pathological="" kidney="" tissues="" of="" mcdk=""><0.0001) and="" cnf=""><>
Kev sib tham
Lub hom phiaj ntawm peb txoj kev tshawb fawb yog txhawm rau tshawb xyuas cov tshuaj tiv thaiv kab mob ntawm a-tubulin, inversion thiab DVL-1 hauv fetal.lub raumcov ntaub so ntswg thiab lub raum postnatal. Tsis tas li ntawd, peb xav tshawb xyuas seb qhov kev qhia thiab qhov staining ntawm a-tubulin, inversion thiab DVL-1 puas cuam tshuam rau cov kab mob raum sib txawv thaum piv rau kev noj qab haus huv. Namely, txawm tias muaj kev txaus siab ntau ntawm lwm cov kws tshawb fawb txog lub luag haujlwm ntawm a-tubulin, inversion thiab DVL-1 thaum lub raum kev loj hlob, feem ntau ntawm cov kev tshawb fawb yav dhau los tau siv cov tsiaj los yog hauv vitro sim ua qauv. Raws li peb tau paub, qhov no yog thawj txoj kev tshawb fawb uas tau qhia txog kev qhia thiab thaj chaw ntawm a-tubulin nrog rau kev hloov pauv thiab DVL-1 hauv cov menyuam hauv plab thiab tom qab yug menyuam hauv lub raum, thiab uas tau tshawb nrhiav cov lus hais txog cov proteins hauv lub raum. kab mob, xws li MCDK, FSGS thiab CNF. Peb kuj tau txheeb xyuas cov kev hloov pauv ntawm cilia ntev thiab cov tsos ntawm lub teeb thiab hluav taws xob microscopy, raws li peb txoj kev tshawb fawb dhau los tau qhia tias kev cuam tshuam ntawm ciliary tuaj yeem cuam tshuam nrog cystogenesis hauv FSGF thiab CNF [19].
Nyob rau hauv txoj kev canonical Wnt-signaling, kev khi ntawm Wnt ligands rau receptors recruits Dvl [37]. Cov txheej txheem ntawm MET thiab txoj hauv kev ntawm cov cell polarity thaum lub sijhawm pib ntawm lub raum morphogenesis yog tswj los ntawm Wnt signaling. Los ntawm kev sib kho ntawm Wnt txoj kev, thawj cilia tswj cell proliferation, sib txawv thiab cov ntaub so ntswg morphogenesis [12] los ntawm kev teeb tsa lub cell cytoskeleton thiab cell orientation nrog rau cov qauv tubular elongation [2,38]. Nyob rau hauv peb txoj kev tshawb no, -tubulin, inversion thiab DVL-1 yog tag nrho cov nyob rau hauv lub raum qauv, thiab tag nrho cov colocalized tsis tau tsuas yog thaum lub sij hawm cev xeeb tub lub raum tab sis kuj nyob rau hauv lub raum cov ntaub so ntswg los ntawm 1.5- thiab {{9 }}cov me nyuam muaj hnub nyoog. Cov kev tshawb pom no tau pom zoo nrog kev ua haujlwm ntawm Wnt-signaling txoj hauv kev uas tau ua pov thawj tau tshwm sim thaum lub sijhawm tubulogenesis [39]. Tsis tas li ntawd, peb cov txiaj ntsig tau qhia tias qhov kev qhia ntawm -tubulin thiab inversion yog reciprocally cuam tshuam nrog DVL-1 thiab hais tias lawv qhia qhov sib txawv tseem ceeb ntawm lawv cov qauv qhia. Qhov ntawd yog nyob rau hauv kev sib raug zoo rau cov qauv kev sim, raws li kev hloov pauv ntawm tag nrho Dvl protein tsev neeg hauv cov nas ua rau tsis muaj cov txheej txheem gastrulation, thaum kev hloov pauv uas tsis suav nrog tag nrho tsev neeg cov protein qhia qhov tsis xws luag hauv nodal cilia qhov chaw nrog rau lub cev tsis xws luag [40] . Cov kev tshawb pom yav dhau los qhia tias kev hloov pauv muaj lub luag haujlwm hauv kev tsiv teb tsaws ntawm tes hauv Xenopus pronephros. Txij li cov ntu ntawd cuam tshuam rau cov tsiaj txhu ntawm Henle thiab cov tubules distal, qhov no tuaj yeem qhia qhov tseem ceeb ntawm kev hloov pauv thaum lub raum loj hlob hauv tib neeg ib yam nkaus [41]. Raws li kev pom zoo nrog qhov chaw, cov kev tshawb fawb yav dhau los tau tshaj tawm tias kev hloov pauv ntawm cov noob inversion tuaj yeem ua rau nephronophthisis hom 2, uas yog kho los ntawm DVL txawv txav -1 qhia [42]. Txawm hais tias lub luag haujlwm ntawm thawj cilium muaj teeb meem hauv kev tswj hwm ntawm Wnt-signaling txoj hauv kev, peb pom tias -tubulin colocalized nrog inversion thiab DVL-1 nyob rau hauv cov tshuaj ntsuam xyuas raum. Tsis tas li ntawd, cov kev tshawb fawb yav dhau los tau taw qhia tias thawj cilium nrog rau kev hloov pauv tswj kev degradation ntawm Dvl los ntawm kev cuam tshuam rau Wnt-signaling txoj kev [43]. Hauv tib neeg lub raum kab mob cuam tshuam nrog kev loj hlob ntawm cov hlwv, qhov txawv txav hauv zos thiab kev ua haujlwm ntawm thawj cilia tau pom [19]. Ib yam li ntawd, txoj kev tshawb fawb tam sim no kuj tau lees paub qhov kev hloov pauv ntawm morphological ntawm thawj cilia tsim hauv cov kab mob xws li CNF, FSGS thiab MCDK. Cov kev tshawb pom yav dhau los tau lees paub tias kev ua kom dhau ntawm txoj hauv kev canonical Wnt tom qab lub raum puas ua rau muaj kev hloov pauv tsis zoo ntawm lub raum cov ntaub so ntswg [44]. Ntawm qhov tsis sib xws, thawj cilium tau muab lub luag haujlwm ntawm kev hloov ntawm canonical mus rau noncanonical Wnt txoj hauv kev pab.lub raumkho. Tsis tas li ntawd, kev ua kom dhau ntawm txoj kev canonical Wnt hauv cov ntaub so ntswg hloov pauv hauv lub raum tau pom tias muaj txiaj ntsig zoo rau lub raum fibrosis [45]. Yog hais tias txoj kev canonical Wnt yeej ntawm tus nqi ntawm noncanonical, nws txhawb txoj kev xav ntawm lub raum fibrosis raws li qhov tshwm sim. Peb qhov kev tshawb pom ntawm qis-tubulin thiab inversion qhia nyob rau hauv CNF thiab FSGS imply inactivity ntawm noncanonical Wnt txoj kev, tshwj xeeb tshaj yog raws li ob qho tib si tej yam kev mob yuav coj mus rau qhov kawg-theem.mob raum.Namely, nws ntseeg hais tias ib txwm localization thiab kev ua haujlwm ntawm thawj cilium tuaj yeem yog qhov tseem ceeb hauv kev tswj hwm txoj hauv kev Wnt tsis tu ncua thiab, yog li ntawd, qhov yuav tsum tau ua ua ntej rau kev loj hlob ib txwm muaj. Hauv qhov sib piv, yog tias Wnt txoj kev txhim kho, nws tuaj yeem ua rau dysregulated cell proliferation thiab sib txawv ua rau carcinogenesis [46]. Raws li tau piav qhia dhau los, txoj hauv kev canonical Wnt yog qhov yuav tsum tau ua rau kev pib ntawm MET thiab tsim cov nephron, thaum nws cuam tshuam yuav ua raulub raumhypoplasia [47]. Peb cov txiaj ntsig tau txhawb nqa lub tswv yim no los ntawm kev nthuav tawm cov lus qhia qis tshaj plaws ntawm DVL-1 nrog cov lus qhia siab tshaj plaws ntawm -tubulin hauv MCDK piv rau kev tswj hwm kev noj qab haus huv. Cov txiaj ntsig no tuaj yeem hais txog qhov ua kom qis tshaj plaws ntawm txoj hauv kev canonical Wnt, uas tuaj yeem yog qhov laj thawj rau qhov tsis muaj nephron tsim. Hauv qhov sib piv, qhov siab tshaj plaws -tubulin qhia nrog qis tshaj DVL-1 kev qhia yuav piav qhia qhov kev tshawb pom ntawm ntau lub hlwv vim tias tsis muaj kev sib koom ua ke thiab cov xov tooj ntawm tes polarization coj los ntawm txoj kev Wnt noncanonical. Cov kev tshawb fawb yav dhau los kuj tau qhia tias kev hloov pauv cuam tshuam rau canonical Wnt signaling txoj hauv kev los ntawm kev tsom Dvl rau degradation [26]. Cov kauj ruam no hauv lawv cov kev sib cuam tshuam tau pom tias yuav tsum tau ua rau inhibit canonical Wnt-signaling nyob rau hauv kev saib xyuas ntawm ib txwm tubular elongation thiab positioning. Inversion mutation ua rau muaj kev tswj hwm ntawm canonical Wnt signaling, uas tom qab provoked txawv txav proliferation hauv tubular hlwb. Cov kauj ruam no tau raug pov thawj tias yog qhov tseem ceeb hauv cystogenesis [42], thaum lub khob ntawm inversion hauv nas ua rau muaj kab mob raum polycystic [48]. Raws li txoj kev xav ntawmlub raumcystogenesis, inversion yog suav hais tias yog "mycoprotein", vim nws qhov chaw nyob rau thawj cilia hauvlub raumtubular hlwb. Hauv peb txoj kev tshawb fawb, thawj cilia tau tshwm sim nyob rau ntawm apical cell nto ntawm tubular hlwb nyob rau hauv tag nrho cov kev soj ntsuam theem, thaum lub sij hawm fetal theem, kev qhia ntawm -tubulin muaj zog tshaj nyob rau hauv lub postnatal lub sij hawm.
CISTANCHE yuav txhim kho lub raum / raum mob
Cov kev tshawb fawb yav dhau los tau pom tias thawj qhov kev ua haujlwm cilia tuaj yeem cuam tshuam los ntawm dysregulation ntawm -tubulin thaum lub sijhawm kev loj hlob, uas tuaj yeem ua rau cystogenesis, kev loj hlob ntawm lub raum txawv txav thiab tuaj yeem mob raum mob rau menyuam yaus [31,49]. Qhov no yog raws li peb qhov kev tshawb pom ntawm luv luv thiab dysmorphic thawj cilia pom hauv MCDK lossis nrog ntau thiab tsis tshua muaj elongated lossis dislocated cilia pom nyob rau hauv dilated tubules ntawm CNF thiab FSGS thaum piv rau kev noj qab haus huv. Txhawm rau txhawb qhov tseeb tias txoj hauv kev canonical thiab noncanonical Wnt-signaling txoj hauv kev, tswj hwm los ntawm thawj cilia, suav tias yog qhov yuav tsum tau ua rau lub raum loj hlob, peb pom muaj qhov qis qis ntawm kev hloov pauv thiab DVL-1 hauv MCDK thaum piv rau kev noj qab haus huv. [31,49]. Cov qauv qhia txawv txawv ntawm -tubulin, inversion thiab DVL-1 thoob plaws hauv lub raum kev loj hlob theem yuav qhia tau tias hloov ntawm txoj kev canonical thiab noncanonical Wnt-cim qhia txoj hauv kev thaum lub raum morphogenesis. Peb hais tias lawv qhov kev sib pauv txiav txim siab kev hloov pauv hauv txoj hauv kev canonical lossis kev txiav txim ntawm cov cell tsiv teb tsaws thiab polarization thaum lub raum kev loj hlob hauv txoj kev tsis yog txoj hauv kev. Lawv qhov sib npaug thiab kev qhia nyob rau hauv txhua qhov kev tshawb xyuas lub raum qhia txog lawv lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm lub raum. Thaum lub raum kev loj hlob ib txwm (los ntawm 13th GW mus rau 38th GW), tag nrho cov lus qhia ntawm -tubulin, inversion thiab DVL-1 yog tsawg zuj zus raws li cov ntaub so ntswg mus rau mature morphology. Hauv 1. Ntawm qhov tsis sib xws, DVL-1 txuas ntxiv nrog txo cov qauv qhia dab tsi yuav ntxiv rau qhov kev txiav txim siab tias txoj hauv kev canonical Wnt tau ntsiag to hauv cov ntaub so ntswg noj qab haus huv. Raws li qhov tshwm sim ntawm cov kab mob hauv lub raum, cov hlwb epithelial ntawm lub raum yuav hnov mob nrog qhov rov tshwm sim ntawm EMT thiab fibrosis [50] cuam tshuam nrog reactivation ntawm canonical Wnt txoj kev [44]. Yog li, kev cuam tshuam ntawm -tubulin, inversion thiab DVL-1 pom nyob rau hauv cov kab mob hauv lub raum tej zaum yuav yog lub hauv paus pathological mechanism thiab tshwm sim los ntawm kev hloov ntawm noncanonical mus rau canonical Wnt txoj hauv kev tsim lub raum alluding rau kev hloov ntawm reversible rau irreversible raum. kev puas tsuaj. Tsis tas li ntawd, kev hloov pauv hauv lawv lub cev xeeb tub thiab tom qab yug menyuamlub raumCov qauv qhia yuav cuam tshuam nrog kev ua haujlwm tsis zoo ntawm lub raum hauv cov neeg laus, ua rau muaj kab mob hauv lub cev thiab lub raum tsis ua haujlwm ntev.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Tib neeg cov qauv
Cov qauv ntawm lub raum fetus tau sau tom qab cev xeeb tub ntawm 14th, 15th, 16th, 22nd thiab 38th GW ntawm Department of Gynecology thiab Obstetrics ntawm University Hospital Center Split. Tag nrho cov khoom siv fetal tau raug tshuaj xyuas los ntawm tus kws kho mob, thiab tsuas yog cov ntaub so ntswg uas tsis muaj cov tsos mob ntawm qhov txawv txav, macerations lossis intrauterine tuag thiab nrog karyogram ib txwm siv rau txoj kev tshawb no. Cov ntaub ntawv kho mob ntawm cov niam tau raug tshuaj xyuas ua ntej kev sau cov qauv thiab yog tias muaj teeb meem kev noj qab haus huv uas yuav cuam tshuam rau kev xeeb tub, cov ntaub so ntswg raum raug tshem tawm los ntawm txoj kev tshawb no. Kev loj hlob tau txiav txim siab los ntawm lub taub hau ncig, lub plab ncig thiab femur ntev [51] nyob rau hauv kev sib raug zoo nrog cov hnub nyoog ntawm cov neeg mob. Cov qauv ntawm 1.5- thiab 7- xyoo-laus cov ntaub so ntswg tau sau tom qab kev tuag tsis zoo. Cov qauv no tau txais ntawm Department of Pathology ntawm University Hospital Center Split. Cov qauv ntawm cov ntaub so ntswg multicystic dysplastic raum (MCDK) tau txais tom qab cev xeeb tub, focal segmental glomerulosclerosis (FSGS) thiab nephrotic syndrome ntawm hom Finnish (CNF) tau txais vim nephrectomy. Tag nrho cov khoom tau txais tau raug tshuaj xyuas thiab tshuaj xyuas los ntawm tus kws kho mob, uas tau txheeb xyuas qhov kev kuaj mob. Txoj kev kawm raws tu qauv tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm University of Split School of Medicine (20 Tsib Hlis 2016) raws li Helsinki Tshaj Tawm thiab nws cov kev hloov tshiab (kev faib tawm tsis yog.: 003-08/16-03/0001, npe: 2181-198-03-04-16-0024, 20 May 2016) [52].
4.2. Immunohistochemistry
Cov ntaub so ntswg cov qauv kho tau ua tiav nrog 4 feem pua ntawm paraformaldehyde hauv phosphate-buffered saline (PBS) rau 24 teev ntawm 22 ◦C. Tom qab lub cev qhuav dej hauv 100 feem pua ethanol, cov qauv tau muab tso rau hauv paraffin, raws li tau piav qhia yav dhau los [53]. Cov qauv tau muab hlais rau hauv 5 µm tuab ntu uas siv lub microtome thiab tom qab ntawd txuas rau ntawm lub tshuab tsom iav. Txhawm rau txheeb xyuas cov ntaub so ntswg khaws cia, txhua ntu ntu 10 tau stained nrog hematoxylin thiab eosin [54]. Depart- feminization thiab immunohistochemistry tau ua raws li tau piav qhia yav dhau los [2,55,56]. Tom qab muab yaug hauv PBS, lub tshuab tsom iav tau muab tso rau ib hmos nrog cov tshuaj tiv thaiv thawj zaug hauv lub chamber ntub ntawm 22 ◦C (StainTray slide staining system; Sigma-Aldrich, St. Louis, MO, USA). Cov tshuaj tiv thaiv tseem ceeb siv tau yog luav monoclonal anti-alpha tubulin antibody (dilution 1: 1000; ab179484, Abcam, Cambridge, UK), Luav Polyclonal Anti-inversion antibody (dilution 1: 100; ab65187, Abcam, Cambridge, UK), nas monoclonal -1 antibody (1:150 dilution; sc-8025, Santa Cruz Biotechnology, Dallas, TX, USA) thiab luav polyclonal anti-gamma tubulin antibody (kom paub tseeb cov cilia tshwj xeeb staining nrog alpha- tubulin, 1: 500 dilution; ab11321, Abcam, Cambridge, UK). Tom qab yaug hauv PBS, cov tshuaj tiv thaiv kab mob thib ob tau muab rau ib teev raws li teev tseg: nees luav tiv thaiv luav IgG H&L, Alexa Fluor 488 (dilution 1:400; ab150073, Abcam, Cambridge, UK) thiab tshis tiv thaiv nas IgG H&L, TRITC (dilution 1:400; ab6786, Abcam, Cambridge, UK) 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) tau siv rau staining ntawm cov nuclei thiab tom qab 2-min incubation, slides tau ntxuav hauv PBS thiab overlaid nrog mounting nruab nrab thiab npog. Tsis yog tshwj xeeb staining raug tiv thaiv los ntawm kev siv cov protein thaiv (ab64226; Abcam, Cambridge, UK) ua ntej thawj daim ntawv thov tshuaj tiv thaiv kab mob. Raws li kev tswj tsis zoo, kev kuaj ua ntej adsorption tau ua, thaum qhov tshwj xeeb ntawm cov tshuaj tiv thaiv kab mob thib ob raug kuaj los ntawm kev tshem tawm thawj cov tshuaj tiv thaiv los ntawm cov txheej txheem staining.
4.3. Electron Microscopy
Fixation ntawmraumCov ntaub so ntswg tau ua hauv 4 feem pua ntawm paraformaldehyde rau 24 teev, tom qab ntawd kho tom qab 1 feem pua osmium tetroxide rau 1 teev. Cov txheej txheem lub cev qhuav dej tau ua nrog ethanol series thiab ua tiav nrog kev sib xyaw hauv LX 112 resin [22]. Ib-micrometer nyias seem tau stained siv toluidine xiav thiab kawm los xaiv ultrathin seem. Ultrathin seem nrog lub thickness ntawm 0.05 micrometers tau kuaj tom qab staining nrog uranyl acetate thiab lead citrate. JEOL 1200 EX microscope tau siv kom tau txais cov duab microphotographs.
4.4. Genetic Analysis
Raws li yav dhau los tau piav qhia [19], siv leukocytes los ntawm cov ntshav peripheral, genomic DNA tau muab rho tawm. Ib homozygous missense mutation nyob rau hauv NPHS1 noob tau pom (c.1096A > C; p.Ser366Arg) dab tsi tau lees paub qhov kev kuaj mob ntawm congenital nephrotic syndrome ntawm Finnish hom (CNF) [57].
CISTANCHE yuav txhim kho lub raum / raum kab mob
4.5. Kev txheeb xyuas cov ntaub ntawv
Kev tshuaj xyuas ntu tau ua tiav ntawm FL fluorescence microscope siv 3 FL fluorescence raws (Olympus BX51, Tokyo, Nyiv). Cov duab tau raug ntes los ntawm lub koob yees duab digital DP71 (Olympus, Tokyo, Nyiv) ntawm lub zog loj (× 40). Tsuas yog covlub raumcortex uas muaj cov tubules sib thooj (pct), cov tubules distal convoluted (dct) thiab glomeruli (g) tau txaus siab. Cov duab tau ua tiav los ntawm ImageJ software (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/ ( nkag mus rau 15 Lub Kaum Hli 2018), 1997-2021. ) thiab Adobe Photoshop (Adobe Inc., San Jose, California, USA) rau kev ntsuam xyuas ntxiv. Ua ntej suav ntawm tes, cov khoom sib cais ImageJ tau siv. Tom qab ntawd, tus thawj immunofluorescence microscopic yees duab raug rho tawm los ntawm liab lossis ntsuab channel (nyob ntawm seb tus channel thawj yog dab tsi) siv lub tshuab xam zauv duab ImageJ cov cuab yeej los tiv thaiv cov teeb liab to. Cov txiaj ntsig hauv qab qib 50 tau suav tias yog qhov tsis zoo. Peb suav cov cim immunoreactive hauv cov hlwb ntawm tsawg kawg 20 cov qauv (pct, dct lossis g) ib theem lossis tag nrho cov cell zoo ntawm ib daim duab micro ntawm cov kab mob multicystic dysplastic.raumcov ntaub so ntswg, FSGS thiab CNF. Peb faib cov hlwb ua qhov zoo yog tias cov teeb liab immunofluorescence tau khaws cia ntawm txhua theem ntawm daim nyias nyias, cytoplasm lossis nucleus siab dua tus nqi 50 ntsuas ntawm ImageJ software siv cov lus txib pib. Cov hlwb tsis zoo tau muab faib ua cov hlwb uas tsis muaj kev tiv thaiv kab mob. Ob tus kws tshawb nrhiav ywj pheej tau tshuaj xyuas cov ntaub ntawv.
4.6. Kev Ntsuam Xyuas Semiquantitative
Qhov kev siv ntawm -tubulin, inversion thiab DVL-1 staining teeb liab tau soj ntsuam los ntawm ob tus kws tshawb fawb ywj pheej siv ImageJ software. Tag nrho cov teeb liab siv tau ntsuas tom qab cov duab tau teeb tsa hauv 8-ntsis hom; Tom qab ntawd, qhov kev siv ntawm cov cim staining raug soj ntsuam hauv 20 pct, dct thiab g los ntawm kev ntsuas thaj tsam ntawm cov qauv. Yog tias cov txiaj ntsig ntawm cov neeg soj ntsuam sib txawv, tus kws tshawb fawb thib peb tau qhia meej qhov tsis ntseeg. Cov teeb liab siab tshaj plaws rau -tubulin yog 84.125 ± 3.214 SD, rau inversion yog 71.50 ± 2.715 SD thiab rau DVL-1 80.916 ± 1.875 SD. Qhov siab tshaj plaws saturation ntawm cov teeb liab xim ntawm cov duab tsom iav tau cim tias yog 3 (66.66–99.99 feem pua ntawm tag nrho cov teeb liab duab siv), ua raws li 2 (33.33–66.66 feem pua) rau qhov nruab nrab siv lub teeb liab thiab 1 (0.33 feem pua – 33.33 feem pua) rau qis teeb liab siv (Table 1.).
4.7. Kev txheeb cais
Rau qhov kev txiav txim siab ntawm qhov sib txawv ntawm cov theem thiab cov qauv, Kruskal–Wallis xeem tau ua tiav, ua raws li Dunn's post hoc siv GraphPad Prism software (Graphpad Software Inc., San Diego California, USA, www.graphpad.com ( nkag mus rau 15th ntawm Lub Kaum Hli. 2018.)). Tus naj npawb ntawm cov hlwb zoo tau qhia nyob rau hauv feem pua ntawm tus nqi nruab nrab ± tus qauv sib txawv (SD), thaum qhov tseem ceeb ntawm cov ntaub ntawv tau lees paub ntawm p <0.05. tus="" naj="" npawb="" ntawm="" cov="" qauv="" txheeb="" xyuas="" yog="" 4200="" hauv="" 35="" cov="" qauv,="" nrog="" rau="" tag="" nrho="" cov="" naj="" npawb="" ntawm="" 135,256="" hlwb="" suav.="" peb="" siv="" 2-txoj="" kev="" anova="" nrog="" sidak's="" post="" hoc="" test="" los="" ntsuas="" qhov="" sib="" txawv="" ntawm="" kev="" qhia="" ntawm="" -tubulin,="" inversion="" thiab="" dvl-1="" nyob="" rau="" hauv="" ntau="" theem="" kev="" loj="" hlob.="" txhawm="" rau="" kawm="" cov="" protein="" qhia="" txog="" lub="" sijhawm="" loj="" hlob,="" peb="" siv="" linear="" regression.="" ib-txoj="" kev="" anova="" ua="" raws="" li="" tukey's="" post="" hoc="" xeem="" tau="" siv="" los="" tshawb="" txog="" qhov="" sib="" txawv="" ntawm="" kev="" qhia="" ntawm="" -tubulin,="" inversion="" thiab="" dvl-1="" hauv="" kev="" noj="" qab="" haus="">raumcov ntaub so ntswg piv rau cov ntaub so ntswg ntawm MCDK, FSGS thiab CNF. Qhov sib txawv ntawm kev qhia cov proteins ntawm MCDK, FSGS thiab CNF tau tshawb xyuas nrog 2- txoj kev ANOVA ua raws li Sidak's post hoc test. Ib txoj kev ANOVA ua raws li Tukey's post hoc test yog siv los ntsuas qhov sib txawv ntawm cilia ntev thiab epithelial cell qhov siab ntawm pct ntawm kev noj qab haus huv thiab kab mob.raumcov ntaub so ntswg. Cov ntaub ntawv tau pom tias tus nqi nruab nrab ± tus qauv sib txawv (SD) nrog cov lej sib txawv tau lees paub ntawm p <>
