Ntu Ⅰ: Muaj peev xwm cuam tshuam ntawm Soluble Luteinizing Hormone Receptor Thaum Kev Loj Hlob Thiab Kev Loj Hlob hauv Cov Tub thiab Txiv Neej
Apr 06, 2023
Cov ntsiab lus yooj yim
Cov tshuaj hormones luteinizing hormone (LH) thiab tib neeg chorionic gonadotropin (hCG), ob qho tib si agonists ntawm luteinizing hormone receptor (LHCGR), yog qhov tseem ceeb rau txiv neej yug me nyuam thaum lub sij hawm loj hlob thiab neeg laus. lHCGR tau nthuav tawm thiab txhawb nqa testosterone ntau lawm los ntawm cov qe ntshav Leydig hlwb. Hauv txoj kev tshawb no, peb pom tau tias LHCGR soluble muaj nyob rau hauv cov ntshav, zis, thiab cov phev ntawm cov tub thiab cov txiv neej noj qab nyob zoo, nrog rau cov neeg mob uas muaj kev sib deev chromosome aberrations. Peb qhia tau hais tias cov theem ntawm sLHCGR cuam tshuam li cas nrog kev loj hlob ntawm pubertal, testicular function, thiab phev zoo, thiab qhia tau tias LHCGR raug tso tawm los ntawm fetal tib neeg cov ntaub so ntswg uas tsis yog-gonadal. HCG raug tso tawm rau hauv cov ntshav ntawm cov qog ntshav thiab lwm yam kabmob, qhia txog lub luag haujlwm ntxiv rau LH lossis hCG hauv cov tub thiab txiv neej.
Abstract
Luteinizing hormone (LH) thiab tib neeg chorionic gonadotropin (hCG) yog agonists ntawm luteinizing hormone receptor (LHCGR), uas tswj cov txiv neej kev ua haujlwm. lHCGR tuaj yeem tso rau hauv cov kua hauv lub cev. Peb xav txiav txim seb puas yog LHCGR soluble yog tus cim ntawm gonadal muaj nuj nqi. Cov kev tshawb fawb hla ntu, ntev, thiab kev cuam tshuam tau ua rau 195 tus tub thiab cov txiv neej noj qab haus huv thiab 396 tus txiv neej uas muaj menyuam tsis taus, tsis muaj peev xwm, lossis Klinefelter syndrome (KS) los txuas LHCGR ntsuas hauv cov ntshav, phev, zis, thiab lub siab / lub raum cov hlab ntsha mus rau kev ua haujlwm ntawm gonadal. . lHCGR tau ntsuas hauv vitro thiab hauv vivo cov qauv ntawm tib neeg cov nqaij mos thiab cov kab ntawm tes, xenograft nas qauv, thiab tib neeg Western blot cov txiaj ntsig tau pom tias LHCGR fragments raug kuaj pom hauv cov ntshav zoo sib xws thiab cov ntaub so ntswg gonadal siv peb cov tshuaj tiv thaiv sib txawv. Txawm tias tom qab tib neeg xenografts, LHCGR-ELISA tsis pom muaj qhov sib txawv ntawm qhov sib txawv lossis tsis muaj kev cuam tshuam tshwj xeeb hauv cov ntshav nas. Hauv qhov sib piv, sLHCGR tau tso tawm rau hauv kab lis kev cai nruab nrab tom qab tib neeg lub raum thiab qog adrenal kab lis kev cai. Serum sLHCGR qis dua hauv cov menyuam yaus noj qab haus huv thaum lub hnub nyoog laus dua (p=0.{13}}001). Hauv cov txiv neej noj qab haus huv, ntshav sLHCGR tsis zoo cuam tshuam nrog qhov sib piv ntawm inhibin B/FSH ( -0.004, p=0.027). Hauv cov txiv neej infertile, phev sLHCGR tsis zoo cuam tshuam nrog cov ntshav FSH (0.006, p=0.009), phev concentration (-3.5, p=0.003), thiab tag nrho cov phev suav. ( -3.2, p=0.007). Kev txhaj tshuaj hCG txo sLHCGR hauv cov ntshav thiab zis ntawm cov txiv neej noj qab haus huv (p <0.01). Hauv kev xaus, sLHCGR raug tso tawm rau hauv cov kua hauv lub cev thiab cuam tshuam nrog kev loj hlob ntawm tus menyuam thiab kev ua haujlwm gonadal. Kev ncig sLHCGR hauv cov txiv neej orchid qhia tias sLHCGR hauv cov ntshav tuaj yeem tshwm sim hauv cov ntaub so ntswg uas tsis yog gonadal thiab tuaj yeem ua haujlwm hauv cov ntaub so ntswg uas tsis yog gonadal. (Clinical Trials: NTC01411527, NCT01304927, NCT03418896).
Ntsiab lus
LH receptor; laus laus; kev loj hlob; tsis muaj menyuam;Cov txiaj ntsig Cistanche; ntxiv-gonadal teebmeem; gonadotropins; fetal raum; fetal qog adrenal; TCAM2; NTUA 2
Nyem qhov no mus yuavHerbal Cistanche Extract
Taw qhia
Luteinizing hormone (LH) thiab tib neeg chorionic gonadotropin (hCG) yog ob qho tib si agonists ntawm luteinizing hormone receptor (LHCGR), uas yog ib qho tseem ceeb rau txiv neej yug me nyuam thaum lub sij hawm loj hlob thiab neeg laus [1]. HCG yog G protein-coupled receptor paub tias yuav tsum tau qhia nyob rau hauv Leydig, granular lutein, thiab membrane hlwb thiab yog ib tug muaj zog regulator ntawm kev sib deev steroid ntau lawm [1]. hCG yog ib tug muaj zog thiab pheej agonist tab sis tsuas yog physiologically tseem ceeb thaum lub sij hawm kev loj hlob ntawm fetal [2]. Tsuas yog qee cov kab mob qog noj ntshav thiab qog nqaij hlav pancreatic, hCG tsis tsim nyob rau hauv cov neeg laus, txawm hais tias txoj kev tshawb fawb ua ntej pom tias pulsatile qis qis ntawm pituitary-hCG hauv cov txiv neej thiab poj niam. hCG yog siv los kho cov poj niam kom ntxias ovulation thaum lub sij hawm kho fertility, rau cov txiv neej los txhawb testosterone synthesis thiab muaj peev xwm spermatogenesis nyob rau hauv cov txiv neej hypogonadal, los yog raws li ib tug kuaj mob rau endogenous testosterone ntau lawm [4]. Kev tshem tawm ntawm tus nas LH receptor (LHCGR) ua rau txo qis hauv testosterone thiab phev zoo [5], thiab ntau pab pawg tshawb fawb tau txheeb xyuas kev hloov pauv thiab SNP hauv LHCGR, qhia txog nws qhov tseem ceeb rau tib neeg kev ua haujlwm ntawm kev ua me nyuam [6-8].
LHCGR noob nyob rau ntawm chromosome 2 thiab muaj 11 exons, ntawm uas exons 1-10 encode extracellular structural domains thiab exon 11 encodes 7 transmembrane thiab intracellular structural domains [9]. Muaj ob peb isoforms ntawm LHCGR, ib qho ntawm cov uas xav tias yuav tsum tau zais vim nws tsis muaj cov thauj tog rau nkoj transmembrane [10]. Lwm qhov ua tau yog tias kev tso LHCGR rau hauv cov kua dej hauv lub cev yog nyob ntawm cov enzymatic cleavage ntawm cov proteins ntawm cov cell membrane, xws li ADAMs, MMPs, thiab lwm yam transmembrane proteins cleaved. Tseeb tiag, hCG tau qhia tshwj xeeb rau kev tswj hwm MMP-2 thiab MMP-9 [11,12], thiab ntau qhov kev tshawb fawb tau pom cov kev cai tom qab hloov pauv ntawm LHCGR [13,14]. Soluble LHCGR (LHC) tau ntsuas hauv poj niam cov ntshav thiab cov kua follicular los ntawm ELISA platform [15,16]. Qhov tshwj xeeb ntawm cov tshuaj tiv thaiv uas siv hauv ELISA qhov kev ntsuam xyuas tau raug lees paub los ntawm kev qhia cov LHCGR sib txawv hauv CHO cov hlwb rau epitope localization thiab kuaj pom kev qhia hauv tib neeg placenta [15-17]. sLHCGR tau kuaj pom hauv cov ntshav los ntawm LC-MS/MS [18]. Cov ntshav ntshav ntau ntawm sLHCGR hauv cov poj niam tau cuam tshuam nrog kev kho mob fertility, preeclampsia, yug ntxov ntxov, thiab cev xeeb tub hauv cov menyuam yaus uas muaj tus mob Down syndrome implantation tus nqi [15,16], tab sis tseem tsis tau lees paub los ntawm lwm pab pawg tshawb fawb txog niaj hnub no. Rau peb txoj kev paub, sLHCGR yeej tsis tau piav qhia hauv cov kua dej hauv lub cev ntawm cov txiv neej noj qab haus huv, tsim cov tub hluas, lossis txiv neej nrog kev sib deev chromosome aberrations. sLHCGR tuaj yeem ua haujlwm hauv cov txiv neej ncig ua tus cim ntawm kev ua haujlwm gonadal los ntawm kev sib koom ua ke ntawm kev thauj mus los lossis kev hloov pauv ntawm LH lossis hCG kev ua haujlwm.
Cov ntaub ntawv thiab cov txheej txheem
1. Cov pawg
Cov Tub Hluas los ntawm Cov Pej Xeem
Hauv tebchaws Denmark, txhua tus tub ntxhais hluas noj qab haus huv uas muaj hnub nyoog 18 xyoo yuav tsum tau kuaj lub cev los txiav txim siab tias lawv tsim nyog rau kev ua tub rog. Txij li xyoo 1996, kwv yees li 300 tus neeg sau npe, sawv cev rau cov pej xeem, tau kuaj xyuas cov phev zoo txhua xyoo. Lawv txhua tus tau muab cov phev kuaj, ua tiav daim ntawv nug, tau kuaj ultrasound ntawm lawv cov noob qes, thiab tau kuaj ntshav. Cov ntshav thiab cov phev kuaj los ntawm biobank, nrog rau kev soj ntsuam ultrasound thiab cov ntaub ntawv ntawm BMI thiab hnub nyoog los ntawm 148 tus txiv neej tshawb fawb hauv 2013 thiab 2014, tau suav nrog hauv txoj kev tshawb fawb tam sim no (Kev pom zoo ntawm Pawg Saib Xyuas Kev Ncaj Ncees: H-KF-289428).
Cov tub ntxhais kawm ua ntej thiab tom qab yug me nyuam noj qab haus huv
Txoj Kev Kawm Copenhagen Adolescence yog ib qho kev sib koom ua ke thiab kev kawm ntev ntawm cov menyuam yaus Danish noj qab haus huv [20]. Los ntawm cov tsev kawm ntawv pej xeem raug xaiv, 6203 tus menyuam raug caw tuaj koom. Tag nrho cov neeg koom yog 2020 cov menyuam yaus. Kev soj ntsuam ntev ntev ntawm 209 tus menyuam yaus (101 tus tub) tau ua txhua 6 lub hlis thiab suav nrog kev kuaj lub cev, kev ntsuas Tanner ntawm cov theem pubertal, thiab kuaj ntshav thiab zis. Peb caug-rau cov tub hluas uas muaj kev ncua ntev tau suav nrog hauv txoj kev tshawb no los ntsuas sLHCGR thaum lub hnub nyoog laus dua (kev sim tshuaj: NTC01411527).
Txiv neej Infertile tsis muaj mob hnyav
Txoj Kev Kawm Copenhagen-Bone-Gonad yog ib qho kev tshawb fawb ntawm 307 tus txiv neej tsis muaj menyuam uas tsis muaj mob hnyav uas tau tuaj koom peb lub tsev kho mob txiv neej los ntawm 2011 txog 2015, suav nrog tag nrho 1427 tus txiv neej [21,22]. Txhua tus txiv neej raug xa mus rau peb lub tsev kho mob vim tias cov phev tsis zoo thiab lub siab xav ua niam txiv. Txhua tus tau kuaj xyuas lub cev, suav nrog kuaj qog nqaij hlav, tau kuaj ntshav, thiab muab cov phev kuaj. Tus kws kho mob tshwj xeeb tau txais lawv cov keeb kwm kho mob ntxaws. Cov txiv neej tau ua raws li 150 hnub los ntawm cov kws tshawb fawb thiab noj cov tshuaj vitamin D lossis placebo raws li tau piav qhia dhau los. Tag nrho cov ntaub ntawv muab rau hauv txoj kev tshawb no yog los ntawm kev mus ntsib thawj zaug ua ntej thiab tag nrho cov kev hloov pauv tau raug txhais ua ntej (kev sim tshuaj: NCT01304927).
Standardized Cistanche
Cov txiv neej thiab cov tub nrog Klinefelter Syndrome thiab cov neeg mob nrog Anorchia
Peb tau txheeb xyuas 64 tus tub thiab cov txiv neej uas muaj Klinefelter syndrome (KS), 47 post-pubertal thiab 16 pre-pubertal, txhais raws li Tanner staging, los ntawm peb lub tsev kho mob txiv neej. Peb muaj cov chromosomal aberrations, keeb kwm kev kho mob, cov xwm txheej pubertal pib, thiab cov ntaub ntawv ntshav muaj rau txhua tus. 7 cov tub ntxhais kawm tau txais kev soj ntsuam ntev ntev nrog kev soj ntsuam kuaj mob thiab kuaj ntshav ua ntej thiab thaum lub sijhawm puberty.
Peb kuj suav nrog 8 tus txiv neej nrog ib txwm chromosome suav uas tau kuaj ob tog raug tshem tawm rau cov xwm txheej dhau los: testicular kab mob cell carcinoma (n=4), diffuse prostate cancer (n=1), ob sab cryptorchidism, thiab pediatric orchiectomy ( n=1), ob sab kab mob ntawm tes neoplasia nyob rau hauv situ (n=1), thiab mob qog noj ntshav siab tom qab tshem tawm cov qe ntshav ntxiv vim raug mob (n=1).
Cov Neeg Laus Tau Txais Kev Ntsuas Splanchnicus Flow Measurement
Peb suav nrog peb tus txiv neej thiab peb tus poj niam postmenopausal nrog lub siab ua haujlwm ib txwm tau txais rau qhov xav tias mesenteric ischemia thiab raug xa mus rau kev ntsuas cov ntshav visceral tab sis tsis pom mesenteric ischemia. Cov ntshav kuaj tau ib txhij los ntawm lub siab, lub raum, femoral vein, thiab cov hlab ntsha sib thooj (Kev pom zoo ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees: H.18048245)
Kev Tshawb Fawb Kev Tshawb Fawb: Cov txiv neej noj qab haus huv raug rau tib neeg Chorion Gonadotropin
Kaum ib tus txiv neej noj qab haus huv tau raug xaiv los koom nrog kev kho mob los ntawm Danish Rigshospitalet tshaj tawm. Cov neeg ua haujlwm pab dawb tau kho nrog 5000 IU tib neeg chorionic gonadotropin thiab lawv qhov siab tshaj plaws ntawm endogenous Testosterone tau ntsuas los ntawm kev sib piv cov ntshav hauv qab thiab cov zis nrog cov qauv ua raws li tau sau tseg 8, 24, thiab 72 teev tom qab hCG txhaj tshuaj (kev sim tshuaj: NCT03418896).
2. Cov nas, tib neeg cov ntaub so ntswg, thiab cov kab cell
Ob hom kab mob qog nqaij hlav qog nqaij hlav hauv tib neeg NTera-2 thiab TCam-2, sawv cev rau embryonal carcinoma thiab seminoma, feem, tau siv thiab coj los ua raws li tau piav qhia yav dhau los [23].
Sera tau sau los ntawm cov nas uas tsis muaj qog nqaij hlav (WT) thiab TCam-2 xenograft cov nas liab qab kho nrog vector, LH, lossis hCG, raws li (Tsib Soj Ntsuam Kev Tshawb Fawb, Copenhagen, Denmark, daim ntawv tso cai naj npawb: 2012-15-2934-00051) .
Thawj peb lub hlis twg tib neeg cov ntaub so ntswg tau sau los ntawm cov poj niam pub dawb cov ntaub so ntswg tom qab kev rho menyuam tawm. Lub raum fetal thiab fetal adrenal cov ntaub so ntswg tau coj mus rau hauv cov qauv ncua kev ncua raws li tau piav qhia yav dhau los thiab qhov nruab nrab tau sau rau kev tshuaj xyuas [24,25] (Kev pom zoo ntawm Pawg Saib Xyuas Kev Ncaj Ncees: H-1-2012-007).
Cov neeg laus testicular specimens tau los ntawm cov txiv neej ovariectomized nrog qog nqaij hlav cancer (Ethics Committee pom zoo: H{{0}}). Cov ntaub so ntswg peritumoral uas tsis muaj qhov tsis zoo tau muab khaws cia rau ntawm - 80◦ C los yog tsau thaum hmo ntuj ntawm 4◦ C hauv formalin lossis hloov kho Stieve 's-fixative (200 mL 37 feem pua formaldehyde, 40 mL acetic acid hauv 1 L 0.05 M phosphate tsis, pH 7.4).
3. Kev tshuaj xyuas biochemical
Kev kuaj pom tag nrho sLHCGR hauv cov ntshav, phev, zis, thiab ntau yam kab lis kev cai xov xwm tau ua nyob rau hauv cov txheej txheem validation siv ELISA cov khoom siv los ntawm New Dutch Biomarker Catalyst Laboratory (NBCL) siv cov tshuaj tiv thaiv LHR029 tiv thaiv tib neeg LHCGR thiab ntsuas tag nrho ntawm no pheej. Qhov kev ntsuam xyuas ELISA muaj qhov txwv tsis pub kuaj pom ntawm 0.01 pmol / mL thiab qhov siab tshaj plaws ntawm 15.55 Rau cov txiv neej los ntawm cov pej xeem uas muaj sLHCGR qib siab tshaj qhov kev txwv siab tshaj ntawm kev kuaj pom tau lawv cov qauv diluted thiab rov ntsuas, qhia qib txog 30 pmol / mL. qhov kev ua tau zoo ntawm ELISA platform tau piav qhia meej yav dhau los [15-17].
Cistanche ntxiv
Peb kuj tau teeb tsa ob qhov sib txawv hauv tsev ELISA, teeb tsa los ntawm peb cov kws tshaj lij hauv chav kuaj, thiab tag nrho cov reagents ntxiv tau muab los ntawm Origin Biomarkers Ltd, UK, raws li lawv tau pom zoo teeb [26]. Ua ntej, circulating sLHCGR khi rau hCG tau kuaj pom siv cov khoom siv Sandwich ELISA nrog cov tshuaj tiv thaiv 5A10C9 (ProMab) thiab am0{{20}}904pun ( OriGene) qhia tawm tsam LHCGR thiab hCG, feem. Qhov thib ob, peb zoo ib yam siv hauv tsev ELISA los txiav txim siab tag nrho daim siab, lub raum, thiab qis qis ntawm cov hlab ntsha thiab cov hlab ntsha los ntawm rau tus neeg laus sLHCGR, qhov uas peb siv tib cov tshuaj tiv thaiv LHR029 (NBCL Holland) tawm tsam sLHCGR. Ob txoj kev sib txawv tau sim. Txhua qhov kev ntsuas sLHCGR tau piav qhia hauv daim ntawv no tau ua tiav siv NBCL ELISA, tshwj tsis yog rau hCG-sLHCGR complex thiab arterial / venous kuaj los ntawm cov kabmob uas tau piav qhia saum toj no. Hauv cov menyuam yaus noj qab haus huv, Testosterone raug tshuaj xyuas siv DPC-coated- suav RIA cov khoom siv (Diagnostic Products, Los Angeles, CA) nrog rau qhov txwv tsis pub kuaj pom (LOD) ntawm 0.23 nmol / L thiab CV ntawm 8.6 feem pua yog siv lub sij hawm daws teeb meem immunofluorescence. (Delfia; Wallac, Turku, Finland), qhov kev kuaj pom yog 0.20 nmol / l nrog CV ntawm 6.4 feem pua. Hauv cov neeg laus, testosterone thiab kev sib deev hormone binding globulin (SHBG) tau soj ntsuam los ntawm chemiluminescence immunoassay (Access, Beckman Coulter, USA) nrog LODs ntawm 0.35 nmol / L thiab 0.33 nmol / L, raws li. Cov ntshav thiab cov zis FSH thiab LH tau ntsuas los ntawm lub sijhawm daws teeb meem immunofluorescence (Delfia; Wallac, Turku, Finland) nrog kev kuaj pom qhov txwv ntawm 0.06 thiab 0.05 IU / L, raws li, thiab CV thiab lt; ob qho tib si ntawm 5 feem pua. Inhibin B assay tau ua los ntawm kev siv ib qho tshwj xeeb ob tog enzyme immunoassay (inhibin B genII, Beckman Coulter, USA) CV <11 feem pua. Rau AMH, peb siv Beckman Coulter enzyme immunoassay (Immunotech, Beckman Coulter, Marseilles, Fabkis) thiab CV <8 feem pua. Thaum kawg, IGF1 thiab IGF-BP3 tau txiav txim siab siv chemiluminescence assay ntawm IDS-iSYS immunodiagnostic system nrog CVs ntawm 10 feem pua thiab 9 feem pua. Dawb estradiol tau suav nrog siv Mazer qhov tsis tu ncua thiab dawb testosterone (FT) tau suav nrog Vermeulen's formula [27,28].
Semen Analysis
Cov txiv neej noj qab haus huv thiab cov neeg laus tsis muaj menyuam tau muab cov qauv phev thiab tau txais cov ntaub ntawv qhia tus kheej txog lub sijhawm ntawm ejaculatory abstinence. Kev soj ntsuam cov phev tau ua los ntawm cov kws tshaj lij uas tau kawm raws li tau piav qhia yav dhau los [19]. Semen ntim tau kwv yees los ntawm qhov hnyav. Rau kev ntsuam xyuas cov phev muaj peev xwm, 10 μ L ntawm cov phev zoo sib xyaw ua ke tau muab tso rau ntawm cov swb thiab tshuaj xyuas hauv qab × 400x lub tshuab ntsuas hluav taws xob ntawm qhov cua sov ntawm 37◦ C. Cov phev tau muab cais ua ib qho advanced viable (WHO class a plus B), non-progressive viable (class C) lossis inactive (class D). Qhov nruab nrab ntawm ob qhov kev ntsuas kev muaj peev xwm raug siv. Txhawm rau ntsuas cov phev concentration, cov qauv tau muab diluted hauv cov dej distilled ntawm 0.6 moL / L NaHCO3 thiab 0.4 feem pua (v/v) formaldehyde thiab tom qab ntawd soj ntsuam siv Bürker-Türk hemocytometer thiab qhov nruab nrab ntawm kev ntsuas tau siv. Tsuas yog spermatozoa nrog tails raug suav. Thaum kawg, smears tau npaj, thiab stained nrog Pap stain, thiab phev morphology raug soj ntsuam nruj me ntsis raws li cov txheej txheem ntawm [29].
Western Blotting
Tib neeg cov ntaub so ntswg yog homogenized nyob rau hauv lysis buffer, diluted nyob rau hauv SDS loading tsis, thiab ces rhuab ntawm 95◦ C rau 5 min. Cov qauv raug thauj mus rau 4-20 feem pua precast polyacrylamide gels (BioRad, cat# 456-8096) thiab khiav ntawm 100 V rau 1 h kom cais cov protein. Txhawm rau kuaj sLHCGR hauv cov ntshav, albumin, thiab IgG raug tshem tawm siv Pierce albumin / IgG tshem tawm cov khoom (Thermo Scientific, Waltham, MA, USA, cat# 89875) ua ntej thauj cov qauv mus rau gel. Tom qab gel electrophoresis, cov protein tau pauv mus rau polyvinylidene difluoride daim nyias nyias siv cov dej ntub dej (BioRad). Membranes raug kaw nyob rau hauv tris-buffered saline thiab 5 feem pua skimmed qhuav mis nyuj rau 1 hr, ces incubated hmo ntuj ntawm 4◦ C nrog thawj cov tshuaj tiv thaiv thiab rau 1 hr ntawm RT nrog lwm yam tshuaj tiv thaiv. Membranes raug ntxuav nrog txhim kho chemiluminescence (Thermo Scientific Super Signal West Femto Maximum Sensitive Substrate, Cat# 34095) thiab photodetector nrog chemiDoc MP imaging system (BioRad, Cat# 17001402). Peb cov tshuaj tiv thaiv sib txawv tau siv los kuaj LHCGR hauv cov ntaub so ntswg thiab ntshav: Aviva System Biology (OASG04237); NBCL Holland, tib cov tshuaj tiv thaiv ib yam li hauv cov khoom siv ELISA (LHR029) thiab Santa Cruz Biotechnology (Dallas, TX, USA, SC- 26341). -2 microglobulin tau siv los tswj kev thauj khoom.
Immunohistochemistry
Immunohistochemistry tau ua raws li tau piav qhia yav dhau los [25]. Luv luv, ntu tau kho nrog hloov kho tawv nqaij, dewaxed, thiab rov ua dej. Antigen extraction tau ua nyob rau hauv ib lub autoclave uas muaj citrate extraction tsis (10 mM, pH 6.0). Endogenous peroxidase tau thaiv nrog 3 feem pua (v/v) H2O2 hauv methanol rau 30 feeb. Nruab nrab ntawm txhua kauj ruam, ntu tau ntxuav hauv TBS. Cov seem tau tsim nrog 0.5 feem pua BSA nees cov ntshav rau 30 feeb, tom qab ntawd los ntawm kev ua haujlwm hmo ntuj nrog ob hom tshuaj tiv thaiv kab mob sib txawv rau 1 teev ntawm chav sov (LHR029 rau WB/ELISA, Santa Cruz Biotechnology SC- 25828). Cov seem tau incubated nrog cov tshuaj tiv thaiv theem nrab rau 30 feeb. Cov duab tau pom nrog cov aminoethyl carbazole (AEC, Invitrogen, Life Technologies / Thermo Fisher Scientific, Waltham, MA, USA). Kev tswj tsis zoo yog tsis muaj cov tshuaj tiv thaiv thawj. Tsis muaj kev tswj tsis zoo tau stained. Kev sib tw tau ua nrog Mayer's hematoxylin thiab mounting tau ua nrog Aquatex. Cov ntu tau soj ntsuam ntawm Nikon Microphot-FXA microscope (Nikon Corporation, Melville, NY, USA). Qhov kev ntsuam xyuas zaum kawg tau ua tiav siv software NDPview version 1.2.36 (Hamamatsu Photonics, Iwata City, Nyiv.
Cistanche extract cov txiaj ntsig rau lub raum
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Li Juel Mortensen1, Mette Lorenzen1, Anne Jørgensen2, Jakob Albrethsen2, Niels Jørgensen2, Sauren Møller3,4, Anna-Maria Andersson2, Anders Juul2,4thiab Martin Blomberg Jensen1,5
1. Pawg ntawm Skeletal, Mineral thiab Gonadal Endocrinology, University Department of Growth and Reproduction, Rigshospitalet, 2100 Copenhagen, Denmark; li.juel.mortensen@regionh.dk (LJM); mette.lorenzen.01@regionh.dk (ML)
2. Lub Tsev Haujlwm Saib Xyuas Kev Loj Hlob thiab Kev Loj Hlob thiab Lub Chaw Thoob Ntiaj Teb rau Kev Tshawb Fawb thiab Kev Tshawb Fawb Kev Kawm hauv Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen, Denmark; Anne.Joergesen.02@regionh.dk (AJ); Jakob.Christian.Albrethsen@regionh.dk (JA); Niels.Joergensen@regionh.dk (NJ); Anna-Maria.Andersson@regionh.dk (A.-MA); Anders.Juul@regionh.dk (AJ)
3. Center for Functional and Diagnostic Imaging and Research, Department of Clinical Physiology and Nuclear Medicine 260, Hvidovre Hospital, 2650 Copenhagen, Denmark; Soeren.Moeller@regionh.dk
4. Department of Clinical Medicine, Kws qhia ntawv ntawm Health Sciences, Copenhagen University, 2200 Copenhagen, Denmark
5. Division of Bone and Mineral Research, Harvard School of Dental Medicine/Harvard Medical School, Boston, MA 02115, USA
