本文選題：干細(xì)胞 + 泌尿系統(tǒng)��； 參考：《重慶醫(yī)科大學(xué)學(xué)報(bào)》2017年05期

【摘要】：目的:分離、培養(yǎng)、鑒定尿液來(lái)源的干細(xì)胞并永生化,為后續(xù)下尿路組織重建的研究提供種子細(xì)胞。方法:收集人新鮮尿液,對(duì)得到的單個(gè)貼壁細(xì)胞進(jìn)行培養(yǎng)。倒置顯微鏡觀察尿源性干細(xì)胞(urine-derived stem cells,USC)形態(tài),免疫熒光以及流式細(xì)胞術(shù)檢測(cè)干細(xì)胞表面標(biāo)記物。RT-PCR檢測(cè)尿路上皮細(xì)胞和平滑肌相關(guān)基因。誘導(dǎo)USC向尿路上皮細(xì)胞和平滑肌細(xì)胞分化。將p SEB-h TERT逆轉(zhuǎn)錄病毒感染USC得到永生化的尿源性干細(xì)胞(immortalized urine-derived stem cells,i USC)。RT-PCR以及Western blot檢測(cè)i USC和USC中h TERT基因和蛋白的表達(dá)并繪制增殖曲線。對(duì)i USC檢測(cè)干細(xì)胞表面標(biāo)記物CD73、CD90、CD146、SSEA-4及免疫熒光檢測(cè)尿溶蛋白Ⅰa和肌間線蛋白。結(jié)果:成功分離培養(yǎng)得到USC,其外觀呈"米粒狀",干細(xì)胞表面標(biāo)記物CD73、CD90、CD146、SSEA-4均呈陽(yáng)性;RT-PCR結(jié)果顯示尿路上皮細(xì)胞表面標(biāo)記物(尿溶蛋白Ⅰa和Ⅲ、細(xì)胞角蛋白-7和13)及平滑肌表面標(biāo)記物(肌間線蛋白和α-平滑肌肌動(dòng)蛋白)表達(dá)陽(yáng)性。RT-PCR中h TERT基因表達(dá)量為41 636.00±4 134.42,同USC(25 452.67±1 586.32)比較具有統(tǒng)計(jì)學(xué)意義(P=0.032);Western blot中h TERT蛋白表達(dá)量為94 479.00±7 102.20,同USC(61 541.67±3 956.54)比較具有統(tǒng)計(jì)學(xué)意義(P=0.017)。i USC能連續(xù)多代培養(yǎng),增殖曲線呈"S"形,且與USC對(duì)比其增殖能力增強(qiáng)(P0 d=0.272,P1 d=0.043,P3 d=0.000,P5 d=0.006,P7 d=0.001,P9 d=0.025),細(xì)胞增殖與天數(shù)(P=0.000,F=219.572)和細(xì)胞類型(P=0.000,F=90.855)均相關(guān);CD34、CD73、CD90、CD146、SSEA-4依次為0.3%、91.4%、15.3%、99.4%、95.3%,表達(dá)無(wú)變化;免疫熒光顯示尿溶蛋白Ⅰa和肌間線蛋白呈陽(yáng)性表達(dá)。結(jié)論:成功從尿液中分離、培養(yǎng)、鑒定尿源性干細(xì)胞并永生化,即i USC為再生醫(yī)學(xué)和組織工程學(xué)提供穩(wěn)定安全的種子細(xì)胞。
[Abstract]:Aim: to isolate, culture, identify and immortalize the urine derived stem cells to provide seed cells for the subsequent study of lower urinary tract tissue reconstruction. Methods: human fresh urine was collected and single adherent cells were cultured. The morphology of urine-derived stem cells was observed by inverted microscope, the surface marker of stem cells was detected by flow cytometry, and the urothelial cells and smooth muscle related genes were detected by flow cytometry. USC was induced to differentiate into urothelial cells and smooth muscle cells. The immorphic urine-derived stem cells were infected with pSEB-hTERT retrovirus to obtain immorphic urine-derived stem cells. RT-PCR and Western blot were used to detect the expression of hTERT gene and protein in IUSC and USC and draw the proliferation curve. CD73, CD90, CD146, SSEA-4, and urolysin I a and myolinear protein were detected by iUSC and immunofluorescence, respectively. Results: USCS were isolated and cultured successfully. The appearance of USCS was "rice granular". The surface markers of stem cell CD73, CD90, CD146 and SSEA-4 were all positive. The results of RT-PCR showed that the surface markers (urolysins 鈪，

本文編號(hào)：2015862