尿源性干细胞的分离、培养、鉴定及永生化

发布时间：2018-06-13 23:04

  本文选题：干细胞 + 泌尿系统　； 参考：《重庆医科大学学报》2017年05期

【摘要】：目的:分离、培养、鉴定尿液来源的干细胞并永生化,为后续下尿路组织重建的研究提供种子细胞。方法:收集人新鲜尿液,对得到的单个贴壁细胞进行培养。倒置显微镜观察尿源性干细胞(urine-derived stem cells,USC)形态,免疫荧光以及流式细胞术检测干细胞表面标记物。RT-PCR检测尿路上皮细胞和平滑肌相关基因。诱导USC向尿路上皮细胞和平滑肌细胞分化。将p SEB-h TERT逆转录病毒感染USC得到永生化的尿源性干细胞(immortalized urine-derived stem cells,i USC)。RT-PCR以及Western blot检测i USC和USC中h TERT基因和蛋白的表达并绘制增殖曲线。对i USC检测干细胞表面标记物CD73、CD90、CD146、SSEA-4及免疫荧光检测尿溶蛋白Ⅰa和肌间线蛋白。结果:成功分离培养得到USC,其外观呈"米粒状",干细胞表面标记物CD73、CD90、CD146、SSEA-4均呈阳性;RT-PCR结果显示尿路上皮细胞表面标记物(尿溶蛋白Ⅰa和Ⅲ、细胞角蛋白-7和13)及平滑肌表面标记物(肌间线蛋白和α-平滑肌肌动蛋白)表达阳性。RT-PCR中h TERT基因表达量为41 636.00±4 134.42,同USC(25 452.67±1 586.32)比较具有统计学意义(P=0.032);Western blot中h TERT蛋白表达量为94 479.00±7 102.20,同USC(61 541.67±3 956.54)比较具有统计学意义(P=0.017)。i USC能连续多代培养,增殖曲线呈"S"形,且与USC对比其增殖能力增强(P0 d=0.272,P1 d=0.043,P3 d=0.000,P5 d=0.006,P7 d=0.001,P9 d=0.025),细胞增殖与天数(P=0.000,F=219.572)和细胞类型(P=0.000,F=90.855)均相关;CD34、CD73、CD90、CD146、SSEA-4依次为0.3%、91.4%、15.3%、99.4%、95.3%,表达无变化;免疫荧光显示尿溶蛋白Ⅰa和肌间线蛋白呈阳性表达。结论:成功从尿液中分离、培养、鉴定尿源性干细胞并永生化,即i USC为再生医学和组织工程学提供稳定安全的种子细胞。
[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，

本文编号：2015862