尿源性干细胞的分离、培养、鉴定及永生化

Isolation,culture,identification and immortalization of urine derived stem cell

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。

Objective：To culture and characterize urine derived stem cells（USC）obtained from the urine,and to establish the immortalized stem cells. Methods：Fresh urine was collected. After urine samples being centrifuged,cell pellets were re-suspended and plated in 24-well tissue culture plates. Only single cells that attached to culture wells by day 2 after plating were used. The morphology of i USC was observed by inverted microscope. Stem cell surface mark CD73,CD90,CD146,SSEA-4 were detected with immunofluorescence and flow cytometry. Urothelial cell surface mark uroplakin Ⅰa,Ⅲ,cytokeratin（CK）-7 and 13 were detected with reverse transcription-polymerase chain reaction（RT-PCR）. Smooth muscle cell surface mark desmin and α-smooth muscle actin were detected with RT-PCR. Part of the cells were selected to induce differentiation of urothelial cells and smooth muscle cells. The human telomerase reverse transcriptase（h TERT）gene was transfected into urine derived stem cell to get the immortalized stem cell. RT-PCR and Western blot were used to detect h TERT. The morphology and the growth curve were observed. Uroplakin Ia and desmin were detected with immunofluorescence. Results：Single,small,compact ＂rice-grain＂like cells was observed. The expression of stem cell surface markers,such as CD73,CD90,CD146,SSEA-4 was positive. RT-PCR findings confirmed that urothelial cell surface marker（uroplakinⅠa,cytokeratin（CK）-7,13）and smooth muscle cell surface marker（desmin,α-smooth muscle actin）were present in USC.The cells were induced to urothelial cells and smooth muscle cells successfully. The expression of h TERT gene detected by RT-PCR and Western blot confirmed that h TERT gene was transfected into USC successfully. The growth curves of USC and i USC showed a ＂S＂shape,but i USC has a faster growth speed compared with USC,and has a statistically significant difference（P=0.043,P=0.000,P=0.006,P=0.001,P=0.025）. The expression of stem cell surface markers,such as CD73,CD90,CD146,SSEA-4 was positive as it presents in USC. i USC had the ability to differentiate into the cell lineages expressing urothelial and smooth muscle cell markers（uroplakin Ⅰa and Desmin）by immunofluorescence analysis. Conclusion：The urine-derived stem cells are successfully isolated and immortalized called immortalized urine-derived stem cells（i USC）. It provides seed cells of stable and safe for regeneration and tissue engineering in vitro studies.