应用原瓶黏附分选表皮干细胞的方法学特点

Methodological characteristics of former plate attachment to isolate epidermal stem cells

目的:探索一种简单易行的方法分离表皮干细胞,为组织工程学的进一步研究奠定基础。方法:实验于2005-07/12在中山大学眼科中心国家眼科学重点实验室完成。健康3个月龄恒河猴2只,分离与培养猴表皮细胞,获取第2～4代的细胞,消化后,将细胞重新放回原瓶中,培养箱中静置20min,获得的贴壁细胞即为快吸附的表皮细胞。①于光学显微镜下观察分选前细胞和快吸附细胞。②对20min快吸附的表皮细胞进行整合素α6和CD71流式细胞仪检测,以未经吸附分选的细胞作为对照。③20min快吸附的表皮细胞和分选后未黏附细胞分别制作3组细胞爬片,进行整合素β1、K15、和K10的免疫组织化学染色。④20min快吸附的表皮细胞和分选后未黏附细胞进行K15、整合素β1和K1(K10对应角蛋白)的mRNA反转录聚合酶链反应的检测。结果:①分选后的细胞体积较小,核浆比例较大。②流式细胞仪的结果显示快吸附细胞以干细胞为主(α6briCD71dim为82.5±1.641),也含有少量的短暂扩增细胞(α6briCD71bri为4.9±2.125),有丝分裂后细胞及终末细胞则检测不到(α6dim)。③免疫组化可见快吸附细胞呈K15和整合素β1阳性,K10阴性;分选后未黏附细胞K10为阳性,而K15和整合素β1均为阴性。④反转录聚合酶链反应示吸附分选后的快吸附细胞高表达K15和β1整合素,K10没有表达;而分选后剩余的细胞只表达K10。结论:原瓶黏附分选方法是一种简单可行的分选表皮干细胞的方法。

AIM： To look for an easy and feasible approach to isolate epidermal stem cells （ESCs）, which may provide a practical source of further researches on tissue engineering. METHODS： From July to December 2005, the research was completed in National Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center of Sun Yat-Sen University. Two rhesus monkeys of 3 months old were adopted to isolate and culture their epidermal cells. The 2^nd-4^th passage cells after digestion were relayed in former plate for 20 minutes to harvest rapid attaching cells.①ESCs before isolation and rapid attaching ceils were observed on a microscope. ② The isolated rapid attaching cells with collagen type IV adhesion were measured by flow cytometry for integrin α6 and CD71, and the cells without attachment were taken as controls.③ Rapid attaching cells and non-attaching cells were respectively put upon three slides, lmmunohistochemistry staining was conducted to detect integrin β1, K10 and K15. ④Reverse transcription polymerase chain reaction （RT-PCR） was used to identify the expression of specific markers （β1, K15 and K1） on the isolated rapid attaching cells and non-attaching cells. RESULTS： ①The rapid attaching cells exhibited small cell size with a high nuclear-to-cytoplasmic ratio. ②The results of flow cytometry showed that putative ESCs （α6^bri CD71^dim=82.5±1.641） were predominant in rapid attaching cells, transient amplifying cells （α6^bri CD71^dim=4.9±2.125） were relatively scarce, and postmitotie differentiating cells and terminally differentiated cells （α6^dim） were not existing. ③The immunohistologic experiments showed that rapid attaching cells isolated by type Ⅳ collagen attachment method expressed K15 and integrin β1 respectively. However, non-attaching cells expressed K10 only. ④RT-PCR showed that the rapid attaching cells were positive for K15 and integrin β1, while the cell after isolation were positive for K10 only. CONCLUSION： Former plate attachment method is an easy and feasible approach to isolate ESCs.