子宫内膜基质干细胞培养方法的比较研究

Comparative study on culture methods of endometrial stromal stem cells

目的通过贴壁纯化技术和磁珠分选法培养人子宫内膜基质干细胞,并对两种方法进行比较,寻求既能方便获得大量子宫内膜基质干细胞,又能减少其分化的理想分离培养方法。方法采用贴壁纯化和磁珠分选法分离培养子宫内膜基质细胞,通过倒置显微镜观察细胞的形态,免疫组化鉴定细胞的来源,流式检测CD146和CD90的表达鉴定基质干细胞。通过比较细胞的克隆形成率和CD146、CD90阳性率的差异,综合评估2种培养方法的优劣。结果倒置显微镜观察2种培养方法所得子宫内膜基质细胞均多呈梭形;波形蛋白免疫组化染色均呈阳性。流式细胞仪检测贴壁纯化培养的基质细胞中CD146、CD90阳性率为51.45%、47.24%;磁珠分选法培养体系为CD146、CD90阳性率为63.53%、77.48%。贴壁纯化和磁珠分选法培养的子宫内膜基质细胞克隆形成率分别为(1.25±0.18)%和(3.3±0.4)%。结论贴壁纯化和磁珠分选培养作为子宫内膜基质干细胞的2种培养方法,均可以获得未分化的干细胞。用磁珠分选培养干细胞的方法比较复杂,但可获得大量的干细胞。用贴壁纯化技术培养干细胞方法简单,但细胞不易贴壁,且数量较少,但也能有效分离出较纯的干细胞,保持较好的细胞活性。

Objective To compare the efficacy of adherent technique and magnetic activated cell sorting（MACS） for endometrial stromal stem cell culture.Methods The morphological features of cultured endometrial stromal stem cells were observed under inverted phase control microscopy.Immunostaining of cell marker vimentin was performed to identify the stromal cell.The stemness of endometrial stromal stem cells was evaluated by colony forming efficiency and proportion of CD146 and CD90 positive cells by flow cytometry.Results Endometrial stromal stem cells were successfully cultured in vitro using two methods.They were both positive for vimentin.The colony forming efficiency of endometrial stromal stem cells cultured by adherent technique and MACS was（1.25±0.18）% and（3.3±0.4）%,respectively.There was significant difference in colony forming efficiency between the two methods（P0.05）.CD146 positive cells proportion was higher by MACS than that by adherent technique（63.53% vs 51.45%,P0.05）.CD90 positive cells proportion was also higher by MACS than that by adherent technique（77.48% vs 47.24%,P0.05）.Conclusion The two methods are applicable to culture endometrial stromal stem cells in vitro.The MACS method is complicated and can propagate a number of stem cells from endometrium,while the adherent method is easier to get pure endometrial stromal stem cells.