子宫内膜基质干细胞的体外分离培养及生物学特性

Endometrial stromal stem cells: in vitro isolation, culture and biological features

背景:从干细胞角度去认识和研究子宫内膜细胞,能够为临床上解决子宫内膜引发的子宫内膜疾病治疗提供新的治疗方案和研究切入点。目的:比较不同方法体外分离、培养子宫内膜基质干细胞的生物学特性。方法:采用3种常用的细胞分离方法(胰蛋白酶法、胶原酶Ⅰ法、胰蛋白酶联合胶原酶Ⅰ法)从切除的子宫中分离培养内膜基质干细胞,通过组织分离培养,对生长5 d的第2代细胞进行锥虫蓝染色,计数活细胞数量。采用间质干细胞标志物CD146联合造血干细胞标志物CD90单克隆抗体,以免疫组化及RT-PCR法对培养获得的子宫内膜基质干细胞进行鉴定;同时取生长对数期细胞进行培养,并绘制细胞生长曲线。结果与结论:(1)培养获得的子宫内膜基质干细胞形态呈现梭形,细胞贴壁生长,排列无极性,具有成纤维细胞形态;(2)子宫内膜基质干细胞标志物CD146和CD90的DNA分别在500 bp和150 bp呈现高表达,而子宫肌层组织对照组不表达;(3)培养所得干细胞的生长曲线呈现S型,3种方法培养的细胞生长周期差异无显著性意义;相对于胰蛋白酶法和胰蛋白酶联合胶原酶Ⅰ法而言,胶原酶Ⅰ法分离培养所得的子宫内膜基质干细胞数量较多,生长速度较快;(4)结果表明,从切除的子宫组织中能够成功分离出子宫内膜基质细胞,且胶原酶Ⅰ法是一种较为高效、实用、稳定的体外子宫内膜基质干细胞培养方法。

BACKGROUND： Understanding and studying endometrial cells from the perspective of stem cells can provide a new therapeutic approach and research entry point for the clinical treatment of endometrial diseases. OBJECTIVE： To compare the biological features of endometrial stromal stem cells isolated and cultured using different methods.METHODS： Three commonly used cell separation methods, including trypsin, collagenase I and their combination, were used to isolate and culture endometrial stromal stem cells from the uterus after removal. Afterwards, passage 2 cells cultured for 5 days were subjected to trypan blue staining and living cell counting. Immunohistochemical staining for CD146 and CD90 and RT-PCR were used to identify harvested endometrial stromal stem cells. Logarithmically growing cells were cultured to draw cell growth curves. RESULTS AND CONCLUSION： Harvested endometrial stromal stem cells presented with spindle, adherent cell growth, nonpolar arrangement, and fibroblast morphology. The DNA of endometrial stem cell marker CD146 andCD90 highly expressed in 500 bp and 150 bp respectively, but did not express in the mesometrium. S-shaped growth curve of cultured cells was found, and there was no difference in the cell cycle of cells cultured using three different methods. Among the three methods, collagenase I method could harvest the highest number of endometrial stromal stem cells that grew fastest. These findings indicate that endometrial stromal stem cells can be successfully isolated from the removed uterine tissues, and collagenase I method is an efficient, practical and stable method for in vitro culture of endometrial stromal stem cells.