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目的：研究人蜕膜组织中子宫内膜干细胞（endometrial stemcells，EnSCs)的分离、培养及鉴定方法。方法：取人工流产患者的蜕膜组织，用胰酶和II型胶原酶消化分离子宫内膜细胞，计数后，以极低密度接种于10 cm培养皿，分离单克隆贴壁细胞。倒置显微镜观察EnSCs 形态变化及克隆形成情况；CCK-8 法绘制EnSCs 生长曲线，计算倍增时间；流式细胞仪分析EnSCs 细胞周期及表面抗原CD29、CD31、CD34、CD44、CD45、CD73、CD90、CD105、CD146表达情况。结果：EnSCs呈长梭形，成纤维细胞样，可见粗细不等的胞质突起，增殖至80-90 %呈漩涡状生长，其克隆形成率分别为3.91 %和9.14 %；EnSCs 生长曲线呈“S”型，接种48 h至 60 h后进入对数生长期，于第9 天达到平台期，倍增时间为37-52 h；流式细胞仪测定EnSCs 表面抗原CD29（99.13 %），CD44 （97.39 %)，CD73（99.68 %)，CD90（96.76 %)，CD105（89.64 %)，CD146（77.96 %）呈阳性表达，而CD31(1.30 %)，CD34(0.68 %)， CD45(1.26 %)呈阴性表达, 有61.79 %的细胞处于G0/G1 期，22.37 %的细胞处于S+G2/M期。结论：采用组织消化法分离单克隆贴壁的子宫内膜细胞，能够获得纯化的EnSCs。

英文摘要:

To investigate isolation, culture and identification of Human Endometrial Stem Cells in decidua. Human endometrial stem cells were digested and isolated from decidua using pancreatin and collagenase II, and cultured with DMEM/F12 in very low density in 10 cm dish, and then specific clones were isolated. Morphology and clone of the Human Endometrial Stem Cell was observed under phase-contrast microscope; the growth curve and doubling time was analysed by MTT assay. Cell cycle percentage and surface antigen was detected by flow cytometry. Endometrial Stem Cells were spindle-like shapes, fibroblast like cells, which had different thicknesses long protuberances, arranged tightly in a vortex-like appearance when grew up to 80-90%. The clone formation rate of the Endometrial Stem Cells was 3.91% to 9.14%. The growth curve was “S”shaped with 48 to 60 hour to logarithmic phase and the ninth days to plateau and the culture doubling time was 37-52 h. Cell cycle analysis showed that 61.79%of the cells was in the G0/G1 phase, 22.37%of the cells was in S+G2/Mphase. Flow cytometry analysis showed that Human Endometrial Stem Cells were positive for CD29(99.13 %), CD44(97.39 %), CD73(99.68 %), CD90(96.76 %), CD105(89.64 %) and CD146( 77.96 %), but they were negative for CD31(1.30 %), CD34(0.68 %), CD45(1.26 %). Human Endometrial Stem Cells were successfully isolated by tissue digestion method.

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