人胚胎滋养细胞和胎盘间充质干细胞的分离与纯化

背景:胎盘的细胞成分较复杂,其中所包含的滋养细胞在母胎免疫耐受过程中起重要作用,胎盘间充质干细胞具有多向分化潜能以及抑制淋巴细胞增殖的特性,常规分离方法通常难以获得大量且纯度较高的上述两种细胞.目的:拟建立一次操作即可同时获得大量、较高纯度的滋养细胞和胎盘间充质于细胞的操作方案.方法:人胎盘组织洗净剪碎,采用胰蛋白酶和DNAse I共同消化,分3个阶段,每个阶段在恒温37℃下180 r/min消化2 min.重悬消化产物,200目筛网过滤后采用Percoll密度梯度分离液分离,分别收集滋养细胞层和胎盘间充质干细胞层.滋养细胞层以差速贴壁法去除成纤维细胞,胎盘间充质干细胞直接接种于75 cm2培养瓶中培养.观察胎盘组织消化情况,计数滋养细胞数量及其细胞角蛋白7的表达,观察胎盘间充质干细胞生长增殖、表型及成骨分化潜能.结果与结论:经胰蛋白酶和DNAse I消化后,胎盘组织仅剩少许残渣.差速贴壁后,滋养细胞数达(5.48±1.98)×10~8个,细胞角蛋白7阳性率为(90+4.36)%.胎盘间充质干细胞接种19～21 d达90%融合,细胞数为(1.96±0.24)×10~6个,强表达CD29,CD44和HLA-ABC,不表达CD34,CD45,CD14和HLA-DR,诱导后茜素红染色呈鲜艳橙红色,可向成骨细胞方向分化.提示采用胰蛋白酶和DNAse I共同消化胎盘组织,并结合Percoll不连续密度梯度分离液分离细胞,可同时一次性获得大量的滋养细胞和较多的胎盘间充质干细胞,细胞纯度和活性均较好.

Isolation and purification of human cytotrophoblasts and placental mesenchymal stem cells

BACKGROUND: Cytotrophoblasts in placental cell components plays an important role in fetal immunological tolerance. Placental mesenchymal stem cells (pMSCs) have potential of multiple differentiation and inhibition of lymphocyte proliferation. However, conventional methods cannot acquire a large amount of purified human cytotrophoblasts or pMSCs. OBJECTIVE: To establish a method to obtain large placenta tissue, and harvest plenty of cytotrophoblasts and pMSCs with high purity and activity. METHODS: Human pl...