论文部分内容阅读
目的分离培养人羊膜间质细胞(hAMCs),初步探讨其向胰岛样细胞的诱导分化。方法羊膜组织经胰蛋白酶消化去除上皮细胞后,采用胶原酶I联合中性蛋白酶的方法分离hAMCs。细胞免疫荧光检测分离得到细胞表面标记Vimen-tin+,SSEA-4+表达;流式细胞仪检测CD29,CD90及CD34,CD45表达;RT-PCR检测ACTG2、ACTA2、MMP2、Cripto、Sox2、LEFTYA、nanog、Oct-4基因表达;添加诱导因子诱导hAMCs向胰岛样细胞分化。结果分离得到的hAMCs可体外长期传代,并保持稳定的遗传学特性;细胞表现vimentin+、SSEA-4+;CD29、CD90表达量分别为91.5%±9.93%和48.7%±9.47%;不表达CD34,CD45;表达ACTG2、ACTA2、MMP2和Cripto、Sox2、LEFTYA、nanog、Oct-4等基因。诱导后的细胞表达insulin、PDX1、ngn3、glucagon等基因,并表现insulin+。结论建立了人羊膜间充质细胞的分离培养方法;在体外可诱导分化为胰岛样细胞。
OBJECTIVE: To isolate and culture human amniotic mesenchymal cells (hAMCs) and to investigate their differentiation into islet-like cells. Methods Amniotic membrane tissue was trypsinized to remove epithelial cells, and then collagenase I and neutral protease were used to separate hAMCs. The expression of Vimen-tin + and SSEA-4 + was detected by immunofluorescence staining. The expressions of CD29, CD90, CD34 and CD45 were detected by flow cytometry. The expressions of ACTG2, ACTA2, MMP2, Cripto, Sox2, LEFTYA and nanog , Oct-4 gene expression; induced by induction of hAMCs into pancreatic islet-like cells. RESULTS: The isolated hAMCs could be passaged in vitro for a long time and maintained stable genetic characteristics. The expression of vimentin +, SSEA-4 +, CD29 and CD90 were 91.5% ± 9.93% and 48.7% ± 9.47%, respectively. CD45; express ACTG2, ACTA2, MMP2 and Cripto, Sox2, LEFTYA, nanog, Oct-4 and other genes. The induced cells expressed insulin, PDX1, ngn3, glucagon and other genes, and showed insulin +. Conclusion The method of isolation and culture of human amniotic membrane mesenchymal cells was established. In vitro, it could be induced to differentiate into islet-like cells.