人羊膜间充质干细胞原代培养及向心肌样细胞分化  

Primary culture and differentiation of human amniotic mesenchymal stem cells into cardiac potential cell

作　　者：杨曲 张卫芳[2] 陈祺 胡锋 李娟[2] 罗军[1] YANG Qu;ZHANG Wei-fang;CHEN Qi;HU Feng;LI Juan;LUO Jun(Department of Rehabilitation Medicine,the Second Affiliated Hospital of Nanchang University,Nanchang 330000,China;Department of Pharmacy,the Second Affiliated Hospital of Nanchang University,Nanchang 330000,China;Department of Cardiology,the Second Affiliated Hospital of Nanchang University,Nanchang 330000,China)

机构地区：[1]南昌大学第二附属医院康复医学科,南昌330000 [2]南昌大学第二附属医院药学部,南昌330000 [3]南昌大学第二附属医院心血管内科,南昌330000

出　　处：《临床与实验病理学杂志》2022年第5期513-519,共7页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金(81760408)。

摘　　要：目的探讨原代人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)体外分离培养的方法及向心肌样细胞分化的能力。方法收集人正常足月顺产或剖宫产后胎盘羊膜,采用机械-酶消化法分离培养hAMSCs,观察其形态学特点。取第3代hAMSCs,通过诱导成骨、成脂分化和流式细胞术检测鉴定其干细胞特性,体内接种法和体外软琼脂克隆法检测其致瘤性。应用5-aza、TGF-β1及Ang-Ⅱ诱导其向心肌样细胞分化(实验组),镜下观察细胞形态学变化;qRT-PCR、Western blot及细胞免疫荧光法分别检测心肌细胞标志基因cTnI、ACTN2、NKX2.5及其蛋白的表达;CCK-8实验检测细胞活性;淋巴细胞增殖实验检测细胞免疫原性。结果分离纯化后的hAMSCs呈梭形,漩涡状生长,可成功诱导向成骨、成脂分化;流式细胞术结果显示hAMSCs高表达CD44、CD90,低表达CD45,不表达HLA-DR;体内外实验未显示致瘤性。诱导hAMSCs向心肌样细胞分化后,细胞呈棒状或长梭形,胞间见细丝样连接结构,与对照组相比,实验组中cTnI、ACTN2、NKX2.5基因及其蛋白水平明显升高,且保持良好的细胞活性及低免疫原性,差异有统计学意义(P<0.05)。结论hAMSCs可在体外分离培养且能够被诱导向心肌样细胞分化,为心肌疾病的再生修复治疗提供了新的种子细胞。Purpose To explore the isolation and culture of primary human amniotic mesenchymal stem cells(hAMSCs)in vitro,and to observe the ability of differentiation into cardiac potential cells.Methods The placental amniotic membrane were taken from full-term normal delivery or cesarean.HAMSCs were isolated by mechanical-enzyme digestion and purified by pancreatin.Then,their morphological change was observed under microscope.When cells were at the 3rd passage,osteogenic induction,adipogenic differentiation and flow cytometry were used to evaluate the character of stem cells.Meanwhile,tumorigenicity was detected by stem cells transplantation in vivo and clone formation test was used to detect oncogenicity in vitro.5-aza,TGF-β1 and Ang-Ⅱwere applied to induce the differentiation of HAMSCs into cardiac potential cell and were used as the experimental group to observe the changes in cell morphology under microscope.The expression of cTnI,ACTN2,NKX2.5 was detected by qRT-PCR,Western blot and cell immunofluorescence.Cell activity was detected by CCK-8 assay and immunogenicity was proved by lymphocyte proliferation test.Results HAMSCs were successfully cultured from human amniotic tissue by mechanical-enzyme digestion method.The isolated and purified hAMSCs grew in spindle shape and arranged in whorled.Under adipogenic and osteogenic differentiation conditions,the hAMSCs were able to differentiate into adipocytes and osteocytes,respectively.Flow cytometry showed that hAMSCs were positive for mesenchymal stem cells markers CD44,CD90,and negative for hematopoietic stem cell CD45,and no expression for the major histocompatibility protein HLA-DR.Tumorigenicity assay demonstrated that hAMSCs had no tumorigenicity both in vitro and vivo.After inducing hAMSCs to differentiate into cardiac potential cells,the cells were spindle and bars with obvious cell-cell junction and intercellular filament-like connections.Compared with the control group,the levels of cTnI,ACTN2,NKX2.5 gene and their proteins were significantly higher in the exp

关 键 词：羊膜间充质干细胞 原代培养 诱导分化 心肌样细胞 

分 类 号：R310.17[医药卫生—基础医学]