[人羊膜上皮细胞的体外培养及分子特征分析]

[In vitro culture and molecular characterization of human amniotic epithelial cells].

作者信息

Liu Xiaoyong, Zhou Qing, Zhang Xiaoling, Qin Xiaoyan, Wang Wenjie, Wang Li, Meng Jing, Chen Jian

机构信息

Department of Ophthalmology, First Affiliated Hospital, Jinan University, Guangzhou 510632, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Dec;30(12):1318-21.

PMID:25481192

Abstract

OBJECTIVE

To investigate the biological and molecular characteristics of human amniotic epithelial cells (HAECs) cultured in vitro.

METHODS

HAECs were isolated by enzyme digestion from amnion after cesarean section. The growth of the cells was observed under an inverted microscope and the curve of cell growth was measured by CCK-8 assay. The colony formation efficiency of HAECs was observed by Giemsa staining. Flow cytometry was used to detect cell cycle distribution and analyze the expressions of CD29, CD31, CD44, CD59, CD105, CD117, CD133, HLA-DR in the second passage of HAECs. The expressions of CD9, E-cadherin, podocalyxin-like protein (PODXL), stage-specific embryonic antigen-4 (SSEA-4), sex determining region Y-box 2 protein (SOX2), SSEA-1, Nanog, octamer-binding protein 3/4 (Oct-3/4) were identified by immunofluorescence staining.

RESULTS

Pure HAECs could be obtained by enzymatic isolation method. The cells grew with adherence and polygonal shape. They entered exponential growth stage from the third day of adherence. The analysis of cell cycle distribution revealed that 66% of the cells were in G0/G1 phase, 31.69% in S phase and 2.31% in G2/M phase. In addition, the colony forming rate of HAECs was (9.33±2.00) %. Flow cytometry and immunofluorescence staining showed that the expressions of CD44, CD29, CD105, CD59, CD9, E-cadherin, PODXL, SSEA-4, SOX2, SSEA-1, Nanog, Oct-3/4 were positive. However, the expressions of CD31, CD117, CD133, HLA-DR were negative.

CONCLUSION

HAECs can be cultured and have proliferation ability for some time in vitro; HAECs express some embryonic stem cell-associated surface molecules.

摘要

目的

研究体外培养的人羊膜上皮细胞（HAECs）的生物学和分子特性。

方法

剖宫产术后，采用酶消化法从羊膜中分离HAECs。在倒置显微镜下观察细胞生长情况，并用CCK-8法检测细胞生长曲线。通过吉姆萨染色观察HAECs的集落形成效率。采用流式细胞术检测第二代HAECs的细胞周期分布，并分析CD29、CD31、CD44、CD59、CD105、CD117、CD133、HLA-DR的表达。通过免疫荧光染色鉴定CD9、E-钙黏蛋白、足突蛋白样蛋白（PODXL）、阶段特异性胚胎抗原-4（SSEA-4）、性别决定区Y盒2蛋白（SOX2）、SSEA-1、Nanog、八聚体结合蛋白3/4（Oct-3/4）的表达。

结果

采用酶分离法可获得纯净的HAECs。细胞贴壁生长，呈多边形。贴壁第3天进入指数生长期。细胞周期分布分析显示，66%的细胞处于G0/G1期，31.69%处于S期，2.31%处于G2/M期。此外，HAECs的集落形成率为（9.33±2.00）%。流式细胞术和免疫荧光染色显示，CD44、CD29、CD105、CD59、CD9、E-钙黏蛋白、PODXL、SSEA-4、SOX2、SSEA-1、Nanog、Oct-3/4表达阳性。然而，CD31、CD117、CD133、HLA-DR表达阴性。