[1]鹿晓燕.小鼠胚胎干细胞条件培养基体外促进人角膜内皮细胞增殖的研究[J].眼科新进展,2016,36(3):231-236.[doi:10.13389/j.cnki.rao.2016.0062]
　LU Xiao-Yan.Proliferation in vitro of human corneal endothelial cells promoted by mouse embryonic stem cell conditioned medium[J].Recent Advances in Ophthalmology,2016,36(3):231-236.[doi:10.13389/j.cnki.rao.2016.0062]
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小鼠胚胎干细胞条件培养基体外促进人角膜内皮细胞增殖的研究
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
36卷
期数:
2016年3期
页码:
231-236
栏目:
实验研究
出版日期:
2016-03-05

文章信息/Info

Title:
Proliferation in vitro of human corneal endothelial cells promoted by mouse embryonic stem cell conditioned medium
作者:
鹿晓燕
450052　河南省郑州市，郑州大学第一附属医院眼科
Author(s):
LU Xiao-Yan
Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052 . Henan Province , China
关键词:
胚胎干细胞; 角膜内皮细胞; 增殖
Keywords:
embryonic stem cell;  corneal endothelial cells ;  proliferation
DOI:
10.13389/j.cnki.rao.2016.0062
文献标志码:
A
摘要:
目的　观察小鼠胚胎干细胞条件培养基（ｍｏｕｓｅｅｍｂｒｙｏｎｉｃｓｔｅｍｃｅｌｌｓｃｏｎｄｉｔｉｏｎｅｄｍｅｄｉｕｍ，ＥＳＣ-ＣＭ）是否可以在体外促进人角膜内皮细胞（ｈｕｍａｎｃｏｒｎｅａｌｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ，ＨＣＥＣｓ）的增殖。方法　利用角膜内皮后弹力层组织块方法进行原代培养Ｐ０ＨＣＥＣｓ。实验组使用含有２５％ＥＳＣ-ＣＭ的培养液进行培养，对照组使用普通角膜内皮细胞培养液（ｃｏｒｎｅａｌｅｎｄｏｔｈｅｌｉｕｍｍｅｄｉ-ｕｍ，ＣＥＭ）进行培养。倒置相差显微镜、反转录聚合酶链反应（ｒｅｖｅｒｓｅ-ｔｒａｎｓｃｒｉｐｔｉｏｎｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＲＴ-ＰＣＲ）鉴定ＨＣＥＣｓ；倒置相差显微镜观察细胞的形态及萌出时间；ＷｅｓｔｅｒｎＢｌｏｔ、免疫组织化学法观察ＨＣＥＣｓ的泵相关功能蛋白（ｚｏｎａｏｃｃｌｕ-ｄｅｎｓｐｒｏｔｅｉｎ-１，ＺＯ-１）及Ｎａ＋-Ｋ＋-ＡＴＰ酶的表达。Ｇｉｅｍｓａ染色细胞克隆实验、免疫组织化学及流式细胞学检测Ｋｉ６７阳性率的方法比较ＨＣＥＣｓ的增殖能力；流式细胞学方法检查细胞周期及细胞凋亡情况。ＷｅｓｔｅｒｎＢｌｏｔ和免疫组织化学方法检测细胞周期负性调节蛋白Ｐ２１的水平，初步探讨其可能的作用机制。结果　原代培养时，２５％ＥＳＣ-ＣＭ组培养的ＨＣＥＣｓＰ２细胞爬出，细胞形态呈典型多角形结构。ＣＥＭ在Ｐ２时细胞形态变大，失去了多角形结构。２５％ＥＳＣ-ＣＭ 组和ＣＥＭ组均表达ＺＯ-１、Ｎａ＋-Ｋ＋-ＡＴＰ酶。２５％ＥＳＣ-ＣＭ组的Ｋｉ６７阳性率、克隆形成数量、进入到细胞周期Ｓ期和Ｇ２期的比例均高于ＣＥＭ组（均为Ｐ＜０．０５）。２５％ＥＳＣ-ＣＭ组的细胞凋亡数量和Ｐ２１阳性率均低于ＣＥＭ组（均为Ｐ＜０．０５）。结论　２５％ＥＳＣ-ＣＭ组可显著促进ＨＣＥＣｓ增殖；其作用可能是通过抑制Ｐ２１蛋白的表达和抑制细胞凋亡实现的，为ＨＣＥＣｓ体外大量扩增提供了一种新方法。
Abstract:
Objective To detenmne whether mouse embryonic stem cell conditioned medium ( ESC-CM) promote the proliferative capacity of human corneal endothelial cells ( HCEC) in vitro. Methods Primary cultures of HCEC were established from explants of the endothelial cell layer , including the Descemet ’ s membrane. Cells were cultured in human corneal endothelium medium ( CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction ( RT-PCR) were used to identify HCEC. The eruption time and HCEC morphology were observed under phase-contrast microscopy. The protein expression of zona occludens protein-l ( ZO-1; a tight junction protein) and Na+-K+-ATPase were detected by Western Blot and immunocytochemistry. To explore the proliferation capacity of HCEC .the colony forming efficiency ( CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein ( Ki-67 ) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor P21 level.was detected by Western Blot analysis and immunocytochemistry. Results In primary culture. HCEC in the 25% ESC-CM group erupted with polygonal appearance on day 2. HCEC in the 25% ESC-CM group could be subcultured until passage 6 without enlargement of cell volume , while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCEC in both the 25% ESC-CM and CEM groups expressed ZO-1 , Na+-K+-ATPase. The number of Ki67 positive cells , CFE , and percentage of cells entering the S and G2 phases in the 25% ESC-CM group were higher than those in the CEM group ( all P < 0. 05) . The number of apoptotic cells and P21 protein expression in the 25% ESC-CM group were both lower than those in the CEM group ( all P < 0. 05 ) . Conclusion Using of 25% ESC-CM significantly increase the number of proliferating cells. These effects may be achieved through inhibition of P21 expression and apoptosis. These results suggested that 25% ESC-CM may be a new tool for cultivating HCEC for transplantation.

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更新日期/Last Update: 2016-03-08