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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:

35卷

期数:

2015年5期

页码:

404-408

栏目:

实验研究

出版日期:

2015-05-05

文章信息/Info

Title:

 Investigation of embryonic stem cell differentiated into retinal progenitor cells in vitro

作者:

 邱莹; 彭广华

 450052　河南省郑州市，郑州大学第一附属医院眼科

Author(s):

 QIU Ying ;  PENG Guang-Hua

 Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052 , Henan Province , China

关键词:

 ; 胚胎干细胞; 视网膜祖细胞; 分化

Keywords:

 ; embryonic stem cell;  retinal progenitor cells ;  differentiation

DOI:

10.13389/j.cnki.rao.2015.0110

文献标志码:

A

摘要:

 目的　探讨小鼠胚胎干细胞（ｍｏｕｓｅｅｍｂｒｙｏｎｉｃｓｔｅｍｃｅｌｌ，ｍＥＳＣ）诱导为视网膜祖细（ｒｅｔｉｎａｌｐｒｏｇｅｎｉｔｏｒｃｅｌｌｓ，ＲＰＣ）的体外培养方法，为致盲性视网膜变性疾病的治疗提供种子细胞。方法　用细胞免疫荧光染色法和流式细胞术对ｍＥＳＣ进行ｍＥＳＣ标记物阶段特异性胚胎抗原１、细胞增殖核抗原Ｋｉ６７、细胞周期和碱性磷酸酶进行鉴定后，在无血清条件下悬浮培养形成拟胚体（ｅｍｂｒｙｏｉｄｂｏｄｉｅｓ，ＥＢ）并添加抑制因子１、左右决定因子Ａ和γ-促分泌酶抑制剂等诱导分化为ＲＰＣ，并对其分化的细胞进行标记物兔来源配对盒基因６（ｐａｉｒｅｄｂｏｘ６，Ｐａｘ６）、兔来源性别决定区Ｙ框蛋白（Ｓｏｘ２）和鼠来源转录因子同源异型框蛋白（ｏｒｔｈｏｄｅｎｔｉｃａｌｈｏｍｅｏｂｏｘ２，Ｏｔｘ２）的检测以及增殖能力和细胞周期的检测。结果　ｍＥＳＣ标记物碱性磷酸酶阳性表达，特异性胚胎抗原１阳性率为（８０.６６±０．６４）％，Ｋｉ６７和细胞周期检测显示有较高增殖能力。定向诱导分化后，ＲＰＣ标记物Ｐａｘ６、Ｓｏｘ２和Ｏｔｘ２阳性率分别为（５０．８７±２．９７）％、（４９．１０±２．６０）％和（３２.０１±３.８７）％，Ｋｉ６７和细胞周期检测均显示诱导后的ＲＰＣ具有一定的增殖能力。结论　在特定的细胞因子作用下，ｍＥＳＣ可以诱导分化为ＲＰＣ。

Abstract:

 Objective To investigate the method of inducing mouse embryonic stem cells ( mESC) differentiation into retinal progerutor cells ( RPC) in vitro , and provide the theory basis of seed cells for blinding retinal degeneration diseases. Methods The specific marker of mESC ,including stage specific embryonic antigen I ( SSEAI ) . Ki67,cell cycle and alkaline phosphatase ( AKP) , were identified by immunofluorescence stairung and flow cytometry. Then mESC were cultured under serum free suspension culture to form embryonic bodies. RPC were induced by Dicckopf-I , Lefty-A and N-[ N- ( 3 , 5-Difluorophenacetyl ) -L-alanyl ] -2-phenylglycine tert-butyl ester, their specific markers Pax6 .Sox2 and Otx2 , multiplication capacity and cell cycle were identified by immunofluorescence staining and flow cytometry. Results The specific markers of AKP for mESC were positively expressed, the positive expressive rate of SSEAI was ( 80. 66 +0. 64 ) % , and Ki67 and cell cycle detection showed the high potential of selfproliferation ability. Induced RPC were positive to Pax6 .Sox2 and Otx2 , and the positive expressive rate were ( 50. 87 +2. 97 ) % . ( 49. 10 +2. 6) % and ( 32. 01 + 3. 87 ) % , the positive expression of Ki67 and cell cycle showed RPC had a potential of self-proliferation ability. Conclusion Under the effect of specific cytokines .mESC can be induced into RPC.

备注/Memo

备注/Memo:

 国家重点基础研究发展计划基金资助（编号：２０１３ＣＢ９６７-００１）；国家自然科学基金项目（编号：３１２７１４００、３１４７００６５）

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更新日期/Last Update: 2015-04-27