表达大鼠融合蛋白NKX2.5-pEGFP的骨髓间充质干细胞的建立

J4 ›› 2011, Vol. 37 ›› Issue (4): 670-674.

表达大鼠融合蛋白NKX2.5-pEGFP的骨髓间充质干细胞的建立

赵东海¹, 朱安峰², 杨淑艳¹, 钟秀宏¹, 张以忠¹, 赵丽微¹

（1.吉林医药学院病理学教研室|吉林吉林132013； 2.吉林省长春市中心医病理科|吉林长春 130051）

收稿日期:2011-04-01 出版日期:2011-07-28 发布日期:2011-07-28
通讯作者: 赵东海 E-mail:（Tel:0432-64560025,E-mail：zdh1027@sina.com）
作者简介:赵东海（1975-）|男|吉林省农安县人|讲师|医学博士|主要从事干细胞工程研究。
基金资助:
林省教育厅“十一五”科学技术研究项目资助课题（2009309）;吉林省吉林市科技发展计划项目资助课题（20090602）

Establishment of |marrow mesenchymal stem cells expressing rat NKX2.5-pEGFP fusion protein

ZHAO Dong-Hai¹, ZHU An-Feng², YANG Shu-Yan¹, ZHONG Xiu-Hong¹, ZHANG Yi-Zhong¹, DIAO Li-Wei¹

1.Department of Pathology,Jilin Medical College,Jilin 132013,China|2.Department of Pathology,Changchun Central Hospital,Changchun　130051,China)

Received:2011-04-01 Online:2011-07-28 Published:2011-07-28

摘要/Abstract

摘要：

目的：构建大鼠NKX2.5融合绿色荧光蛋白真核表达质粒NKX2.5-pEGFP,并筛选达
大鼠融合蛋白NKX2.5- pEGFP 的骨髓间充质干细胞(MSCs)，为后续性细胞和基因治疗心肌梗死提供理论基础。方法:提取胎鼠心肌细胞总RNA,RT-PCR扩增获得大鼠NKX2.5基因,将目的基因克隆到pEGFP-N3载体并获得重组后NKX2.5-pEGFP质粒。采用密度梯度离心法联合贴壁法分离骨髓单个核细胞并获得MSCs。将重组质粒NKX2.5-pEGFP 以 Lipo2000 转染到大鼠MSCs，G418筛选2周。蛋白免疫印记和荧光检测重组后质粒在骨髓间充质干细胞中的表达情况。结果：电泳检测证实经RT-PCR获得 NKX2.5 基因大小为981 bp，与理论值相符。NKX2.5- pEGFP重组质粒经BamHⅠ、Sal Ⅰ双酶切，PCR鉴定得到2条大小约为981和4 700 bp的酶切片段，鉴定结果与预期结果完全一致。蛋白免疫印记检测到转染重组质粒的MSCs中有相对分子质量为65 000的蛋白表达，与理论值相符。荧光检测NKX2.5-pEGFP重组质粒转染的MSCs中可见绿色荧光，证实表达阳性。结论：成功建立表达大鼠NKX2.5-pEGFP基因的MSCs。

关键词: 骨髓间充质干细胞；NKX2.5基因；增强型绿色荧光蛋白

Abstract:

Abstract:Objective
To construct the rat NKX2.5-pEGFP fusion eukaryotic expression plasmid and screen bone marrow mesenchymal stem cells（MSCs） xpressing rat fusion protein NKX2.5-pEGFP and provide theoretical foundation for the research of cell and gene therapy of myocardial infarction. Methods The total RNAs were extracted from myocardial cells of embryo rats,RT-PCR amplification was performed to produce rat gene of NKX2.5.The recombinant plasmid NKX2.5-pEGFP was obtained
by inserting the aim gene into pEGFP-N3.The MSCs derived from bone arrow mononuclear cells were obtained by gradient centrifugation and adherent culture.The recombinant plasmids NKX2.5-pEGFP were ransfected into rat MSCs by Lipo2000 and screened with G418 for 2 eeks.The expression of recombinant plasmid in MSCs was detected by Western blotting and fluoroscope analysis.Results The length of NKX2.5 gene obtained by RT-PCR was 981 bp which was consistent with theoretical numerus.The recombinant plasmid NKX2.5- pEGFP was identified by endonuclease digestion and PCR,which could be digested into two fragments with BamHⅠ and SalⅠ,about 981 and 4 700 bp,the results were coincident with the anticipated result.The protein with molecular weight of 65 000 was found in MSCs transfected with the recombinant plasmid NKX2.5- pEGFP by Western blotting which was coincident with theoretical numerus. Green fluorescence was positive expression in MSCs transfected with the recombinant plasmid NKX2.5-pEGFP by fluorescence detection.
Conclusion The rat MSCs expressing NKX2.5-pEGFP fusion protein is successfully constructed.

Key words: mesenchymal stem cells；NKX2.5；enhancement type green fluorescent protein

中图分类号:

R361

赵东海, 朱安峰, 杨淑艳, 钟秀宏, 张以忠, 赵丽微. 表达大鼠融合蛋白NKX2.5-pEGFP的骨髓间充质干细胞的建立[J]. J4, 2011, 37(4): 670-674.

ZHAO Dong-Hai, ZHU An-Feng, YANG Shu-Yan, ZHONG Xiu-Hong, ZHANG Yi-Zhong, DIAO Li-Wei. Establishment of |marrow mesenchymal stem cells expressing rat NKX2.5-pEGFP fusion protein[J]. J4, 2011, 37(4): 670-674.