中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (18): 3337-3340.doi: 10.3969/j.issn.1673-8225.2012.18.024

• 移植与免疫 transplantation and Immunology • 上一篇    下一篇

大鼠CD4+CD25-T细胞的Foxp3基因转染及其表达***★◆

黄  洋1，李永翔1，沈继龙2，罗庆礼2，陆  威2，霍星星2   

  1. 1安徽医科大学第一附属医院，安徽省合肥市230032；2安徽病原生物学省级实验室(安徽医科大学)，人兽共患病安徽省重点实验室(安徽医科大学)，安徽省合肥市  230032
  • 收稿日期:2011-12-31 修回日期:2012-01-25 出版日期:2012-04-29 发布日期:2012-04-29
  • 通讯作者: 李永翔，教授，主任医师，博士，博士生导师，安徽医科大学第一附属医院，安徽省合肥市230032
  • 作者简介:黄洋★，男，安徽省阜阳市人，汉族，安徽医科大学第一附属医院在读硕士，主要从事Foxp3基因介导的胰岛干细胞移植研究。 yanghuang6699@yahoo.com.cn
  • 基金资助:

    国家自然科学基金面上项目(30871207)；安徽省科技厅重点科研计划项目(7020304101)；安徽省教育厅重点科研项目(KJ2008A154)。

Transfection of rat CD4+CD25- T cells with lentiviral vector containing Foxp3 gene and its expression

Huang Yang1, Li Yong-xiang1, Shen Ji-long2, Luo Qing-li2, Lu Wei2, Huo Xing-xing2   

  1. 1First Affiliated Hospital of Anhui Medical University, Hefei  230032, Anhui Province, China; 2 Key Laboratory of Microbiology and Parasitology (Anhui Medical University), the Key Laboratory of Zoonoses (Anhui Medical University), Hefei  230032, Anhui Province, China
  • Received:2011-12-31 Revised:2012-01-25 Online:2012-04-29 Published:2012-04-29
  • Contact: Li Yong-xiang, Doctor, Professor, Chief physician, Doctoral supervisor, First Affiliated Hospital of Anhui Medical University, Hefei 230032, Anhui Province, China yongxiangli@yahoo.com.cn
  • About author:Huang Yang★, Studying for master’s degree, First Affiliated Hospital of Anhui Medical University, Hefei 230032, Anhui Province, China yanghuang6699@yahoo.com.cn
  • Supported by:

    the National Natural Science Foundation of China, No.30871207*; Key Scientific Research Projects of Anhui Science and Technology Agency, No.7020304101*; Key Scientific Research Projects of Anhui Education Department, No.KJ2008A154*

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摘要：

背景：调节性T细胞在维持机体免疫应答稳态和免疫耐受方面具有非常重要的作用，但外周血中CD4+CD25+调节性T细胞含量极低，且增殖能力较差。
目的：以携带Foxp3基因的慢病毒EGFP+载体转染大鼠CD4+CD25- T细胞，观察其在大鼠CD4+CD25- T细胞中的表达。
方法：以免疫磁珠两步法分选大鼠CD4+CD25- T细胞，用携带大鼠Foxp3基因的慢病毒载体体外转染分选的细胞，以转染Foxp3基因的CD4+CD25- T细胞为实验组，EGFP空白质粒组及CD4+CD25- T细胞为阴性对照组，CD4+CD25+ T细胞为阳性对照组。荧光显微镜和RT-PCR分别从蛋白和mRNA水平检测Foxp3基因的表达。
结果与结论：成功完成了免疫磁珠的分选，获得了纯度较高的CD4+CD25-T细胞和CD4+CD25+ T细胞，细胞存活率为(94±2)%，慢病毒转染的CD4+CD25- T细胞高表达Foxp3基因。表明以携带Foxp3基因的慢病毒载体系统可有效介导Foxp3基因在大鼠CD4+CD25- T细胞中高表达。

关键词: 调节性T细胞, CD4+CD25- T细胞, 慢病毒, Foxp3, 磁珠分选

Abstract:

BACKGROUND: Regulatory T cells play an important role in maintaining immune response homeostasis and immune tolerance, but the CD4+CD25+T cells are less in peripheral blood and with low proliferation ability in vitro.
OBJECTIVE: To observe the expression of transfected rat Foxp3 gene in rat CD4+CD25- T cells using lentiviral vector carrier EGFP+.
METHODS: Rat CD4+CD25-T cells were sorted by magnetic activated cell sorting, and transfected by lentiviral vector containing rat Foxp3 gene in vitro. The whole observation was divided into three groups. Transfected rat CD4+CD25- T cells as the experimental group, the blank plasmid EGFP+ and rat CD4+CD25-T cells as the negative control group, rat CD4+CD25+T cells as the positive control group. The expression of Foxp3 gene was assayed by fluorescence microscope and RT-PCR from the level of protein and mRNA respectively.
RESULTS AND CONCLUSION: The high purity CD4+CD25- and CD4+CD25+ T cells were obtained and the survival rate of cells was (94±2)%. Significantly high expression of rat Foxp3 gene was detected in lentiviral vector transfected CD4+CD25- T cells. The lentiviral vector system carrying rat Foxp3 gene can efficiently mediate the highly expression of rat Foxp3 gene in the rat CD4+CD25- T cells.

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