中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (5): 848-853.doi: 10.3969/j.issn.1673-8225.2010.05.021

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

小鼠白细胞介素10重组腺病毒载体构建及对树突状细胞的基因修饰

许  飞，陈传辉，林耀广，张  伟   

  1. 南昌大学第一附属医院呼吸内科，江西省南昌市   330006
  • 出版日期:2010-01-29 发布日期:2010-01-29
  • 通讯作者: 张 伟，教授，主任医师，南昌大学第一附属医院呼吸内科，江西省南昌市 330006 zhangweiliuxin@163.com
  • 作者简介:许 飞☆，男，1973年生，安徽省含山县人，汉族，博士，副教授，副主任医师，主要从事哮喘的免疫及基因治疗方面的研究。 hiphopchen1984@sina.com
  • 基金资助:

    国家科技支撑计划(2008BAI68B00)资助。

Construction of mouse interleukin-10 recombinant adenovirus vector and gene modification for dendritic cells

Xu Fei, Chen Chuan-hui, Lin Yao-guang, Zhang Wei   

  1. Department of Respiratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang  330006, Jiangxi Province, China
  • Online:2010-01-29 Published:2010-01-29
  • Contact: Zhang Wei, Professor, Chief physician, Department of Respiratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China zhangweiliuxin@163.com
  • About author:Xu Fei☆, Doctor, Associate professor, Associate chief physician, Department of Respiratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China hiphopchen1984@ sina.com
  • Supported by:

    National Key Technology R&D Program, No. 2008BAI68B00*

PDF

619

被引次数

12

摘要：

背景：对于抗原递呈细胞树突状细胞及其分泌的细胞因子白细胞介素10在气道高反应性和炎症中的作用国内外文献报道较少。
目的：构建小鼠白细胞介素10腺病毒重组体Ad-mIL-10，获得小鼠白细胞介素10基因修饰的树突状细胞，以期为下一步基因治疗的动物实验打下基础。
方法：通过人工合成获得小鼠白细胞介素10基因，根据白细胞介素10基因序列及腺病毒载体的多克隆位点，合成包括酶切位点的基因序列，连接到pMD18-T载体并测序鉴定。用基因工程的方法将小鼠白细胞介素10基因克隆到BD Adeno-XTM腺病毒载体，于人胚肾293细胞中包装、扩增病毒并测定白细胞介素10蛋白表达，转染到体外培养的小鼠骨髓来源的树突状细胞。
结果与结论：成功构建了小鼠Ad-mIL-10重组腺病毒载体并高包装、扩增成功，测定高表达白细胞介素10蛋白，体外成功培养小鼠骨髓来源的小鼠树突状细胞并顺利转染Ad-mIL-10。提示用基因工程方法构建小鼠Ad-mIL-10重组腺病毒载体并转染小鼠骨髓来源的树突状细胞是可行的，可为进行相关基因治疗的可能性提供更充足的理论依据。

关键词: 白细胞介素10基因, 小鼠, 重组腺病毒, 树突状细胞, 基因修饰

Abstract:

BACKGROUND: Few reports concern the effects of dendritic cells-a kind of antigen presenting cells, and interleukin-10 (IL-10) on airway hyperreactivity or inflammation.
OBJECTIVE: To construct mice IL-10 recombinant adenovirus vector Ad-mIL-10 to acquire the dendritic cells modified by mIL-10, which can provide a foundation for the further study.
METHODS: Mouse IL-10 (mIL-10) gene comprise of enzyme cutting spot was synthesized according to the mIL-10 gene sequence and multiclone spot of adenovirus vector, connected to pMD18-T vector and sequenced. MIL-10 was subcloned to BD Adeno-XTM vector, packed and augmented in HEK 293 cells, following determine the protein expression, and the vector was transfected to mice bone marrow-derived dendritic cells.
RESULTS AND CONCLUSION: Recombinant adenovirus vector Ad-mIL-10 was successfully synthesized, packed and augmented, which could highly express protein IL-10. Bone marrow-derived dendritic cells were successfully cultured and transduced in vitro. It suggested that it is feasible to transfect mice dendritic cells by Ad-mIL-10 adenovirus vector. The study can provide more sufficient theoretic evidence for the possibility of correlative gene therapy.

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