中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (27): 5028-5033.doi: 10.3969/j.issn.2095-4344.2012.27.017

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

骨髓间充质干细胞中大鼠白细胞介素10复制缺陷型重组腺病毒的表达

李 爽1，毕良宽2，朱 斌3，唐 冰3，朱家源3   

  1. 1南方医科大学附属第三医院普通外科，广东省广州市 510630；
    2中山大学附属第二医院泌尿外科，广东省广州市 510120；
    3中山大学附属第一医院烧伤外科，广东省广州市 510080
  • 收稿日期:2011-12-03 修回日期:2011-12-03 出版日期:2012-07-01 发布日期:2013-11-01
  • 通讯作者: 朱家源，教授，中山大学附属第一医院烧伤外科，广东省广州市 510080 Zhujiayuan@163.net
  • 作者简介:李爽★，女，1974年生，安徽省蚌埠市人，汉族，2006年中山大学附属第一医院毕业，硕士，讲师，主要从事外科感染的基因治疗研究。 crystaljasmine0302@yahoo.com.cn
  • 基金资助:

    罗氏青年基金资助项目

Expression of replication-deficient adenovirus expressing rat interleukin-10 in bone marrow mesenchymal stem cells

Li Shuang1, Bi Liang-kuan2, Zhu Bin3, Tang Bing3, Zhu Jia-yuan3   

  1. 1Department of General Surgery, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China;
    2Department of Urinary Surgery, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China;
    3 Department of Burns, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
  • Received:2011-12-03 Revised:2011-12-03 Online:2012-07-01 Published:2013-11-01
  • Contact: Zhu Jia-yuan, Professor, Department of Burns, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China Zhujiayuan@163.net
  • About author:Li Shuang★, Master, Lecturer, Department of General Surgery, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China crystaljasmine0302@yahoo.com.cn

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摘要：

背景：研究显示，白细胞介素10在脓毒症等炎性疾病发挥了抑制炎症反应、促进疾病转归的重要作用。
目的：构建大鼠白细胞介素10重组腺病毒，并以骨髓间充质干细胞为基因载体，观察外源基因在细胞中的表达情况。
方法：采用反转录-聚合酶链反应方法从SD大鼠新鲜脾脏组织中提取出的总RNA中克隆大鼠白细胞介素10基因，通过AdEasy系统成功包装、扩增并纯化出含目的基因的重组腺病毒颗粒。取大鼠骨髓细胞，利用密度梯度离心法和贴壁法分离并扩增骨髓间充质干细胞，并使用流式细胞仪进行表型鉴定。以不同感染倍数的Ad.rIL-10/Ad.GFP感染骨髓间充质干细胞，在荧光显微镜下观察感染效率，并用反转录-聚合酶链反应和Weatern blot方法检测目的基因在大鼠骨髓间充质干细胞中的表达。
结果与结论：成功构建了重组腺病毒Ad.rIL-10，计算病毒滴度为6.0×1010 pfu/mL。重组腺病毒Ad.rIL-10对骨髓间充质干细胞有较高的转染效率，当感染复数=200时为70%。提示含外源基因大鼠白细胞介素10的骨髓间充质干细胞持续高效地表达目的基因，是基因治疗的良好细胞载体。

关键词: 白细胞介素10, 腺病毒载体, 骨髓间充质干细胞, 目的基因, 脓毒症

Abstract:

BACKGROUND: Studies have demonstrated that interleukin-10 (IL-10) plays an important role in anti-inflammatory reaction and promoting prognosis of diseases such as sepsis.
OBJECTIVE: A recombinant adenovirus containing rat IL-10 was constructed and bone marrow mesenchymal stem cells were as gene vectors to investigate exogenous gene expression in cells.
METHODS: Total RNA was extracted from SD rat fresh spleen tissue by reverse transcription-polymerase chain reaction and rat IL-10 gene was cloned. After package, amplification and purified by AdEasy system, recombinant adenovirus particles containing target gene were harvested. Rat bone marrow mesenchymal stem cells were isolated and amplified using density gradient centrifugation and adherence. Phenotype identification was performed through the use of flow cytometer. Bone marrow mesenchymal stem cells were infected using different multiplicities of infection of Ad.rIL-10/Ad.GFP. Then efficiency of infection was observed under fluorescent microscope. The expression of target gene in rat bone marrow mesenchymal stem cells was detected by reverse transcription-polymerase chain reaction and western blot method.
RESULTS AND CONCLUSION: The recombinant adenovirus Ad.rIL-10 was successfully constructed. The titer was calculated as 6.0×1010 pfu/mL after amplification and purification of adenovirus. Ad.rIL-10 effectively infected 70% of rat bone marrow mesenchymal stem cells with the multiplicity of infection value of 200. These findings suggest that rat bone marrow mesenchymal stem cells with rat IL-10 recombinant adenovirus can express target gene effectively and persistently. Rat bone marrow mesenchymal stem cells are optimal and promising vector for gene therapy.

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