Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (27): 5028-5033.doi: 10.3969/j.issn.2095-4344.2012.27.017

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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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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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