Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (23): 4186-4190.doi: 10.3969/j.issn.1673-8225.2010.23.002

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Construction and expression of an adenoviral vector encoding human interleukin-12 in human bone marrow mesenchymal stem cells

Chen Yuan-yuan1,2, Tan Xiao-hua1, Ma Jing-ying1   

  1. 1Biotherapy Center, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing  100700, China;
    2Shanxi Medical University, Taiyuan  030001, Shanxi Province, China
  • Online:2010-06-04 Published:2010-06-04
  • Contact: Tan Xiao-hua, Doctor, Chief physician, Master’s supervisor, Biotherapy Center, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China xiaohua_t@hotmail. com
  • About author:Chen Yuan-yuan, Studying for master’s degree, Biotherapy Center, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China; Shanxi Medical University, Taiyuan 030001, Shanxi Province, China yuanyuan840725@126.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30972804*

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Abstract:

BACKGROUND: Previous studies have suggested that interleukin-12 (IL-12) is a powerfull anti-tumor cell factor. Bone marrow mesenchymal stem cells (BMSCs) are prone to introduction and expression of exogenous gene, and have weak immunizing antigen and immunological regulation functions. Therefore, mesenchymal stem cells (MSCs) carrying IL-12 gene possess great prospects for tumor treatment.

OBJECTIVE: To construct an adenoviral (Ad) vector encoding human IL-12 (hIL-12) gene, infect human BMSCs, and detect the expression of IL-12.

METHODS: The total RNA of human dendritic cells (DCs) was obtained. hIL-12 p35 and p40 cDNA were amplified from the total RNA by RT-PCR, and p35 and p40 fragments were linked by internal ribosomal entry sites (IRES), to construct the shuttle vector pDC515 p35/IRES/p40. Using Ad MaxTM adenovirus vector system, pDC515 p35/IRES/p40 and pBHGfrt△E1, 3FLP were cotransfected into 293 cells, and Ad hIL-12 was generated by FLP recombinase-mediated site-specific recombination. After Ad hIL-12 infection, hBMSCs were irradiated with γ-ray to lose proliferative activity. IL-12 levels were determined in cell supernatants utilizing hIL12p70 enzyme linked immunosorbent assay.

RESULTS AND CONCLUSION: The sequences of p35 and p40 fragments were identical with those provided by GenBank NM_000882 (762 bp) and NM_002187 (987 bp), respectively. Ad MaxTM was an efficiently and quickly packaging system of adenoviral vectors. hIL-12 gene in which the two subunits p35 and p40 were linked by IRES can efficiently express the protein of IL-12p70. The high expression of IL-12 is consecutively found after the infection of human BMSCs with Ad hIL-12, suggesting a potential application to the gene therapy of human BMSCs as a carrier.

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