›› 2011, Vol. 31 ›› Issue (5): 538-.doi: 10.3969/j.issn.1674-8115.2011.05.003

• Original article (Basic research) • Previous Articles     Next Articles

Effects of human amelogenin gene transduction on proliferation and synthesis of alkaline phosphatase of human bone marrow stromal cells

HU Jing-chao, SHU Rong, SONG Zhong-chen, CHENG Lan   

  1. Department of Periodontology, the Ninth People's Hospital, College of Stomatology, |Shanghai Jiaotong University School of Medicine, Shanghai Research Institute of Stomatology, Shanghai 200011, China
  • Online:2011-05-28 Published:2011-05-27
  • Supported by:

    National Natural Science Foundation of China, 30672315, 30801292

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Abstract:

Objective To investigate the effects of lentivirus-mediated human amelogenin (hAm) gene transduction on proliferation and synthesis of alkaline phosphatase (ALP) of human bone marrow stromal cells (hBMSCs). Methods RT-PCR was adopted to obtain hAm encoding gene, and recombinant lentivirus vector plasmid FUAmW was constructed by connection of amplified products with lentivirus vector plasmid (FUGW) carrying green fluorescent protein (GFP). Recombinant lentivirus was prepared from 293T cells by polyethylenimine(PEI)-mediated transient cotransfection, and hBMSCs were infected with generated lentivirus (target gene transduction group). hBMSCs with FUGW infection and those without virus infection were served as control gene transduction group and blank control group, respectively. The infection efficiency of lentivirus was analysed by flow cytometry, and the expression of hAm in cells was detected by RTPCR. Cell proliferation was determined by MTT colorimetric assay. ALP staining was observed with inverted phase contrast microscopy 7 d after lentivirus infection. The expression of ALP mRNA in hBMSCs was detected by RT-PCR 4 d, 7 d and 10 d after infection. Results In target gene transduction group, the infection efficiency of FUAmW was 40.29%, 540 bp strap of target gene was detected by RT-PCR, and cell proliferation was significantly higher than that in control gene transduction group and blank control group (P<0.05). The number of cells with positive ALP staining in target gene transduction group was significantly smaller than those in control gene transduction group and blank control group 7 d after lentivirus infection, and the expression of ALP mRNA in target gene transduction group was significantly lower than that in control gene transduction group and blank control group 4 d, 7 d and 10 d after infection. Conclusion Lentivirusmediated transduction with Am gene may enhance the proliferation of hBMSCs, while reduce the expression of ALP mRNA in cells.

Key words: bone marrow stromal cell, recombinant lentivirus vector, amelogenin, gene transduction