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Published on: October 16, 2014
[Construction and expression of recombinant plasmid pcDNA3.1(+) -hHGF available in osteoblast]
Hong-sheng Dang1, Wei Li, Bin Shen
1Department of Orthopaedics West China Hospital, Sichuan University, Chengdu 610041, China.
Objective:
To construct a plasmid carrying human hepatocyte growth factor (hHGF) gene and determine the effects of the hepatocyte growth factor (HGF) gene on proliferation and differentiation of osteoblast in vitro.
Methods:
The full length cDNA of hHGF, which was amplified from human liver mRNA by RT-PCR, was cloned into pcDNA3.1(+) vector to construct pcDNA3.1(+) -hHGF recombinant plasmid. With lipofectamine, the recombinant plasmid pcDNA3.1(+) -hHGF was used to transfect the culture osteoblasts. After pcDNA3.1(+) - hHGF transfection, the positive cell clones were selected with G418. The stable transfection and expression of hHGF in the osteoblasts were measured by immunohistochemical staining and RT-PCR respectively. The effect of pcDNA3.1(+) -hHGF transfection on osteoblast proliferation was measured by MTT colorimetric assay. Flow cytometer was used to determine the effect of pcDNA3.1(+) -hHGF transfection on cell cycle of osteoblast. The quantitive detection of hHGF expression was performed through ELISA. Alkaline phosphatase (AP) was also detected using enzyme kinetics.
Results:
The recombinant plasmid pcDNA3. 1(+) -hHGF was identified by restriction endonuclease digestion and nucleotide sequencing. Osteoblasts could be effectively transfected by pcDNA3.1(+) -hHGF with lipofectamine in vitro. The stable expression of hHGF in pcDNA3.1(+) -hHGF transfection osteoblast was confirmed. Human HGF protein was detected by immunohistochemical staining and RT-PCR in osteoblasts 4 weeks after cell clone selected with G418. pcDNA3.1(+) -hHGF gene transfer responsible to improve the proliferation was proved by MTT assay, flow cytometer showed cellular proportion in "S" phase obviously increased. AP activity in transfected cells was increased significantly.
Conclusions:
Osteoblasts which express stable and high-level hHGF was established successfully, exogenous hHGF gene could stimulate the proliferation, differentiation and function of osteoblast and could be applied further to gene therapy.
