摘要
目的构建人肝细胞生长因子(hHGF)真核表达质粒,探讨重组质粒对体外培养胎兔成骨细胞增殖分化的影响,为转基因治疗骨科疾病提供实验基础。方法RT-PCR扩增人肝脏中hHGF全长cDNA片段,克隆入pcDNA3.1(+)真核表达载体中,用脂质体法将pcDNA3.1(+)-hHGF质粒转染成骨细胞,通过G418筛选获得阳性克隆,用免疫荧光染色检测hHGF基因在成骨细胞内的表达;MTT法和FCM检测pcDNA3.1(+)-hHGF转染后对细胞增殖和细胞周期的影响;采用RT-PCR和ELISA方法检测hHGF在成骨细胞中的表达。NPP法检测碱性磷酸酶合成情况。结果成功构建hHGF真核表达质粒,免疫组化和RT-PCR检测显示转染成骨细胞后细胞内hHGFmRNA呈现高水平表达;MTT法和FCM检测显示重组质粒转染后能促进成骨细胞的增殖,S期细胞比例增多;ELISA法检测到hHGF在细胞中有分泌性表达。转染重组质粒的成骨细胞合成碱性磷酸酶能力显著提高。结论成骨细胞经基因转染后可以表达hHGF,外源性hHGF基因能够刺激成骨细胞增殖、分化及成骨活性。
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 plasmld. 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.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2007年第6期929-933,共5页
Journal of Sichuan University(Medical Sciences)
