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目的 将重组正义hTERT基因荧光真核表达载体导入人胚胎成纤维细胞 (hEF)中 ,探讨外源性hTERT基因转染对hEF细胞胶原含量变化的影响。方法 应用脂质体法将正义重组pIRES2 -EGFP -hTERT质粒及空载pIRES2 -EGFP质粒分别转染至原代培养hEF中 ,westernblot检测转染细胞和未转染细胞中Ⅰ、Ⅲ型胶原表达变化 ;放免法检测转染细胞和未转染细胞培养液上清中Ⅰ、Ⅲ型胶原含量。结果 外源性hTERT基因转染细胞 (hEF -hTERT)较未转染细胞 (hEF)和空载体转染细胞 (hEF -EGFP)中Ⅰ、Ⅲ型胶原表达明显增加 ;hEF -hTERT细胞培养上清液中Ⅰ、Ⅲ型胶原含量也较hEF和hEF -EGFP细胞有显著增加。结论 外源性hTERT基因转染能使原代培养的hEF细胞胶原合成增加 ,细胞增殖能力增强
OBJECTIVE: To introduce recombinant eukaryotic expression vector of hTERT gene into human embryonic fibroblasts (hEF) and investigate the effect of exogenous hTERT gene transfection on the changes of collagen content in hEF cells. Methods The positive recombinant pIRES2-EGFP-hTERT plasmid and empty pIRES2-EGFP plasmid were transfected into primary culture hEF by liposome method. The expression of type Ⅰ and type Ⅲ collagen in transfected and non-transfected cells was detected by western blot The contents of type Ⅰ and type Ⅲ collagen in the supernatants of transfected cells and untransfected cells were detected by radioimmunoassay. Results The expression of type Ⅰ and type Ⅲ collagen in hEF-hTERT transfected cells (hEF-hTERT) and hEF-EGFP transfected cells (hEF-EGFP) was significantly increased; hEF-hTERT cell culture supernatant The content of type I and type III collagen in liquid also increased significantly compared with hEF and hEF-EGFP cells. Conclusion Exogenous hTERT gene transfection can increase the collagen synthesis of primary cultured hEF cells and enhance the cell proliferation ability