人基质细胞衍生因子1α和hVEGF_(165)基因转染成肌细胞的实验研究

A EXPERIMENTAL STUDY ON TRANSFECTING HUMAN STROMAL CELL-DERIVED FACTOR 1α AND HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR 165 GENES INTO MYOBLASTS IN VIRTO

目的探索转染hVEGF165基因、人基质细胞衍生因子1α(human stromal cell-derived factor 1α,hSDF-1α)基因的大鼠成肌细胞在体外mRNA和蛋白表达,为进一步检测这两种基因对血管生成的协同效应奠定基础。方法取新生1日龄雄性SD大鼠,采用胰蛋白酶消化法体外分离培养、纯化成肌细胞,并采用结蛋白免疫细胞化学染色鉴定。将质粒增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)-hVEGF165和EGFP-hSDF-1α转染第3代成肌细胞(转染组),以未转染质粒的细胞作为对照组。体外培养后各时间点行RT-PCR、Western blot、ELISA法检测两种质粒mRNA及蛋白的表达。结果培养的细胞经鉴定确定为成肌细胞。荧光显微镜观察示成功转染hSDF-1α和hVEGF165进入成肌细胞,可见绿色荧光表达。RT-PCR检测示转染组转染后2、4、6、8 d均可见hVEGF165和hSDF-1αmRNA的表达,Western blot检测示转染组转染后2、4、6、8 d成肌细胞内均有hVEGF165和hSDF-1α蛋白表达,对照组均无相关表达。ELISA检测示对照组hSDF-1α和hVEGF165均稳定表达,呈较低水平。转染组转染2 d时hSDF-1α和hVEGF165表达均达较高水平,之后呈逐渐下降趋势;转染后各时间点间hSDF-1α和hVEGF165表达比较差异均有统计学意义(P<0.05)。转染组转染后各时间点与对照组比较差异均有统计学意义(P<0.05)。结论转染hSDF-1α和hVEGF165进入大鼠成肌细胞后在体外均有表达,为下一步研究体内是否表达提供了实验依据。

Objective To explore the human stromal cell-derived factor 1α（hSDF-1α） and human vascular endothelial growth factor 165（hVEGF165） mRNA expressions of the transfected cells after hSDF-1α gene and hVEGF165 gene were transfected into rat myoblasts in vitro so as to lay a foundation for further study on the synergistic effects of 2 genes on tissue engineered skeletal muscle vascularization.Methods The myoblasts of 1-day-old Sprague Dawley rats were cultured and purified by trypsin digestion assay in vitro and were identified by immunohistochemistry staining of Desmin.Approximately 70%-80% of confluent myoblasts were transfected with enhanced green fluorescent protein（EGFP）-hSDF-1α and EGFP-hVEGF165 genes in vitro（transfected group） and were not transfected（control group）.The expressions of hSDF-1α and hVEGF165 mRNA and protein in the transfected cells were detected by RT-PCR,ELISA,and Western blot respectively.Results The cultured cells were identified as myoblasts by immunohistochemistry staining of Desmin.The expression of green fluorescent protein was observed in transfected cells,indicating that hSDF-1α and hVEGF165 genes were transfected into myoblasts successfully.The mRNA and protein expressions of the 2 genes were positive in the transfected group by RT-PCR and Western bolt assay at 2,4,6,and 8 days after transfection,and were negative in the control group.The expressions of hSDF-1α and hVEGF165 showed a stable low level in the control group,but the expressions of the proteins increased at 2 days and then showed gradual downtrend with time in the transfected group by ELISA assay.There were significant differences in the expressions of hSDF-1α and hVEGF165 proteins between different time points in the transfected group,and between 2 groups（P〈 0.05）.Conclusion hSDF-1α and hVEGF165 genes are successfully transfected into myoblasts in vitro,and mRNA and proteins of hSDF-1α and hVEGF165 can be expressed in the transfected myoblasts,which may provide the experimental evidence for the expressions of hSDF-1α and hVEGF165 mRNA and proteins in vivo successfully.