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目的 为提高体外转基因 (exvivo)治疗帕金森病大鼠模型的疗效 ,本实验拟比较两种基因表达载体介导的外源基因在两种不同运载细胞的瞬时表达效率。 方法 分别将酪氨酸羟化酶 (TH)基因的重组表达质粒和重组逆转录病毒导入原代培养的大鼠成纤维细胞和脑胶质细胞 ,用免疫组织化学检测TH的表达效率 ;Westernblot法检测样品中TH特异性条带并转换成A值 ,以比较其在不同细胞中表达的相对含量。 结果 免疫组织化学检测表明 ,表达质粒的转染率为 5 %～ 7% ,逆转率病毒的感染率为 18%～ 2 0 % ;根据GDNF标准品蛋白条带含量与Westernblot结果的相应A值的标准曲线 ,推断出质粒转染和逆转率病毒感染的成纤维细胞中单个阳性细胞表达的TH相对平均含量分别为 4 76× 10 - 2 A和 4 35× 10 - 2 A ,质粒转染和逆转率病毒感染的脑胶质细胞分别是 5 4 5×10 - 2 A和 5 0 5× 10 - 2 A。 结论 不论是质粒载体还是逆转录病毒载体所携带的外源基因在两种细胞的单个阳性细胞中的表达无显著性差异。但由于逆转录病毒载体的感染率高于质粒载体 ,对于原代培养的成纤维细胞和脑胶质细胞 ,逆转率病毒载体是一种更好的表达载体。
Objective To improve the therapeutic effect of exvivo in the rat model of Parkinson’s disease in vitro, this experiment compared the transient expression efficiency of two kinds of gene expression vectors in two different carrier cells. Methods Recombinant plasmids containing tyrosine hydroxylase (TH) gene and recombinant retrovirus were respectively introduced into primary cultured rat fibroblasts and glial cells. The expression of TH was detected by immunohistochemistry. Western blotting The TH-specific bands in the samples were detected and converted to A values to compare their relative amounts expressed in different cells. Results The results of immunohistochemistry showed that the transfection rate of expression plasmids was between 5% and 7%, and the infection rate of retrovirus was between 18% and 20%. According to the corresponding A value of protein band of GDNF standard and Western blot results Standard curve, the relative average levels of TH expression in single positive cells in plasmid-transfected and retroviral-infected fibroblasts were 4 76 × 10 -2 A and 4 35 × 10 -2 A, respectively. The plasmid was transfected and reversed Rate virus-infected glial cells were 545 x 10-2 A and 5 05 x 10-2 A, respectively. Conclusion There is no significant difference in the expression of exogenous genes carried by either plasmid vector or retroviral vector in single positive cells of both cells. However, retroviral vector is a better expression vector for primary cultured fibroblasts and glial cells due to the higher infection rate of retroviral vector than plasmid vector.