大鼠骨髓间充质干细胞转染人TH基因的实验研究

Research on rat bone mesenchymal stem cells transfected with human tyrosine hydroxylase gene in vitro

目的 评价酪氨酸羟化酶(tyrosine hydroxylase,TH)基因修饰骨髓间充质干细胞(mesenchymalstem cells,MSCs)后TH的表达情况及转染TH基因对MSCs的影响。方法 构建含人TH基因的pCMV/hTH质粒,用脂质体转染原代培养的大鼠MSCs;Western blot及免疫细胞化学染色鉴定TH基因的表达及MSCs的分化情况;MTT比色法测定转染后的MSCs细胞活性,并与未转染的MSCs进行细胞活性比较。结果 构建的pCMV/hTH质粒经ECoRI酶切后产生1.9 kb和5.3 kb的片段,与回收的目的基因及载体基因片段大小相符;转基因后的MSCs Western blot及免疫细胞化学染色显示TH染色阳性;转染后MSCs未见有NeuN,GFAP的表达;MTT比色法测定细胞活性,未转染与转染者差异无显著性。结论 构建的TH基因能在体外培养的鼠MSCs中较好的表达,转染不会诱导MSCs向神经样细胞分化,对MSCs细胞活性无明显影响。

Objective To evaluate the expression efficiency of TH (tyrosine hydroxylase) gene after it being transduced into rat mesenchymal stem cells (rMSCs) and the influence of the transfection. Methods Plasmid containing human TH gene was constructed by using the cytomegalovirus (CMV) promoter to drive its expression, and then transfect the primary cultured rMSCs by Lipofect Amine; We verify the expression and the differentiation using Western blot and immunocytochemical staining, and compare the transfected MSCs activity with the untransfected MSCs by MTT colorimetric metrod. Results The pCMV/hTH can produce 1.9 kb and 5.3 kb fragment after cutting by restriction enzyme ECoRI which was in accordance with anticipated result; Western blot and immunhisto-chemical staining of post-transgeneic rMSC transfeced showed TH positive and NeuN, GFAP Negative; MTT colorimetric metrod showed no significant difference between transfected MSCs and untransfected MSCs. Conclusions Rat mesenchymal stem cells constructed by gene transfection can express human TH gene in vitro. The TH gene-cannot induce MSCs' differentiation and has no significant influence on the cells's activity.