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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 of TH after tyrosine hydroxylase (TH) gene modified mesenchymal stem cells (MSCs) and the effect of TH gene transfection on MSCs. Methods Plasmid pCMV / hTH containing human TH gene was constructed and transfected into primary cultured rat MSCs by lipofectamine. The expression of TH gene and the differentiation of MSCs were identified by Western blot and immunocytochemistry. The post-staining MSCs activity was compared with that of untransfected MSCs. Results The constructed pCMV / hTH plasmid was digested by ECoRI to produce fragments of 1.9 kb and 5.3 kb, which was consistent with the size of the target gene and vector fragment recovered. Transgenic MSCs were positive for TH staining by Western blot and immunocytochemistry staining. There were no NeuN and GFAP expression in MSCs after staining. The activity of cells was determined by MTT colorimetric assay. There was no significant difference between untransfected and transfected MSCs. Conclusion The constructed TH gene can be expressed well in MSCs cultured in vitro. Transfection does not induce differentiation of MSCs into neuron-like cells and has no significant effect on the activity of MSCs.