重组NIS基因腺病毒的制备及在心肌细胞中的特异表达

目的 构建肌凝蛋白轻链-2(MLC-2v)启动子调控的NIS腺病毒载体,探讨外源基因在心肌细胞中的特异性表达和NIS作为报告基因的可行性.方法 将MLC-2v启动子和NIS基因亚克隆至腺病毒穿梭载体,再与含有骨架质粒(pAdEasy-1)的大肠杆菌(BJ5183)同源重组,经人胚肾细胞(HEK293)包装并扩增得到重组腺病毒Ad-MLC-NIS,同时构建巨细胞病毒(CMV)启动子调控的腺病毒Ad-CMV-NIS、仅含有启动子的腺病毒Ad-MLC和仅含有NIS基因片段的腺病毒Ad-NIS作为对照组.腺病毒体外感染心肌细胞和非心肌细胞,通过Western blot检测NIS蛋白的表达,行动态摄碘及NaClO4摄碘抑制实验观察NIS蛋白的功能和特性.通过台盼蓝染色实验检测腺病毒感染及NIS摄碘情况对心肌细胞活力和增殖的影响.结果 成功构建重组NIS腺病毒.Western blot检测示感染Ad-CMV-NIS的心肌细胞和非心肌细胞均呈NIS蛋白高表达；感染Ad-MLC-NIS的非心肌细胞出现微量蛋白表达,而心肌细胞中NIS蛋白高表达.动态摄碘实验示感染Ad-MLC-NIS的H9C2细胞、A549细胞、U87细胞摄碘峰值分别为5844.0、833.6、846.0放射性计数·min-1.H9C2细胞的摄碘被NaClO4抑制.感染复数(MOI)为100的腺病毒感染心肌细胞,感染和摄碘实验对心肌细胞活力和增殖的影响较小.结论 MLC-2v启动子调控的腺病毒载体可使外源基因NIS在心肌细胞内特异性表达,表达的NIS蛋白具有摄碘功能,为NIS在心肌中作为报告基因的研究奠定了基础.

Preparation of a recombinant adenoviral encoding human NIS gene and its specific expression in cardiomyocytes

Objective To construct a recombinant adenovirus vector containing the human NIS gene with the myosin light chain-2（MLC-2v） gene as the promoter and evaluate its specific expression and feasibility as a reporter gene in cardiomyocytes. Methods MLC-2v promoter and NIS were subcloned into an adenovirus shuttle vector, and forwarded by homologous recombination in the bacteria BJ5183 containing AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the HEK293 cells to obtain recombinant adenovirus Ad-MLC-NIS. Ad-cytomegaoviyns （CMV）-NIS with cyto- megalovirus as the promoter, Ad-MLC without NIS and Ad-NIS without promoter were constructed as the controls. Cardiomyocytes and non-cardiomyoeytes were then infected by the adenovirus. The protein expres- sion was tested by Western blot analysis. The function and features of NIS protein were evaluated by dynam- ic iodide uptake and NaCIO4 iodine uptake inhibition test in vitro. The viability and proliferation of cardio- myocytes after adenovirus transfection and radioiodine incubation were checked by trypan blue staining. Re- suits Recombinant NIS adenovirus was successfully constructed. Western blot analysis showed that the NIS protein was highly expressed in cardiomyocytes transfected with Ad-MLC-NIS, and all cells transfected with Ad-CMV-NIS. However, in non-cardiomyocytes transfected with Ad-MLC-NIS, little NIS protein was detec- ted. Dynamic iodine uptake tests showed that the peaks of iodide uptake of the three different cell lines （H9C2, A549, U87 cell） transfected with Ad-MLC-NIS were 5844.0, 833.6 and 846.0 counts . min-1respectively. The iodide uptake function of H9C2 was inhibited by NaC104. There was almost no change in cell viability and proliferation when the MOI was 100. Conclusions Ad-MLC-NIS allows myocardial spe- cific expression of an external gene, and the cardiomyocytes with NIS expression are capable of iodine up- take. Further research of NIS as a reporter gene in myocardium is needed.