重组HIF—1α及NIS慢病毒表达载体的构建及功能鉴定

目的构建肌凝蛋白轻链-2（MLC-2v）启动下HIF-1α及人NIS慢病毒真核表达载体，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。方法应用基因重组技术构建慢病毒（Lv）-延长因子（EF）1-HIF-1α-内部核糖体进入位点（IRES）-NIS及Lv-MLC-HIF-1α-IRES-NIS慢病毒表达载体。对重组质粒进行酶切、测序鉴定后，采用脂质体转染法将重组质粒转入Hela细胞。细胞免疫荧光及Westernb1a分别验证HIF-1α及NIS蛋白在细胞中的定位及定量表达；制备重组病毒，分别以不同的感染复数（MOI）感染H9C2大鼠心肌细胞、NIH-3T3小鼠胚胎成纤维细胞和L6大鼠骨骼肌成肌细胞，Westernb1a确定最佳MOI并验证MLC·2v启动子的特异性；分别行H9C2细胞动态摄碘和NaClO4抑制实验。采用两样本t检验分析数据。结果成功构建Lv-EFl-HIF-1a-IRES-NIS及Lv-MLC-HIF-1α-IRES-NIS慢病毒表达载体。2种质粒分别转染Hela细胞，免疫荧光显示HIF-1α蛋白表达于胞质，NIS蛋白表达于胞膜；Westernb1a显示EFl启动下2种蛋白质的表达量多于MLC-2v启动下相应表达。Westernb1a蛋白检测后测定蛋白灰度值，阳性对照组NIS及HIF-1α灰度值分别为69-8和71-9，EF1启动下分别为109-4和92-7，MLC-2v启动下分别为141-9和132-4。Lv-MLC-HIF-1α-IRES-NIS病毒感染H9C2细胞，MOI为20时NIS蛋白表达最高，为最佳MOI。2种病毒以MOI为20分别感染H9C2、NIH-3T3及L6细胞，Westernb1a显示EF1启动下NIS蛋白在3种细胞中均有表达，灰度值分别为23.4、29.8和28.6，相差较小。MLC-2v启动下仅H9C2细胞中有大量NIS蛋白表达，NIS蛋白在H9C2、L6及NIH-3T3细胞的灰度值分别为157.9、178.8和2173。H9C2细胞的摄碘高峰时间为40min，峰值为4287.2计数·min-1，是阴性对照组（254.g计数·min-1）的16.85倍（t=5.34，P〈0.01）。摄碘可被NaC国O4抑制，抑制率达85.5％（t=21.3，P〈0.01）。结论MLC-2v可启动治疗基因HIF-1α及报告基因NIS在心肌细胞中特异性表达，表达的NIS蛋白具有摄碘功能，为NIS作为报告基因监测外源基因的靶向治疗奠定了基础。

Construction of recombinant HIF-la and NIS lentiviral expression plasmid and its functional identi fication

Objective To construct a recombinant lentivirus vector containing the human NIS gene and HIF-la with the myosin light chain-2v（MLC-2v） as a promoter and to investigate the specific expres sion and feasibility of NIS as a reporter gene in cardiomyocytes. Methods The target gene HIF-la and NIS were subcloned into the lentivirus （Lv）-elongation factor （EF） 1-HIF-la-internal ribosome entry site （IRES）-NIS and Lv-MLC-HIF-lcMRES-NIS lentivirus vectors. The recombinated vectors were transfected into Hela ceils by lipofectamine 2000. The expression of HIF-la and NIS in the transfected Hela cells was detected by indirect immunofluorescence and Western blot. The H9C2 cells were exposed to different multi- plicities of infection （MOI; 5, 10, 20, 40） with packaged virus particles. The infection efficiency was de-tected by Western blot. MOI 20 was used for H9C2, NIH-3T3 and L6 cell lines and the specificity of the MLC-2v promoter was detected by the count of NIS protein in the 3 different cell lines with Western blot. The function and features of NIS protein were evaluated by dynamic iodine uptake and NaC104 iodine uptake inhibition tests in vitro. Two-sample t test was used to analyze the data. Results The two recombinant lenti- virus vectors were constructed successfully. The HIF-Iot protein was expressed in the cytoplasm and the NIS protein was expressed on the cell membrane in Hela cells. The grey levels of NIS and HIF-1a proteins in the positive control were 69.8 and 71.9, respectively, which were 109.4 and 92.7 after being prompted by EF1, and 141.9 and 132.4 by MLC-2v. The expression of these proteins was much higher by EF1 promoter than that by MLC-2v promoter. The optimal MOI for the Lv-MLC-HIF-la-IRES-NIS virus to infect H9C2 ceils was 20. With the MOI of 20, the grey levels of NIS protein promoted by EF1 were 23.4, 29.8 and 28.6 for H9C2, NIH-3T3 and L6 cells infected with Lv-EF1-HIF-loMRES-NIS virus, respectively. The expression of NIS protein promoted by MLC-2v was much higher in H9C2 cells than the other two cell lines. The grey level of NIS protein was 157.9 in H9C2 cells, 178.8 in L6 cells and 217.3 in NIH-3T3 ceils. The NIS protein ex- pressed in infected H9C2 ceils showed high radioiodine uptake. The peak of iodine uptake was 4 287.2 counts min-1 at 40 min which was 16.85 times of the control group （254.4 counts min-1） （t=5.34, P〈 0.01）. The inhibition rate of iodine uptake was up to 85.5% （3 666.4/4 287.2, t=21.3, P〈0.01） by NaC104. Conclusions MLC-2v promoter allows specific expression of the external gene HIF-la and NIS in myocar dium. The cardiomyocytes transfected with NIS gene acquires the function of iodine uptake. Therefore, NIS may have a potential to be the reporter gene to monitor the external gene therapy in ischemic cardiomyopathy.