大鼠RNAi-A2aR慢病毒载体的构建及鉴定

Construction and identification of RNAi-A2aR lentiviral vector in rats

目的构建大鼠RNAi-腺苷A2a受体(A2aR)慢病毒载体并进行鉴定。方法首先构建3对短发夹RNA(shRNA)-A2aR序列(shRNA-A2aR 1、shRNA-A2aR 2、shRNA-A2aR 3),在shRNA慢病毒载体中分别插入3对双链shRNA oligo,shRNA慢病毒重组质粒构建成功后,将重组质粒、包装载体、穿梭载体共转染293T细胞,从而获得病毒液。实验Ⅰ采用随机数字表法将大鼠原代心肌细胞分为3组(n=6):空载组(V组)、shRNA-A2aR 1组和shRNA-A2aR 3组,各组分别转染MOI为10的相应病毒液,转染48 h时,Western blot法检测A2aR表达水平,确定干扰效率,以筛选最佳慢病毒载体。实验Ⅱ采用随机数字表法将大鼠原代心肌细胞分为5组(n=36):空载组(V组)、MOI5组、MOI10组、MOI15组和MOI20组,各组分别转染相应MOI的病毒液(最佳慢病毒载体),于转染24、48和72 h时,采用CCK-8法测定细胞存活率,荧光显微镜下观察细胞活力和细胞死亡情况,Western blot法检测A2aR表达水平,确定干扰效率。结果实验Ⅰ成功构建了2种shRNA-A2aR慢病毒载体(shRNA-A2aR 1、3)。shRNA-A2aR 3病毒液滴度为3.5×10^8 TU/ml。与V组和shRNA-A2aR 1组比较,shRNA-A2aR 3组心肌细胞A2aR表达下调(P<0.01),shRNA-A2aR 3慢病毒载体干扰效率73%。实验Ⅱ选择shRNA-A2aR 3慢病毒载体转染24 h时,各组细胞存活率均>85%;转染48 h时,MOI5组和MOI10组细胞存活率>80%;转染72 h各组细胞存活率<70%。倒置荧光显微镜下可见,MOI5组荧光密度稍低,MOI10组转染48 h及MOI20组转染24 h时,荧光密度较高且细胞状态较好;转染72 h时各组心肌细胞活力明显下降、死亡细胞增加。Western blot显示:MOI10组转染48 h、MOI15组转染48 h、MOI20组转染24和48 h时干扰效率均>70%。结论成功构建了大鼠shRNA-A2aR慢病毒载体,且MOI为10、转染48 h或MOI为20、转染24 h为最佳转染方案。

Objective To construct and identify the lentiviral vector of adenosine RNAi-adenosine A2a receptor(A2aR)in rats.Methods Three pairs of short hairpin RNA(shRNA)-A2aR sequences(shRNA-A2aR 1,shRNA-A2aR 2,shRNA-A2aR 3)were designed,and three pairs of double-stranded shRNA oligos were respectively inserted into the shRNA virus vector to gain three kinds of shRNA lentiviral recombinant plasmid.The recombinant plasmid,packaging vector,and shuttle vector were co-transfected into 293T cells to obtain virus liquid.The experiment was performed in two parts.PartⅠThe rat primary cardiomyocytes were divided into 3 groups(n=6 each)by a random number table method:vehicle group(V group),shRNA-A2aR 1 group and shRNA-A2aR 3 group.Each group was transfected with virus solution of MOI 10 for 48 h.The expression of A2aR was detected by Western blot to select the most efficient lentivirus vector.PartⅡThe cardiomyocytes were randomly divided into 6 groups(n=36 each):vehicle group(V group),MOI5 group,MOI10 group,MOI15 group and MOI20 group.Each group was transfected with the corresponding MOI virus liquid(the most effective lentivirus vector).At 24,48,and 72 h of transfection,the cell viability and cell death were observed with a fluorescent microscope,and the A2aR expression was detected by Western blot to determine the interference efficiency.Results PartⅠTwo types of shRNA-A2aR lentiviral vectors(shRNA-A2aR 1,3)were successfully constructed,among which shRNA-A2aR 3 virus solution with a titer of 3.5×10^8 TU/ml had the best effect.Compared with group V and group shRNA-A2aR 1,the expression of A2aR in cardiomyocytes was significantly down-regulated(P<0.01),and the interference efficiency of shRNA-A2aR 3 was 73%in shRNA-A2aR 3 group.PartⅡshRNA-A2aR 3 was selected to screen out the transfection plan.The cell survival rate in each group was more than 85%at 24 h of transfection,the cell survival rate was more than 80%at 48 h of transfection in MOI5 and MOI10 groups;the cell survival rate in each group was less than 70%at 72 h of transfection.Under an inverted fluorescent microscope,a slightly lower fluorescence density was found in MOI5 group,the fluorescent density was higher and the cell condition was better at 48 h of transfection in MOI10 group and at 24 h of transfection in MOI20 group,and the cardiomyocyte viability was significantly decreased,and dead cells were increased at 72 h of transfection in each group.The results of Western blot showed that the interference efficiency at 48 h of transfection in MOI10 group,48 h in MOI15 group,24 and 48 h in MOI20 group was all>70%.Conclusion MOI of 10,transfection for 48 h or MOI of 20,transfection for 24 h is the optimal transfection protocol.