慢病毒介导的RNA干扰对大鼠神经元PKCγ mRNA及蛋白表达的影响

目的 探讨慢病毒介导的RNA干扰(RNAi)对大鼠神经元PKCymRNA及蛋白表达的影响.方法 孕16 d的SD大鼠,原代培养大鼠胚胎皮层神经元,随机分为3组,每组6孔,对照组(C组):不行任何处理;阴性对照组(NC组):每孔加入阴性对照慢病毒3×105个;RNAi组:每孔中加入编码PKCγ shRNA的慢病毒载体(LV-PKCγ shRNA)颗粒3×105个.5 d后采用RT-PCR和Western blot法检测PKCγ mRNA和蛋白的表达水平,并计算干扰效率.结果 与C组比较,RNAi组大鼠神经元PKCγ mRNA及其蛋白表达水平均降低(P<0.05),NC组上述指标差异无统计学意义(P>0.05).基因干扰效率99.3%,蛋白干扰效率85.2%.结论 慢病毒介导的RNAi可有效地下调大鼠原代神经元PKCγ基因及其蛋白的表达.

Effect of lentivirus-mediated RNA interference on expression of

Objective To investigate the effect of lentivirus-mediated RNA interference(RNAi) on the expression of PKCγ mRNA and protein in rat neurons.Methods Primary cultured cortical neurons from SD rat (16 days of pregnency) embryos were randomly divided into 3 groups with 6 wells in each group:control group (group C),negative group(group NC)and RNAi group.Group C received no treatment.Each well in group NC was given negative lentivirus 3 × 105.Each well in group RNAi was given the recombinant lentiviral vector containing PKCγ shRNA(LV-PKC7 shRNA).The expression of PKCT mRNA and protein in rat neurons was detected by RT-PCR and Western blot 5 days later.The interference efficiency was ealculated.Results Compared with group NC,the expression of PKCγ mRNA and protein was down-regulatedin group RNAi(P<0.05),but no significant change Was found in group NC(P>0.05).The interference efficiency of gene and protein were 99.3%and 85.2%respectively.Conclusion Lentivirus-mediated RNAi can down restate the expression of PKC7 gene and protein in rat neurons.