双链小片段干扰RNA抑制缺氧条件下乳鼠心肌细胞缺氧诱导因子1α表达

Inhibition of the expression of cardiomyocytic hypoxic induction factor-1α during hypoxic state by double chain siRNA

目的 构建含缺氧诱导因子 1α(HIF 1α)小片段干扰RNA(siRNA)靶序列的U6启动子表达框结构 ,观察其对缺氧条件下乳鼠心肌细胞HIF 1α表达的影响。 方法 分离培养乳鼠心肌细胞 ,分为常规培养液对照组、RNA干扰 (RNAi)组 (转染无效干扰序列Ⅳ )、RNAi抑制组 (转染有效干扰序列并按下游引物不同分为Ⅰ、Ⅱ、Ⅲ组 )。设计、合成 3对 (Ⅰ、Ⅱ、Ⅲ )含HIF 1α编码基因片段(正、反义 )和 1对 (Ⅳ )随机序列 (正、反义 )的PCR下游引物。PCR法构建U6启动子表达框及相应正、反序列表达框 ,同时转染心肌细胞。每组每时相点 5皿细胞。于缺氧 1h后 ,用蛋白免疫印迹法(Western blot)测定其蛋白水平表达 ,免疫组织化学法检验干扰效果。缺氧 6h后采用逆转录 聚合酶链反应 (RT PCR)法检测HIF 1αmRNA的表达。 结果 筛选出的最佳抑制片段为Ⅱ组序列。缺氧 1h,对照组、RNAi组心肌细胞HIF 1α蛋白水平显著增高 ,Ⅰ、Ⅱ、ⅢRNAi抑制组HIF 1α蛋白水平较对照组明显降低 ,其中Ⅱ组降低最为显著 (P <0.0 1);缺氧 6h,RNAi组心肌细胞HIF 1αmRNA水平较常氧条件下明显增高 (P <0.0 1);RNAi抑制Ⅱ组未见明显增高 (P >0.0 5 )。 结论 构建的HIF 1αⅡ组表达框能有效地抑制缺氧乳鼠心肌细胞HIF

Objective To construct hypoxic induction factor-1α(HIF-1α) siRNA expression cassette containing U6 promoter,α HIF-1α sense or antisense target sequence,and to observe its influence on the expression of cardiomyocytic HIF-1α during hypoxic state. Methods Neonatal murine cardiomyocytes cultured in the mixed gas were employed as the hypoxic model and were divided into normal control (cultured in normal oxygen), RNAi control (invalidated transfection interference sequence Ⅳ) and RNAi effective inhibition (effective transfection interference sequence, which was further divided into Ⅰ,Ⅱ and Ⅲ groups according to the difference of downstream primer ) groups. Three pairs (Ⅰ,Ⅱ and Ⅲ) of PCR downstream primer containing HIF-1α encoded gene fragments (sense and antisense) and one pair of randomize sequence (Ⅳ) PCR downstream primer were designed and synthesized. U6 starter expression frame was constructed by PCR method. The cardiomyocytes were transfected simultaneously by sense and antisense sequence expression frame. Five plates of the cells were set at each time points in each group. The expression of HIF-1α mRNA was detected by RT-PCR at 6 hours of hypoxia. The change in the protein expression level at 1 hour of hypoxia was determined by Western blot, and the interference effects were monitored by immunohistochemistry. Results The best inhibition fragment screened was group Ⅱsequence. After the transfection and hypoxic culture, it was found that the cardiomyocytic HIF1α mRNA and protein levels in RNAi effective inhibition group were evidently lower than those in normal control and RNAi control groups (P<0 01). While the protein inhibition rate (60%-80%) between the former group and normal and RNAi control groups was no difference (P>0.05). Conclusion The expression of the HIF1α in hypoxic rat cardiomyocytes could be effectively inhibited by our constructed HIF1α siRNA expression cassette group Ⅱ.