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目的研究自组装核酸纳米材料携带的mTOR小干扰RNA(self-assembled nucleic acid nanoparticles loaded mTOR small interfering RNA,siRNA-NPs)对大鼠肺动脉平滑肌细胞(rat pulmonary arterial smooth muscle cells,RPASMCs)自噬和增殖的影响。方法设计并合成DNA序列m T-2A、m T-1A3以及5’端标记Cy3的mTOR siRNA,共设计mTOR siRNA序列3条,选其中最佳序列,将上述核酸材料按照操作步骤自组装合成三角板型的核酸纳米材料。实验分4组:核酸纳米材料携带的mTOR siRNA组(siRNA-NPs组)、单纯纳米材料组(NPs组)、单独的siRNA组和空白对照组。各组材料分别转染RPASMCs 24 h,激光共聚焦显微镜观察RPASMCs对核酸纳米材料携带的mTOR siRNA的摄取情况;应用RT-PCR检测各组细胞中mTOR mRNA的表达水平,应用Western blot检测p-mTOR、LC3B蛋白的表达水平;激光共聚焦、透射电镜检测细胞自噬水平;MTT及3H-TdR检测各种材料处理后对细胞的生长的影响。结果激光共聚焦显微镜下可见siRNA-NPs组细胞质内均匀分布大量呈颗粒状的红色荧光物质,其荧光强度均显著高于单独的siRNA组(P<0.05);而且siRNA-NPs组处理的mTOR mRNA和p-mTOR蛋白表达显著低于单独的siRNA组、NPs组及空白对照组(P<0.05);而LC3B蛋白表达显著高于其他各组(P<0.05),激光共聚焦检测显示siRNA-NPs组标记自噬小体的绿色荧光明显强于单独的siRNA组、NPs组及空白对照组(P<0.05),电镜结果显示siRNA-NPs能明显诱导细胞自噬小体形成;MTT及3H-TdR检测siRNA-NPs和单独siRNA处理RPASMCs后明显抑制其生长,且siRNA-NPs组细胞生长抑制率又显著高于单独的siRNA组(P<0.01)。结论 siRNA-NPs能被RPASMCs有效摄取并明显抑制靶基因的表达,进而诱导细胞自噬并且抑制其细胞增殖。
Objective To investigate the effects of self-assembled nucleic acid nanoparticles loaded with mTOR small interfering RNA (siRNA-NPs) on autophagy and proliferation of rat pulmonary arterial smooth muscle cells (RPASMCs) Impact. Methods Three mTOR siRNA sequences were designed and synthesized by mT-2A, m T-1A3 and 5 ’Cy3 mTOR siRNAs. The optimal sequences were designed and the above-mentioned nucleic acid materials were self-assembled and synthesized according to the procedure. Type of nucleic acid nanomaterials. The experiment was divided into 4 groups: mTOR siRNA group (siRNA-NPs group), simple nano-material group (NPs group), siRNA group alone and blank control group carried by nucleic acid nanomaterials. The RPMIMCs were transfected with RPASMCs for 24 h respectively. The uptake of mTOR siRNA by nucleic acid nanomaterials was detected by laser confocal microscopy. The expression of mTOR mRNA in each group was detected by RT-PCR. The expression of p-mTOR , LC3B protein expression levels were detected by laser scanning confocal microscope and transmission electron microscope. MTT and 3H-TdR were used to detect the effect of various materials on cell growth. Results Laser confocal microscopy showed that a large amount of granular red fluorescent substance was uniformly distributed in the cytoplasm of siRNA-NPs group, and its fluorescence intensity was significantly higher than that of the siRNA alone group (P <0.05). Furthermore, mTOR mRNA (P <0.05), while the expression of LC3B protein was significantly higher than that of other groups (P <0.05). The results of confocal laser scanning showed that siRNA-NPs NPs group and blank control group (P <0.05). The results of electron microscopy showed that siRNA-NPs could significantly induce the formation of autophagic bodies. The MTT and 3H-TdR The siRNA-NPs and RPMIMCs alone treatment significantly inhibited the growth of the cells, and the cell growth inhibition rate of siRNA-NPs group was significantly higher than that of siRNA alone group (P <0.01). Conclusion siRNA-NPs can be effectively uptake by RPASMCs and significantly inhibit the expression of the target gene, thereby inducing autophagy and inhibiting cell proliferation.