水溶性纳米凝脂聚合物运载siRNA沉默PC12细胞N-甲基-D-天冬氨酸受体1基因的实验研究

Water soluble lipopolymer can deliver siRNA targeting N-methyl-D-aspartate receptor 1 in vitro efficiently

目的探讨离体条件下水溶性纳米凝脂聚合物（water-soluble lipopolymer，WSLP）运载小干涉RNA（small interference RNA，siRNA）沉默N-甲基-天冬氨酸受体1（N-methyl—D-aspartate receptor 1，NMDAR1）基因的可行性，为在体条件下研究WSLP运载siRNA沉默NMDAR1治疗慢性疼痛等疾病打下基础。方法先合成WSLP并与NMDAR1siRNA连接成WSLP／siRNA复合物，观察其在血清中的稳定性及其对PC12细胞的毒性；然后将PC12细胞通过随机数字表法分为阴性转染组（单纯siRNA转染PC12细胞）、对照转染组（WSLP/乱序siRNA复合物转染PC12细胞）及WSLP转染组（WSLP／siRNA转染PC12细胞），通过实时-聚合酶链反应（real time polymerase chain reaction，RT-PCR）和免疫蛋白印迹法（western-blot）检测各组NMDAR1转录水平及蛋白水平基因表达的变化。结果WSLP／NMDAR1／siRNA在血清中的稳定性高，对PC12细胞几乎无毒性。与阴性转染组（0．69±0．18、4．36±1．02）相比，WSLP转染组（0．35±0．21、1．96±0．48）转录水平NMDAR1的基因表达降低50％，蛋白表达水平降低55％，差异均有统计学意义（P〈0．01），阴性转染组和对照转染组（0．64±0．13、4．32±1．09）之间的基因表达水平差异无统计学意义。结论离体条件下WSLP可有效运载siRNA沉默NMDAR1基因的表达。

Objective To examine the potential application of a non-viral genecarrier, water soluble lipopolymer （WSLP） for delivering siRNA targeting N-methyl-D-aspartate receptor 1 （NMDAR1） in vitro. Methods WSLP was complexed with siRNA designed to inhibit NR1 expression. Following serum stability and cytotoxicity observation, WSLP/siRNA （scrambled siRNA as a control ） complexes were transfected in PC12 cells and then siRNA delivery efficiency of the complexes was evaluated by gene expression level assay using reverse transcriptive polymerase chain reaction （RT-PCR） and western-blot technique. Results WSLP protected siRNAs from enzymatic degradation in serum conditioned media, and the complexes of WSLP/siRNA had little cytotoxicity to cultured PC12 cell. NMDARI expression of PC12 cell was efficiently inhibited by WSLP/siRNA complexes, while complexes of WSLP with scrambled siRNA （0.64±0.13,4.32 ±1.09） did not show this inhibitory effect compared to unmodified siRNA （0.69±0.18,4.36±1.02） neither by transcriptional level nor protein level. WSLP/siRNA complexes reduced NR1 transcriptional level by 50% （0.35±0.21）and protein level by 55% （1.96±0.48） when compared to unmodified siRNA. Conclusion Our data suggest that Water soluble lipopolymer can deliver siRNA targeting NMDA receptor 1 in vitro efficiently and safely.