国际麻醉学与复苏杂志
國際痳醉學與複囌雜誌
국제마취학여복소잡지
INTERNATIONAL JOURNAL OF ANESTHESIOLOGY AND RESUSCITATION
2011年
5期
554-557
,共4页
于英妮%陆建华%施冲%曾因明
于英妮%陸建華%施遲%曾因明
우영니%륙건화%시충%증인명
NMDA受体1%水溶性凝脂聚合物%小干涉RNA
NMDA受體1%水溶性凝脂聚閤物%小榦涉RNA
NMDA수체1%수용성응지취합물%소간섭RNA
NMDA receptor 1%Water soluble lipopolymer%siRNA
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NMDAR1 siRNA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(0.640.13、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.
目的 探討離體條件下水溶性納米凝脂聚閤物(water-soluble lipopolymer,WSLP)運載小榦涉RNA(small interference RNA,siRNA)沉默N-甲基-天鼕氨痠受體1(N-methyl-D-aspartate receptor 1,NMDAR1)基因的可行性,為在體條件下研究WSLP運載siRNA沉默NMDAR1治療慢性疼痛等疾病打下基礎.方法 先閤成WSLP併與NMDAR1 siRNA連接成WSLP/siRNA複閤物,觀察其在血清中的穩定性及其對PC12細胞的毒性；然後將PC12細胞通過隨機數字錶法分為陰性轉染組(單純siRNA轉染PC12細胞)、對照轉染組(WSLP/亂序siRNA複閤物轉染PC12細胞)及WSLP轉染組(WSLP/siRNA轉染PC12細胞),通過實時-聚閤酶鏈反應(real time polymerase chain reaction,RT-PCR)和免疫蛋白印跡法(western-blot)檢測各組NMDAR1轉錄水平及蛋白水平基因錶達的變化.結果 WSLP/NMDAR1/siRNA在血清中的穩定性高,對PC12細胞幾乎無毒性.與陰性轉染組(0.69±0.18、4.36±1.02)相比,WSLP轉染組(0.35±0.21、1.96±0.48)轉錄水平NMDAR1的基因錶達降低50％,蛋白錶達水平降低55％,差異均有統計學意義(P＜0.01),陰性轉染組和對照轉染組(0.640.13、4.32±1.09)之間的基因錶達水平差異無統計學意義.結論 離體條件下WSLP可有效運載siRNA沉默NMDAR1基因的錶達.
목적 탐토리체조건하수용성납미응지취합물(water-soluble lipopolymer,WSLP)운재소간섭RNA(small interference RNA,siRNA)침묵N-갑기-천동안산수체1(N-methyl-D-aspartate receptor 1,NMDAR1)기인적가행성,위재체조건하연구WSLP운재siRNA침묵NMDAR1치료만성동통등질병타하기출.방법 선합성WSLP병여NMDAR1 siRNA련접성WSLP/siRNA복합물,관찰기재혈청중적은정성급기대PC12세포적독성；연후장PC12세포통과수궤수자표법분위음성전염조(단순siRNA전염PC12세포)、대조전염조(WSLP/란서siRNA복합물전염PC12세포)급WSLP전염조(WSLP/siRNA전염PC12세포),통과실시-취합매련반응(real time polymerase chain reaction,RT-PCR)화면역단백인적법(western-blot)검측각조NMDAR1전록수평급단백수평기인표체적변화.결과 WSLP/NMDAR1/siRNA재혈청중적은정성고,대PC12세포궤호무독성.여음성전염조(0.69±0.18、4.36±1.02)상비,WSLP전염조(0.35±0.21、1.96±0.48)전록수평NMDAR1적기인표체강저50％,단백표체수평강저55％,차이균유통계학의의(P＜0.01),음성전염조화대조전염조(0.640.13、4.32±1.09)지간적기인표체수평차이무통계학의의.결론 리체조건하WSLP가유효운재siRNA침묵NMDAR1기인적표체.
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.NMDAR1 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 NR 1 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.
