摘要
目的探讨离体条件下细胞穿透肽Tat-LK15运载小干扰RNA(small interference RNA,siRNA)沉默RGC-5视神经节细胞(retinal ganglion cell line,RGC-5)神经元型一氧化氮合酶(neuronal nitric oxide synthase,n NOS)基因的可行性,为在体条件下研究Tat-LK15运载siRNA沉默n NOS表达治疗神经病理性疼痛提供理论依据。方法 1通过凝胶阻滞分析测定Tat-LK15与siRNA的最佳交联比。流式细胞术检测Tat-LK15/FAM-siRNA以最佳交联比转染RGC-5细胞的转染效率;不同剂量Tat-LK15(1、2.5、5、10和20μg)孵育RGC-5细胞24 h,流式细胞术检测细胞凋亡率。2制备n NOS高表达的RGC-5细胞模型。3将RGC-5细胞随机分为5组:对照组、模型组、Tat-S组(Tat-LK15运载n NOS/siRNA转染模型细胞)、Lipo-S组(LipofectamineTMRNAi MAX运载n NOS/siRNA转染模型细胞)及Tat-N组(Tat-LK15运载NCsiRNA转染模型细胞),通过Q-PCR及Western blot检测各组nN OS表达水平。结果 Tat-LK15与siRNA质量比为2∶1时可完全包裹siRNA,达到最佳交联,此时其转染效率为(84.4±3.9)%。当Tat-LK15剂量为20μg(6.1μmol·L-1)时才出现一定细胞毒性,细胞凋亡率高于对照组[(10.3±1.1)%vs(7.4±0.9)%,P<0.05]。造模后RGC-5细胞nN OS表达水平明显升高(P<0.05)。与模型组相比,Tat-S组nN OS mRNA及蛋白表达水平降低(P<0.05),Tat-S组与Lipo-S组相比无差异(P>0.05)。结论Tat-LK15能高效转染siRNA,细胞毒性低,离体条件下可有效运载siRNA沉默nN OS的基因表达。
Aim To investigate the potential application of a non-viral gene carrier Tat-LK15 for delivering siRNA targeting n NOS in vitro,which provides evidence of Tat-LK15 mediating siRNA targeting n NOS in vivo for treatment of neuropathic pain. Methods 1.Tat-LK15 was mixed with siRNA,then the mixture was analyzed the best ratio by Gel retardation. The transfection efficiency of FAM-siRNA mediated by TatLK15 on RGC-5 cells was examined by Flow Cytometry. The apoptosis ratio of RGC-5 was identified by Flow Cytometry 24 h after treated with the different doses of Tat-LK15( 1,2. 5,5,10 and 20 μg). 2. The model of RGC-5 cell overexpression of n NOS protein was prepared. 3. RGC-5 cells were randomly divided into 5 groups: control group,model group,Tat-S group( Tat-LK15 mediate n NOS /siRNA transfection model cell),Lipo-S group( LipofectamineTMRNAi MAX mediate n NOS / siRNA transfection model cell) and Tat-N group( Tat-LK15 mediate NCsiRNA transfection model cell). Real-time Quantitative polymerase chain reaction( Q-PCR) and Western blot were used to evaluate n NOS expression level assay. Results It indicated that the Tat-LK15 / siRNA complex completely formed at the weight ratio of 2 ∶ 1( μg / μg),and the transfection efficiency was( 84. 4 ± 3. 9) %. It caused cotytoxicity when Tat-LK15 dose was 20 μg( 6. 1 μmol ·L- 1),and the apoptosis rate more than control group[( 10. 3 ± 1. 1) % vs( 7. 4 ± 0. 9) %,P < 0. 05]. The n NOS protein level of RGC-5 cells was significantly elevated after modeling. Compared with that of model group,Tat-LK15 / siRNA efficiently inhibited the expression of n NOS at transcriptional level or protein leve1 of Tat-S group( P < 0. 05),and there was no significant difference of the efficiency inhibited between Tat-S group and Lipo-S group. Conclusions TatLK15'advantage is with high efficiency,low cytotoxicity. The Tat-LK15 can deliver siRNA targeting n NOS in vitro efficiently and safely.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2015年第2期278-283,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81100817
81371233)
