mPEG-CS纳米粒介导livin与survivin小干扰RNA杀伤结肠癌细胞

Livin and survivin small interfering RNAs mediated by mPEG-CS nanoparticles have an anti-coloncarcinoma effect

目的以mPEG-CS纳米粒作为livin基因和survivin基因的干扰RNA的载体,转染人结肠癌细胞HT-29,观察livin和survivin沉默对结肠癌细胞增殖及凋亡的影响。方法利用离子交联法,制备粒径约60nm的mPEG-CS纳米粒。利用静电吸附法,以纳米粒混悬液与基因溶液配比为3∶1的条件,构建mPEG-CS-livin shRNA纳米粒、mPEG-CS-survivin shRNA纳米粒以及mPEG-CS-(livin shRNA+surviving shRNA)纳米粒,之后,分别转染进人结肠癌HT-29细胞中。利用RT-PCR和Western Blot分别检测livin、survivin基因和蛋白的表达变化;利用MTT法检测livin、survivin基因表达下调对HT-29细胞增殖的抑制效率;利用Hoechst染色检测livin、survivin基因沉默对HT-29细胞凋亡的诱导。结果各干扰组均能在mRNA和蛋白水平上有效抑制HT-29细胞中livin、survivin的表达。MTT和Hoechst染色结果显示,联合干扰livin和survivin基因可有效抑制人结肠癌HT-29细胞的增殖,并促进细胞凋亡,且效果均强于单独干扰livin或sur-vivin基因。结论 mPEG-CS纳米粒介导的livin和survivin双基因干扰能有效降低人结肠癌HT-29细胞中livin和survivin基因的表达,抑制HT-29细胞的增殖,并促进HT-29细胞的凋亡,其效果强于单独干扰livin或survivin基因。双基因联合干扰具有协同增强效应。

As a carrier of the livin shRNA and survivin shRNA,MPEG-CS nanoparticles were used to transfect the human coloncarcinoma HT-29 cells, and then to observe the impact of livin and survivin silencing on the proliferation and apoptosis of HT-29 ceUs. [ Methods ] Ion cross-linking method was applied to construct the mPEG-CS nanoparticles, the particle size of which was about 60 nm. With the volume ratio of nanoparticle suspen- sion and genetic solution 3：1, electrostatic adsorption was used to construct the mPEG-CS livin shRNA nanoparti- cles, the mPEG-CS survivin shRNA nanoparticles and the mPEG-CS-（livin shRNA＋surviving shRNA）, which was transfected into human coloncarcinoma HT-29 cells, separately. RT-PCR and Western Blot were performed to detect the mRNA as well as the protein expression of livin and survivin. MTY was used to detect the inhibitive efficiency of HT-29 cells proliferation caused by the down-regulated livin and survivin. Hoechst staining was applied to analyze the apoptosis of HT-29 cells induced by the silence of livin and survivin. [ Results ] The mRNA and protein levels of livin and survivin in HT-29 ceils were effectively decreased in all interference groups. The results of MTr and Hoechst staining showed that the joint interference of livin and survivin can inhibit the proliferation of human coloncarcinoma HT-29 cells and promote their apoptosis, its effect better than the separate interference livin or sur- vivin gene. [ Conclusion ] The joint interference of livin and survivin mediated by mPEG-CS nanoDarticles could ef-fectively reduce the expression of livin and survivin in human coloncarcinoma HT-29 cells, inhibit the proliferation of HT-29 ceils and promote their apoptosis, the efficiency of which was better than that of the separate interference of livin or survivin. The joint Interference of two genes has a synergistic enhancement effect.