摘要
目的用siRNA干扰技术沉默小鼠胰岛素瘤NIT-1细胞血管紧张素Ⅱ1型受体(AT1R)基因,探讨其对链脲佐菌素(STZ)诱导的胰岛素瘤细胞凋亡的影响。方法针对小鼠AT1R基因,设计并合成3对siRNA序列,脂质体法转染至NIT-1细胞,荧光显微镜观察荧光强度检测转染效率,Real-time PCR检测AT1R mRNA表达量判断不同干扰序列及干扰时间的基因沉默效果;分4组:空白组不予任何干预,STZ组予5 mmo L/L STZ干预30 min,si-AT1R组予40 nmol/L si-AT1R转染24 h,si-AT1R+STZ组予40 nmo L/L si-AT1R转染24 h后5mmo L/L STZ干预30 min;Real-time PCR检测Caspase-3 mRNA表达量,Hoechst33342染色荧光显微镜和Annexin V-FITC/PI流式细胞术检测细胞凋亡率。结果 40 nmol/L siRNA转染的荧光强度最强,转染后AT1R mRNA表达量明显下降,si-AT1R-2转染24 h的沉默效果最佳(92.20%);Caspase-3 mRNA表达量:STZ组与空白组比增加了2.37倍(P<0.05),si-AT1R+STZ组与STZ组比减少了11.28%,但差异无统计学意义;细胞凋亡率:STZ组与空白组比增加了3.27倍(P<0.05),si-AT1R+STZ组与STZ组比减少了26.82%(P<0.05)。结论本研究建立了稳定的胰岛细胞AT1R基因沉默模型;RNA干扰沉默胰岛细胞AT1R基因可通过下调Caspase-3mRNA表达量降低STZ诱导的细胞凋亡率,提示AT1R介导了STZ诱导的胰岛细胞凋亡过程。
Objective To investigate the effects of silencing angiotensin Ⅱ type 1 receptor （AT1 R） in apoptosis induced by STZ in mice islet cells NIT - 1. Methods Three pairs of siRNA targeting AT1R were designed and synthesized. They were transfected into NIT - 1 by liposome transfection. Fluorescence microscopy was used to examine the effect of different siRNA concentration on the transfection efficiency. AT1R mRNA expression was detected with real - time PCR to compare silencing effects among the three siRNA sequence and different durations of interference. NIT - 1 cells were allocated into 4 groups. The control group did not receive intervention. The STZ group was treated with 5 mmol/L STZ for 30 min. The si - AT1R group was transfected with 40 nmol/L si - AT1R and detected 24 h later. The si - AT1R ＋ STZ group was transfected with 40 nmol/L si -AT1R and 24 h later treated with 5 mmol/L STZ for 30 min. Caspase -3 mR- NA expression was detected by Real -time PCR. Apoptosis rate was measured by Hoechst33342 staining microscopy and Annexin V - FITC/PI - labeled flow cytomety. Results Transfection with 40 nmol/L siRNA had the greatest fluorescence. The maximum inhibitory effects of AT1R siRNA transfection on AT1R expression was 40 nmol/L at 24 h with si - AT1R -2. The expression of AT1R mRNA was markedly down regulated by 92. 20% compared with that in the control group （P 〈 0. 05 ）. The expression of caspase - 3 mRNA in the STZ group increased by 2. 37 times compared with the control group （P 〈 0. 05）. The expression of caspase- 3 mRNA in the si- AT1R ＋ STZ group decreased by 11.28% compared with the STZ group, but the difference was not statistically significant. Apoptosis rate in the STZ group increased by 3.27 times compared with the control group （P 〈 0. 05）. The cells treated with both si - AT1R and STZ had a significantly decreased apoptosis rate （by 26. 82% ） compared with the STZ group （P 〈 0. 05）. Conclusion This study suc- cessfully silenced AT1R in NIT - 1 cells and obtained
出处
《广东医学》
CAS
北大核心
2016年第11期1593-1597,共5页
Guangdong Medical Journal
基金
广西自然科学基金资助项目(编号:2013GXNSFAA019202,2013GXNSFAA019253)
