HSP70反义寡脱氧核苷酸促进MRC-5细胞衰老的实验研究

发布时间：2018-06-19 02:11

本文选题：HSP70 + 反义寡脱氧核苷酸　；参考：《四川大学》2005年硕士论文

【摘要】：【目的】通过人类HSP70(heat shock protein 70,热休克蛋白70)反义寡脱氧核苷酸,阻断MRC-5细胞(人胚肺二倍体成纤维细胞)HSP70蛋白表达,观察不同时间点HSP70蛋白水平、细胞凋亡及G1期细胞比例的变化,探讨HSP70与细胞衰老及细胞凋亡的关系。【方法】 1)根据不同OND(oligodeoxynucleotides,寡脱氧核苷酸)及脂质体浓度设计ODN组、脂质体组、ODN-脂质体复合物组及空白对照组共21个组别,用MTT法测定各处理方式对MRC-5细胞增殖的影响。2)用第一步实验确定的正义寡脱氧核苷酸和反义寡脱氧核苷酸(anti-senseoligodeoxynucleotides,AS-ODN)及脂质体的最佳作用浓度分别转染两组细胞,于转染后12小时、1天、3天和5天,用流式细胞仪、免疫荧光显微镜检测HSP70蛋白的荧光强度、细胞凋亡及细胞周期。3)设立正义、反义和空白对照三个组,分别于转染后2天、4天和6天取各组细胞爬片,行衰老相关的β半乳糖苷酶(senescence-associated β-Gal,SA-β-Gal)染色检测蓝染细胞。【结果】 1)各时间点反义组蛋白表达均低于正义组(P0.05),且其抑制作用在转染后12小时最强。反义和正义组细胞在转染后均出现蛋白表达高峰,但反义组高峰(转染后第1天)较正义组高峰(转染后第3天)提前。2)各时间点反义组细胞凋亡率均高于正义组(P0.05),两组细胞凋亡率均在转染后第1天达到高峰,其后逐渐下降。3)各组细胞随着观
[Abstract]:[objective] to block the expression of antisense oligodeoxynucleotides (antisense oligodeoxynucleotides) of human HSP70 shock protein 70 (HSP70) in human embryonic lung diploid fibroblasts, and to observe the level of HSP70 protein at different time points. The changes of apoptosis and the proportion of cells in G1 phase, To investigate the relationship between HSP70 and cell senescence and apoptosis. [methods] 1) the ODN group was designed according to different OND oligodeoxynucleotides (oligodeoxynucleotides) and liposome concentrations. In liposome group, there were 21 groups in ODN- liposome complex group and blank control group. MTT assay was used to determine the effect of different treatments on the proliferation of MRC-5 cells. The first step experiment was used to determine the optimal concentration of sense oligodeoxynucleotides and antisense oligodeoxynucleotides (AS-ODNs) and liposomes. At 12 hours after transfection, 3 and 5 days after transfection, the fluorescence intensity, apoptosis and cell cycle of HSP70 protein were detected by flow cytometry and immunofluorescence microscopy. After transfection, 4 and 6 days after transfection, the cells of each group were taken. Senescence-associated 尾 -galactosidase-associated 尾 -galactosidase 尾 -galactosidase staining was used to detect the expression of antisense histone in blue-stained cells. The inhibitory effect was strongest at 12 hours after transfection. After transfection, both antisense and sense cells showed peak protein expression. However, the apoptosis rate of the antisense group was higher than that of the sense group on the first day after transfection. The apoptosis rate of the antisense group was higher than that of the sense group on the first day after transfection, and the apoptosis rate of both groups reached the peak on the first day after transfection, and the apoptosis rate of the antisense group was higher than that of the sense group on the third day after transfection. Then gradually decreased. 3) the cells in each group followed the observation.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

【参考文献】