發(fā)布時(shí)間：2018-06-19 02:11

  本文選題：HSP70 + 反義寡脫氧核苷酸�。� 參考：《四川大學(xué)》2005年碩士論文

【摘要】：【目的】 通過人類HSP70(heat shock protein 70,熱休克蛋白70)反義寡脫氧核苷酸,阻斷MRC-5細(xì)胞(人胚肺二倍體成纖維細(xì)胞)HSP70蛋白表達(dá),觀察不同時(shí)間點(diǎn)HSP70蛋白水平、細(xì)胞凋亡及G1期細(xì)胞比例的變化,探討HSP70與細(xì)胞衰老及細(xì)胞凋亡的關(guān)系。 【方法】 1)根據(jù)不同OND(oligodeoxynucleotides,寡脫氧核苷酸)及脂質(zhì)體濃度設(shè)計(jì)ODN組、脂質(zhì)體組、ODN-脂質(zhì)體復(fù)合物組及空白對照組共21個(gè)組別,用MTT法測定各處理方式對MRC-5細(xì)胞增殖的影響。2)用第一步實(shí)驗(yàn)確定的正義寡脫氧核苷酸和反義寡脫氧核苷酸(anti-senseoligodeoxynucleotides,AS-ODN)及脂質(zhì)體的最佳作用濃度分別轉(zhuǎn)染兩組細(xì)胞,于轉(zhuǎn)染后12小時(shí)、1天、3天和5天,用流式細(xì)胞儀、免疫熒光顯微鏡檢測HSP70蛋白的熒光強(qiáng)度、細(xì)胞凋亡及細(xì)胞周期。3)設(shè)立正義、反義和空白對照三個(gè)組,分別于轉(zhuǎn)染后2天、4天和6天取各組細(xì)胞爬片,行衰老相關(guān)的β半乳糖苷酶(senescence-associated β-Gal,SA-β-Gal)染色檢測藍(lán)染細(xì)胞。 【結(jié)果】 1)各時(shí)間點(diǎn)反義組蛋白表達(dá)均低于正義組(P0.05),且其抑制作用在轉(zhuǎn)染后12小時(shí)最強(qiáng)。反義和正義組細(xì)胞在轉(zhuǎn)染后均出現(xiàn)蛋白表達(dá)高峰,但反義組高峰(轉(zhuǎn)染后第1天)較正義組高峰(轉(zhuǎn)染后第3天)提前。2)各時(shí)間點(diǎn)反義組細(xì)胞凋亡率均高于正義組(P0.05),兩組細(xì)胞凋亡率均在轉(zhuǎn)染后第1天達(dá)到高峰,其后逐漸下降。3)各組細(xì)胞隨著觀
[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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R363

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