HSP70沉默对缺氧诱导神经母细胞瘤细胞凋亡的影响及机制研究

发布时间：2018-01-23 04:33

本文关键词： HSP70 SH-SY5Y 缺血缺氧细胞凋亡　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的采用RNA干涉技术沉默神经母细胞瘤细胞系(SH-SY5Y)热休克蛋白70(HSP70)的表达,观察HSP70沉默对缺氧诱导的SH-SY5Y细胞凋亡及BAG-1、NF-κB、caspase-3表达的影响。方法培养SH-SY5Y细胞至对数生长期,采用RNA干涉技术沉默HSP70,将SH-SY5Y细胞分为4组,分别为细胞对照组(正常的SH-SY5Y细胞)、慢病毒对照组(感染含GFP但不含靶向HSP70 siRNA的慢病毒)、慢病毒感染组Ⅰ(感染含GFP及靶向HSP70 siRNA-Ⅰ的慢病毒)、慢病毒感染组Ⅱ(感染含GFP及靶向HSP70 siRNA-Ⅱ的慢病毒)。采用蛋白质免疫印迹试验(Western blotting)检测缺氧处理后HSP70蛋白表达水平;实时荧光定量反转录-聚合酶链反应(q RT-PCR)检测HSP70 m RNA转录水平。4组细胞分别缺氧4h、8h、12h后再复氧24h,采用CCK-8法检测SH-SY5Y细胞生长与增殖情况。选择8h为最佳缺氧时间,流式细胞仪检测分析SH-SY5Y细胞凋亡率和细胞周期;q RT-PCR检测缺氧8h时SH-SY5Y细胞BAG-1 m RNA转录水平;Western blotting检测缺氧8h时SH-SY5Y细胞BAG-1、NF-κB、caspase-3蛋白表达水平。结果1.重组慢病毒siRNA-Ⅰ感染组和重组慢病毒siRNA-Ⅱ感染组HSP70 m RNA转录水平分别低于细胞对照组和慢病毒对照组(2-△△ct为1.00±0.04,0.98±0.12,0.22±0.03,0.15±0.01),差异均有统计学意义(P0.05)。重组慢病毒siRNA-Ⅰ感染组和重组慢病毒siRNA-Ⅱ感染组HSP70蛋白表达水平分别低于细胞对照组和慢病毒对照组(灰度值分别为1.31±0.23,1.26±0.17,0.77±0.10,0.80±0.17),差异均有统计学意义(P0.05)。2.随缺氧时间的延长,4组细胞的细胞活性先增强,在缺氧8h时细胞活性达高峰,随后各组细胞活性均有下降的趋势。缺氧8h时,重组慢病毒siRNA-Ⅰ感染组及重组慢病毒siRNA-Ⅱ感染组细胞活性均低于细胞对照组及慢病毒对照组(OD值分别为0.85±0.02,0.84±0.03,0.23±0.03,0.25±0.02,P0.05)。3.与细胞对照组和慢病毒对照组比较,重组慢病毒siRNA-Ⅰ感染组及重组慢病毒siRNA-Ⅱ感染组细胞凋亡均显著升高(细胞凋亡率(%)分别为11.17±1.12,11.40±0.40,68.97±6.82,58.10±3.16,P0.05)。4.与细胞对照组和慢病毒对照组比较,重组慢病毒siRNA-Ⅰ感染组及重组慢病毒siRNA-Ⅱ感染组BAG-1 m RNA转录水平无显著差异(2-△△ct为1.00±0.06,0.94±0.07,0.92±0.05,0.91±0.09,P0.05);重组慢病毒siRNA-Ⅰ感染组及重组慢病毒siRNA-Ⅱ感染组与细胞对照组和慢病毒对照组比较,BAG-1蛋白表达水平无显著差异(BAG-1L灰度值分别为0.96±0.10,0.99±0.03,1.03±0.02,1.02±0.07,P0.05;BAG-1M灰度值分别0.95±0.03,0.98±0.05,1.02±0.09,1.00±0.01,P0.05;BAG-1S灰度值分别1.02±0.04,1.00±0.07,1.03±0.07,0.96±0.04,P0.05);NF-κB蛋白表达水平显著升高(灰度值分别为1.00±0.00,1.01±0.08,1.95±0.22,2.03±0.16,P0.05);caspase-3蛋白表达水平显著升高(灰度值分别为0.82±0.04,0.83±0.03,1.19±0.04,1.21±0.04,P0.05)。结论HSP70沉默可促进缺血缺氧SH-SY5Y细胞凋亡,降低SH-SY5Y细胞活力,增加NF-κB、caspase-3的蛋白表达水平,但对BAG-1蛋白表达及m RNA转录水平无明显影响。因此,HSP70沉默可增加SH-SY5Y细胞对缺氧耐受的敏感性,其机制可能是通过上调NF-κB及caspase-3表达促进细胞凋亡实现。
[Abstract]:The purpose of interference silencing neuroblastoma cell line by RNA (SH-SY5Y) heat shock protein 70 (HSP70) expression, observation of HSP70 silencing on SH-SY5Y cell apoptosis and BAG-1 induced by hypoxia, NF- kappa B, caspase-3 expression. Methods SH-SY5Y cells were cultured to the logarithmic growth phase, using RNA interference technology to silence HSP70, will SH-SY5Y cells were divided into 4 groups, respectively. Cells in the control group (normal SH-SY5Y cells), control group (infected with lentivirus containing GFP but not containing HSP70 targeting siRNA lentivirus), lentivirus infection (infection group 1 containing GFP and HSP70 targeting siRNA- I lentiviral lentiviral infection), group II (infection containing GFP and HSP70 targeting siRNA- II lentivirus). Using Western blotting test (Western blotting) HSP70 protein expression after hypoxia treatment detection; real time fluorescence quantitative reverse transcription polymerase chain reaction (Q RT-PCR) HSP70 m RNA.4 transcription level detection Group 4H cells were 8h, 12h after hypoxia and reoxygenation 24h, CCK-8 method was used to detect SH-SY5Y cell growth and proliferation. 8h is the best choice of hypoxia time, flow cytometry analysis of SH-SY5Y cell apoptosis rate and cell cycle; Q RT-PCR 8h SH-SY5Y BAG-1 cells to detect hypoxia m RNA transcription level of SH-SY5Y cells; BAG-1 Western blotting detection of hypoxia 8h and NF- K B, the level of caspase-3 protein. Results the expression of 1. siRNA- recombinant lentiviral infection of recombinant lentivirus group and siRNA- group HSP70 m RNA transcription level II infection were lower than those of control group cells and slow virus control group (2- delta CT 1 + 0.04,0.98 + 0.12,0.22 + 0.03,0.15 + 0.01), the differences were statistically significant (P0.05). The recombinant lentiviral siRNA- 1 infection group and siRNA- recombinant lentiviral infection group II protein expression level of HSP70 cells were lower than those of control group and slow virus control group (gray value was 1.3 1 + 0.23,1.26 + 0.17,0.77 + 0.10,0.80 + 0.17), the differences were statistically significant (P0.05.2.) with the duration of hypoxia, cells of 4 groups were first enhanced in hypoxia 8h cell activity reached the peak, then each cell activity was decreased. After 8h, the recombinant lentiviral infection group and siRNA- I the recombinant lentiviral infection activity of cells in siRNA- group were lower than control group cells and lentivirus control group (OD value were 0.85 + 0.02,0.84 + 0.03,0.23 + 0.03,0.25 + 0.02, P0.05) and.3. cells in the control group and the lentivirus control group, recombinant virus siRNA- 1 infection group and siRNA- recombinant lentiviral infection group II cells apoptosis increased significantly (apoptosis rate (%) were 11.17 + 1.12,11.40 + 0.40,68.97 + 6.82,58.10 + 3.16, P0.05) and.4. cells in the control group and the lentivirus control group, recombinant lentivirus infected with recombinant siRNA- I group and chronic disease SiRNA- II BAG-1 m virus infection group, the transcription level of RNA had no significant difference (2- delta CT 1 + 0.06,0.94 + 0.07,0.92 + 0.05,0.91 + 0.09, P0.05); recombinant lentivirus siRNA- 1 infection group and siRNA- control group and recombinant lentiviral lentiviral infection cell group and the control group II, BAG-1 expression had no significant difference (level BAG-1L gray values were 0.96 + 0.10,0.99 + 0.03,1.03 + 0.02,1.02 + 0.07, P0.05; BAG-1M gray value of 0.95 + 0.03,0.98 + 0.05,1.02 + 0.09,1.00 + 0.01, P0.05; BAG-1S gray value of 1.02 + 0.04,1.00 + 0.07,1.03 + 0.07,0.96 + 0.04, P0.05); the expression of NF- kappa B protein significantly increased (gray value respectively. 1 + 0.00,1.01 + 0.08,1.95 + 0.22,2.03 + 0.16, P0.05); the expression level of caspase-3 protein was significantly increased (gray values were 0.82 + 0.04,0.83 + 0.03,1.19 + 0.04,1.21 + 0.04, P0.05). Conclusion HSP70 silencing can promote hypoxia ischemia S The apoptosis of H-SY5Y cells, SH-SY5Y decreased cell viability, increased NF- kappa B, the expression level of caspase-3 protein, but had no obvious effect on BAG-1 protein expression and M transcription level of RNA. Therefore, HSP70 silencing can increase the sensitivity of SH-SY5Y cells to hypoxia tolerance, the mechanism may be achieved by promoting apoptosis on NF- kappa B and Caspase-3 expression.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

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