本文關(guān)鍵詞： HSP70 SH-SY5Y 缺血缺氧 細胞凋亡　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的采用RNA干涉技術(shù)沉默神經(jīng)母細胞瘤細胞系(SH-SY5Y)熱休克蛋白70(HSP70)的表達,觀察HSP70沉默對缺氧誘導(dǎo)的SH-SY5Y細胞凋亡及BAG-1、NF-κB、caspase-3表達的影響。方法培養(yǎng)SH-SY5Y細胞至對數(shù)生長期,采用RNA干涉技術(shù)沉默HSP70,將SH-SY5Y細胞分為4組,分別為細胞對照組(正常的SH-SY5Y細胞)、慢病毒對照組(感染含GFP但不含靶向HSP70 siRNA的慢病毒)、慢病毒感染組Ⅰ(感染含GFP及靶向HSP70 siRNA-Ⅰ的慢病毒)、慢病毒感染組Ⅱ(感染含GFP及靶向HSP70 siRNA-Ⅱ的慢病毒)。采用蛋白質(zhì)免疫印跡試驗(Western blotting)檢測缺氧處理后HSP70蛋白表達水平;實時熒光定量反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(q RT-PCR)檢測HSP70 m RNA轉(zhuǎn)錄水平。4組細胞分別缺氧4h、8h、12h后再復(fù)氧24h,采用CCK-8法檢測SH-SY5Y細胞生長與增殖情況。選擇8h為最佳缺氧時間,流式細胞儀檢測分析SH-SY5Y細胞凋亡率和細胞周期;q RT-PCR檢測缺氧8h時SH-SY5Y細胞BAG-1 m RNA轉(zhuǎn)錄水平;Western blotting檢測缺氧8h時SH-SY5Y細胞BAG-1、NF-κB、caspase-3蛋白表達水平。結(jié)果1.重組慢病毒siRNA-Ⅰ感染組和重組慢病毒siRNA-Ⅱ感染組HSP70 m RNA轉(zhuǎn)錄水平分別低于細胞對照組和慢病毒對照組(2-△△ct為1.00±0.04,0.98±0.12,0.22±0.03,0.15±0.01),差異均有統(tǒng)計學(xué)意義(P0.05)。重組慢病毒siRNA-Ⅰ感染組和重組慢病毒siRNA-Ⅱ感染組HSP70蛋白表達水平分別低于細胞對照組和慢病毒對照組(灰度值分別為1.31±0.23,1.26±0.17,0.77±0.10,0.80±0.17),差異均有統(tǒng)計學(xué)意義(P0.05)。2.隨缺氧時間的延長,4組細胞的細胞活性先增強,在缺氧8h時細胞活性達高峰,隨后各組細胞活性均有下降的趨勢。缺氧8h時,重組慢病毒siRNA-Ⅰ感染組及重組慢病毒siRNA-Ⅱ感染組細胞活性均低于細胞對照組及慢病毒對照組(OD值分別為0.85±0.02,0.84±0.03,0.23±0.03,0.25±0.02,P0.05)。3.與細胞對照組和慢病毒對照組比較,重組慢病毒siRNA-Ⅰ感染組及重組慢病毒siRNA-Ⅱ感染組細胞凋亡均顯著升高(細胞凋亡率(%)分別為11.17±1.12,11.40±0.40,68.97±6.82,58.10±3.16,P0.05)。4.與細胞對照組和慢病毒對照組比較,重組慢病毒siRNA-Ⅰ感染組及重組慢病毒siRNA-Ⅱ感染組BAG-1 m RNA轉(zhuǎn)錄水平無顯著差異(2-△△ct為1.00±0.06,0.94±0.07,0.92±0.05,0.91±0.09,P0.05);重組慢病毒siRNA-Ⅰ感染組及重組慢病毒siRNA-Ⅱ感染組與細胞對照組和慢病毒對照組比較,BAG-1蛋白表達水平無顯著差異(BAG-1L灰度值分別為0.96±0.10,0.99±0.03,1.03±0.02,1.02±0.07,P0.05;BAG-1M灰度值分別0.95±0.03,0.98±0.05,1.02±0.09,1.00±0.01,P0.05;BAG-1S灰度值分別1.02±0.04,1.00±0.07,1.03±0.07,0.96±0.04,P0.05);NF-κB蛋白表達水平顯著升高(灰度值分別為1.00±0.00,1.01±0.08,1.95±0.22,2.03±0.16,P0.05);caspase-3蛋白表達水平顯著升高(灰度值分別為0.82±0.04,0.83±0.03,1.19±0.04,1.21±0.04,P0.05)。結(jié)論HSP70沉默可促進缺血缺氧SH-SY5Y細胞凋亡,降低SH-SY5Y細胞活力,增加NF-κB、caspase-3的蛋白表達水平,但對BAG-1蛋白表達及m RNA轉(zhuǎn)錄水平無明顯影響。因此,HSP70沉默可增加SH-SY5Y細胞對缺氧耐受的敏感性,其機制可能是通過上調(diào)NF-κB及caspase-3表達促進細胞凋亡實現(xiàn)。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743

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