腺病毒介导的HSP70在神经元和胶质细胞中的抗缺氧研究

发布时间：2018-03-25 10:15

本文选题：热休克蛋白70　切入点：腺病毒载体　出处：《青岛大学》2009年硕士论文

【摘要】： 目的初步探讨不同缺氧时间刺激对神经细胞中外源性HSP70基因表达的影响及观察HSP70表达对神经元和胶质细胞缺氧再复氧损伤的保护作用。方法①采用重组腺病毒vAd-HSP70感染体外原代培养的神经细胞,检测靶细胞中外源性HSP70的表达。48h后给予感染细胞不同缺氧时间(缺氧Oh,0.5h,1h,2h,3h,4h)刺激,再复氧处理后,用RT-PCR和Western blotting检测重组腺病毒介导的HSP70基因在神经元和胶质细胞中的转录表达。②重组腺病毒vAd-HSP70感染体外培养的神经元和胶质细胞,vAd-HSP70感染组24h、48h、72h和vAd-GFP感染组的细胞经缺氧再复氧处理后,分别对细胞活力、细胞培养上清中乳酸脱氢酶(LDH)活力及线粒体和胞浆内细胞色素C进行检测分析。结果①重组腺病毒vAd-HSP70感染的神经细胞可检测到外源性HSP70基因的转录表达。②经缺氧再复氧处理后,随着缺氧时间延长,HSP70转录水平和蛋白表达都增加,缺氧1h时最高(1.1539±0.0315,0.9699±0.0023),其次是缺氧2h时(1.0398±0.0723,0.9622±0.0026),随后依次降低,缺氧4h组(0.7477±0.0328,0.9335±0.0034)HSP70表达最低,与其他各组比较有统计学意义(P0.05)。③经缺氧再复氧处理后,vAd-HSP70感染组细胞活力较对照组明显增强(P0.01);细胞培养上清中乳酸脱氢酶活力vAd-HSP70感染24h、48h和72h分别为1480±121,1023±106,1132±197,明显低于vAd-GFP感染组(1976±190,P0.01)。实验各组线粒体中细胞色素C含量(0.9856±0.0121,1.0277±0.0073,1.0136±0.0078)高于对照组(0.9695±0.0031,P0.05),而胞浆中的含量(0.9868±0.0076,0.9600±0.0049,0.9645±0.0028)低于对照组(1.0109±0.0049,P0.01),以作用48h最为理想。结论①缺氧2h内给予神经细胞再复氧处理外源性HSP70表达水平较高,有利于神经细胞功能的恢复。②腺病毒介导的外源性HSP70表达可保护神经元和胶质细胞抵抗缺氧再复氧损伤,具有明确的细胞保护作用。
[Abstract]:Objective to investigate the effects of hypoxia time stimulation on exogenous HSP70 gene expression in neurons and to observe the protective effect of HSP70 expression on hypoxia reoxygenation injury of neurons and glial cells. Methods 1Recombinant adenovirus vAd-HSP70 was used to infect the primary cultured neurons in vitro. The expression of exogenous HSP70 in target cells was detected for 48 h. The cells were stimulated with different hypoxia time (1 h ~ 2 h ~ 2 h ~ 3 h ~ 4 h) after reoxygenation. Transcriptional expression of HSP70 Gene mediated by Recombinant adenovirus in neurons and glial cells by RT-PCR and Western blotting. 2 Recombinant adenovirus vAd-HSP70 infection of cultured neurons and glial cells in vitro. After oxygen reoxygenation, Cell viability, lactate dehydrogenase (LDH) activity and cytochrome C in mitochondria and cytoplasm were determined. Results 1Transcriptional expression of exogenous HSP70 gene was detected in neurons infected with recombinant adenovirus vAd-HSP70. 2. After hypoxia and reoxygenation, the transcription level and protein expression of HSP70 increased with the prolongation of hypoxia. The highest level was 1.1539 卤0.0315U 0.9699 卤0.0023C at 1 h, followed by 1.0398 卤0.0723U 0.9622 卤0.0026m at 2h hypoxia, and then decreased in turn. The lowest expression of 0.7477 卤0.03280.335 卤0.0034)HSP70 was observed in the hypoxia group for 4 h. Compared with other groups, the activity of vAd-HSP70 in the cell culture supernatant was significantly higher than that in the control group after anoxic reoxygenation, and the activity of lactate dehydrogenase in the supernatant of cell culture was 1480 卤121 卤1023 卤106 1132 卤197, which was significantly lower than that in the control group, which was significantly lower than that in the control group, which was significantly lower than that in the control group at 24 h and 72 h after hypoxia reoxygenation and hypoxia reoxygenation, respectively. The content of cytochrome C in mitochondria of the experimental group was 0.9856 卤0.0121 卤1.0277 卤0.0073 卤1.0136 卤0.0078), which was higher than that of the control group (0.9695 卤0.0031), while the content of cytoplasm was 0.9868 卤0.0070.9600 卤0.0049-0.9645 卤0.0028), which was better than that of the control group for 48h. Conclusion (1) the expression of exogenous HSP70 after reoxygenation is higher within 2 h after hypoxia, which is beneficial to the recovery of nerve cell function. 2 Adenovirus-mediated exogenous HSP70 expression can protect neurons and glial cells from hypoxia and reoxygenation injury. It has definite cell protective effect.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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