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氧糖剥夺再灌注后Hsp20的神经保护作用及其机制

发布时间:2018-01-28 04:54

  本文关键词: 氧糖剥夺再灌注 Hsp20 高尔基体 线粒体 凋亡 Hsp20 突变体 质粒 磷酸化 Hsp20 氧糖剥夺再灌注 神经保护 Ser16磷酸化 线粒体 出处:《中南大学》2011年博士论文 论文类型:学位论文


【摘要】:第一章氧糖剥夺再灌注后Hsp20的表达变化 目的:探讨氧糖剥夺再灌注后细胞活力、细胞凋亡及Hsp20的表达变化,并观察氧糖剥夺再灌注后线粒体、高尔基体等细胞结构的变化,为下一步研究Hsp20的神经保护作用及其机制奠定基础。 方法:小鼠脑神经瘤N2a细胞经氧糖剥夺再灌注后,采用MTT法检测细胞活力,流式细胞技术检测细胞凋亡率的变化;并采用免疫荧光技术研究高尔基体、线粒体等细胞结构的变化,采用Western blot及实时定量PCR检测Hsp20与高尔基体蛋白GM130的蛋白基因表达变化。 结果:1.氧糖剥夺4小时并再灌注12及24小时后,N2a细胞的活力明显下降(P0.01)。同时,氧糖剥夺4小时并再灌注6、12及24小时后,N2a细胞的凋亡率明显增高(P0.05)。 2.氧糖剥夺4小时并再灌注0小时及6小时后,Hsp20的蛋白及基因表达水平与基础水平比较明显下降(P0.05)。再灌注12小时及24小时后,则回到基础水平。 3.氧糖剥夺4小时并再灌注0小时及6小时后,丝氨酸磷酸化Hsp20蛋白与总Hsp20蛋白的比值,与基础水平比无显著性差异。再灌注12小时及24小时后,其比值则比基础水平明显增高(P0.05)。 4.氧糖剥夺再灌注后,高尔基体蛋白GM130的蛋白及基因表达、高尔基体形态均未见明显变化,而线粒体则发生了碎裂,相互间紧密连接消失。结论:氧糖剥夺再灌注后,N2a细胞的活力明显受损,凋亡率增高,Hsp20及磷酸化Hsp20的表达受氧糖剥夺再灌注的调节,同时线粒体发生了碎裂,但高尔基体形态及GM130的表达并未发现明显变化。 第二章Hsp20野生型及其突变体的构建和表达 目的:构建Hsp20野生型、Ser16磷酸化突变体Hsp20s16D及Ser16去磷酸化突变体Hsp20s16A的表达质粒,为进一步研究Hsp20的神经保护作用及其机制做好前期准备工作。 方法:使用小鼠脑神经瘤N2a细胞抽提RNA,逆转录成cDNA后,利用特异性Hsp20引物和带突变位点的长引物进行PCR,以获得Hsp20的CDS序列,将带有酶切位点的PCR产物,连接到1pEGFP-N1表达载体中,经过酶切鉴定和测序证实其正确性。将构建好的载体转染到N2a细胞中,通过免疫荧光和免疫印迹,观察Hsp20野生型、Ser16磷酸化突变体Hsp20s16D及Ser16去磷酸化突变体Hsp20s16A表达质粒在细胞中的表达。 结果:通过PCR法成功获得小鼠Hsp20 CDS序列,并成功连接到pEGFP-N1表达载体中,经测序和酶切鉴定正确。免疫荧光证实Hsp20野生型、Ser16磷酸化突变体Hsp20S16D及Ser16去磷酸化突变体Hsp20S16A表达分布相同,免疫印迹显示构建的Hsp20野生型和突变体能成功表达且分子量大小正确。 结论:成功构建了Hsp20野生型、Ser16磷酸化突变体Hsp20s16D及Ser16去磷酸化突变体Hsp20s16A的表达质粒,为下一步研究Hsp20的神经保护作用及其机制奠定了基础。 第三章Hsp20的神经保护作用及其机制 目的:研究氧糖剥夺再灌注后Hsp20的神经保护作用并探讨其可能机制。 方法:将构建成功的Hsp20野生型、Ser16磷酸化突变体Hsp20S16D及Serl6去磷酸化突变体Hsp20S16A的表达质粒转染小鼠脑神经瘤N2a细胞,各组细胞经历氧糖剥夺再灌注后,采用MTT法、流式细胞技术及免疫荧光技术,检测细胞活力、凋亡率及线粒体的变化,并采用Western blot技术检测Bax, Bcl-2及细胞色素C的释放变化,探讨Hsp20在保护神经细胞线粒体凋亡通路中的作用。 结果:1.转染Hsp20野生型及Ser16磷酸化突变体Hsp20s16D的细胞经氧糖剥夺4小时并再灌注12及24小时后,与空载体相比,细胞活力增高(P0.05),细胞凋亡率下降(P0.01) 2.转染Ser16去磷酸化突变体Hsp20s16A的细胞,经氧糖剥夺再灌注后,其细胞活力及细胞凋亡率与空载体组相比没有显著性差异。 3.转染Hsp20野生型及Ser16磷酸化突变体Hsp20s16D的细胞经氧糖剥夺再灌注后,线粒体的碎裂程度明显降低,而转染Serl6去磷酸化突变体Hsp20s16A的细胞,其细胞内线粒体碎裂程度较空载体组则并未见明显区别。 4.转染Hsp20野生型及Serl6磷酸化突变体Hsp20s16D的细胞经氧糖剥夺再灌注后,Bcl-2的表达水平增高,Bax的表达水平下降,从线粒体释放到胞浆中的细胞色素C减少(P0.05),而转染Ser16去磷酸化突变体Hsp20S16A细胞中Bax,Bcl-2及细胞色素C的表达,与空载体组相比则没有显著性差异。 结论:1.Hsp20在氧糖剥夺再灌注中具有神经保护作用。 2. Hsp20在氧糖剥夺再灌注中的神经保护作用与Serl6磷酸化有关,阻断Ser16磷酸化则其不再具有神经保护作用。 3. Hsp20的神经保护作用与保护线粒体结构稳定有关。 4. Hsp20的神经保护作用与抑制线粒体凋亡通路有关。
[Abstract]:The change of expression of Hsp20 after oxygen deprivation and reperfusion in the first chapter
Objective: To investigate the changes of cell viability, apoptosis and Hsp20 expression after oxygen glucose deprivation reperfusion, and observe the changes of cell structure of mitochondria and Golgi apparatus after oxygen glucose deprivation and reperfusion, so as to lay a foundation for further research on the neuroprotective effect and mechanism of Hsp20.
Methods: mouse brain neuroma N2a cells with oxygen glucose deprivation after reperfusion, cell viability was determined by MTT method, flow cytometry apoptosis rate; and the Golgi immunofluorescence technique, changes of mitochondria structure, the protein expression of Western gene using blot and real-time quantitative PCR detection of Hsp20 and Golgi protein GM130.
Results: after 1. hours of oxygen glucose deprivation and 4 hour reperfusion, 12 hours and 24 hours after reperfusion, the viability of N2a cells decreased significantly (P0.01). Meanwhile, the apoptosis rate of N2a cells increased significantly after 4 hours of oxygen glucose deprivation and reperfusion for 6,12 and 24 hours (P0.05).
After 2. hours of oxygen glucose deprivation for 4 hours and reperfusion for 0 hours and 6 hours, the protein and gene expression level of Hsp20 decreased significantly compared with basal level (P0.05). After reperfusion for 12 hours and 24 hours, it returned to basal level.
After 3. hours of oxygen glucose deprivation for 4 hours, and 0 hours and 6 hours after reperfusion, there was no significant difference in the ratio of serine phosphorylated Hsp20 protein to total Hsp20 protein. Compared with basal level, the ratio of serine phosphorylated protein to basal level was significantly higher than that of basal level after reperfusion for 12 hours and 24 hours (P0.05).
4. oxygen glucose deprivation after reperfusion, and the expression of golgiosome protein GM130, there were no obvious changes of Golgi body morphology, and mitochondria occurred between fragmentation, tight junction disappeared. Conclusion: the reperfusion after oxygen and glucose deprivation, N2a cell viability was significantly impaired, the apoptosis rate increased, the expression of Hsp20 and phosphate Hsp20 by oxygen glucose deprivation and reperfusion regulation, mitochondria were broken, but the expression of the Golgi morphology and GM130 did not show significant change.
The construction and expression of the second chapter Hsp20 wild type and its mutant
Objective: to construct Hsp20 wild type, Ser16 phosphorylation mutant Hsp20s16D and Ser16 dephosphorylation mutant Hsp20s16A expression plasmid, so as to further study the neuroprotective effect and mechanism of Hsp20, prepare for early preparations.
Methods: using mouse brain neuroma N2a cell RNA was extracted and reverse transcribed into cDNA, using specific Hsp20 primers and primers with long mutation PCR, with sequence of CDS Hsp20, PCR products with restriction sites, connected to the expression vector 1pEGFP-N1. After enzyme digestion and sequencing confirmed the correctness of the. The constructed vector was transfected into N2a cells, by immunofluorescence and Western blot, observe the wild type Hsp20, Ser16 phosphorylation and dephosphorylation of Ser16 mutant Hsp20s16D mutant Hsp20s16A expression plasmid in cells.
Results: the PCR method successfully obtained CDS sequence of mouse Hsp20, and successfully connected to the expression vector pEGFP-N1. After sequencing and restriction enzyme digestion. Immunofluorescence showed that the wild type Hsp20, Ser16 phosphorylation and dephosphorylation of Ser16 mutant Hsp20S16D mutant Hsp20S16A expression of the same distribution, Western blot showed that the construction of the wild type and Hsp20 mutant can succeed the expression and the molecular weight of the correct size.
Conclusion: we successfully constructed the expression plasmid of Hsp20 wild type, Ser16 phosphorylation mutant Hsp20s16D and Ser16 dephosphorylation mutant Hsp20s16A, which laid the foundation for further research on the neuroprotective effect and mechanism of Hsp20.
The neuroprotective effect and mechanism of the third chapter Hsp20
Objective: To study the neuroprotective effect of Hsp20 after oxygen deprivation and reperfusion and to explore its possible mechanism.
Methods: the successful construction of wild type Hsp20, Ser16 phosphorylation and dephosphorylation of Serl6 mutant Hsp20S16D mutant Hsp20S16A expression plasmid was transfected into mouse brain tumor N2a cells, the cells were undergoing oxygen glucose deprivation after reperfusion by MTT method, flow cytometry and immunofluorescence technique to detect cell viability, apoptosis rate and change mitochondria, and the detection of Bax Western blot, Bcl-2 and cytochrome C release changes, to explore the role of Hsp20 in apoptosis pathway protect mitochondria in nerve cells.
Results: 1.. After transfection of Hsp20 wild type and Ser16 phosphorylation mutant Hsp20s16D cells, after 4 hours of oxygen glucose deprivation and 12 and 24 hours of reperfusion, the cell viability increased (P0.05) and the apoptosis rate decreased (P0.01).
2., after transfection of Ser16 to dephosphorylate mutant Hsp20s16A cells, the viability and apoptosis rate of cells after oxygen glucose deprivation and reperfusion were not significantly different from those of empty load group.
3. transfection of wild type Hsp20 and phosphorylation of Ser16 mutant Hsp20s16D cells after oxygen glucose deprivation after reperfusion, the extent of fragmentation of mitochondria decreased significantly, while transfection of Serl6 dephosphorylation of Hsp20s16A mutant cells, the intracellular mitochondrial fragmentation degree compared with the empty vector group and no significant difference.
4. transfection of wild type Hsp20 and phosphorylation of Serl6 mutant Hsp20s16D cells after oxygen glucose deprivation after reperfusion, increased the expression level of Bcl-2, the expression level of Bax decreased cytochrome C release from mitochondria to the cytoplasm decreased (P0.05), and transfected the dephosphorylation of Ser16 mutants in Hsp20S16A cells the expression of Bcl-2 and Bax. Cytochrome C, and empty vector group compared with no significant difference.
Conclusion: 1.Hsp20 has neuroprotective effect in oxygen glucose deprivation reperfusion.
The neuroprotective effect of 2. Hsp20 in oxygen glucose deprivation reperfusion is related to Serl6 phosphorylation, and blocking the phosphorylation of Ser16 no longer has neuroprotective effect.
The neuroprotective effect of 3. Hsp20 is related to the protection of the structural stability of mitochondria.
The neuroprotective effect of 4. Hsp20 is related to the inhibition of mitochondrial apoptosis pathway.

【学位授予单位】:中南大学
【学位级别】:博士
【学位授予年份】:2011
【分类号】:R363

【共引文献】

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2 赵国华;CMT2L型致病基因的克隆与CMT的SIMPLE、RAB7、LMNA和MTMR2基因突变分析[D];中南大学;2006年

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1 陈兰;沙鼠脑缺血再灌注损伤后Hsp22和HIF-1α表达的变化及神经保护作用[D];中南大学;2007年

2 郭晓倩;沙鼠脑缺血再灌注后Caspase-3和Hsp20的表达变化及西比灵干预的影响[D];中南大学;2007年

3 曾六旺;沙鼠脑缺血再灌注和美满霉素干预后高尔基体与TGF-β1的变化及TGF-β1在高尔基体定位的研究[D];中南大学;2007年

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