發(fā)布時間：2018-01-28 04:54

  本文關(guān)鍵詞： 氧糖剝奪再灌注 Hsp20 高爾基體 線粒體 凋亡 Hsp20 突變體 質(zhì)粒 磷酸化 Hsp20 氧糖剝奪再灌注 神經(jīng)保護(hù) Ser16磷酸化 線粒體　出處：《中南大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：第一章氧糖剝奪再灌注后Hsp20的表達(dá)變化 目的：探討氧糖剝奪再灌注后細(xì)胞活力、細(xì)胞凋亡及Hsp20的表達(dá)變化,并觀察氧糖剝奪再灌注后線粒體、高爾基體等細(xì)胞結(jié)構(gòu)的變化,為下一步研究Hsp20的神經(jīng)保護(hù)作用及其機(jī)制奠定基礎(chǔ)。 方法：小鼠腦神經(jīng)瘤N2a細(xì)胞經(jīng)氧糖剝奪再灌注后,采用MTT法檢測細(xì)胞活力,流式細(xì)胞技術(shù)檢測細(xì)胞凋亡率的變化；并采用免疫熒光技術(shù)研究高爾基體、線粒體等細(xì)胞結(jié)構(gòu)的變化,采用Western blot及實(shí)時定量PCR檢測Hsp20與高爾基體蛋白GM130的蛋白基因表達(dá)變化。 結(jié)果：1.氧糖剝奪4小時并再灌注12及24小時后,N2a細(xì)胞的活力明顯下降(P0.01)。同時,氧糖剝奪4小時并再灌注6、12及24小時后,N2a細(xì)胞的凋亡率明顯增高(P0.05)。 2.氧糖剝奪4小時并再灌注0小時及6小時后,Hsp20的蛋白及基因表達(dá)水平與基礎(chǔ)水平比較明顯下降(P0.05)。再灌注12小時及24小時后,則回到基礎(chǔ)水平。 3.氧糖剝奪4小時并再灌注0小時及6小時后,絲氨酸磷酸化Hsp20蛋白與總Hsp20蛋白的比值,與基礎(chǔ)水平比無顯著性差異。再灌注12小時及24小時后,其比值則比基礎(chǔ)水平明顯增高(P0.05)。 4.氧糖剝奪再灌注后,高爾基體蛋白GM130的蛋白及基因表達(dá)、高爾基體形態(tài)均未見明顯變化,而線粒體則發(fā)生了碎裂,相互間緊密連接消失。結(jié)論：氧糖剝奪再灌注后,N2a細(xì)胞的活力明顯受損,凋亡率增高,Hsp20及磷酸化Hsp20的表達(dá)受氧糖剝奪再灌注的調(diào)節(jié),同時線粒體發(fā)生了碎裂,但高爾基體形態(tài)及GM130的表達(dá)并未發(fā)現(xiàn)明顯變化。 第二章Hsp20野生型及其突變體的構(gòu)建和表達(dá) 目的：構(gòu)建Hsp20野生型、Ser16磷酸化突變體Hsp20s16D及Ser16去磷酸化突變體Hsp20s16A的表達(dá)質(zhì)粒,為進(jìn)一步研究Hsp20的神經(jīng)保護(hù)作用及其機(jī)制做好前期準(zhǔn)備工作。 方法：使用小鼠腦神經(jīng)瘤N2a細(xì)胞抽提RNA,逆轉(zhuǎn)錄成cDNA后,利用特異性Hsp20引物和帶突變位點(diǎn)的長引物進(jìn)行PCR,以獲得Hsp20的CDS序列,將帶有酶切位點(diǎn)的PCR產(chǎn)物,連接到1pEGFP-N1表達(dá)載體中,經(jīng)過酶切鑒定和測序證實(shí)其正確性。將構(gòu)建好的載體轉(zhuǎn)染到N2a細(xì)胞中,通過免疫熒光和免疫印跡,觀察Hsp20野生型、Ser16磷酸化突變體Hsp20s16D及Ser16去磷酸化突變體Hsp20s16A表達(dá)質(zhì)粒在細(xì)胞中的表達(dá)。 結(jié)果：通過PCR法成功獲得小鼠Hsp20 CDS序列,并成功連接到pEGFP-N1表達(dá)載體中,經(jīng)測序和酶切鑒定正確。免疫熒光證實(shí)Hsp20野生型、Ser16磷酸化突變體Hsp20S16D及Ser16去磷酸化突變體Hsp20S16A表達(dá)分布相同,免疫印跡顯示構(gòu)建的Hsp20野生型和突變體能成功表達(dá)且分子量大小正確。 結(jié)論：成功構(gòu)建了Hsp20野生型、Ser16磷酸化突變體Hsp20s16D及Ser16去磷酸化突變體Hsp20s16A的表達(dá)質(zhì)粒,為下一步研究Hsp20的神經(jīng)保護(hù)作用及其機(jī)制奠定了基礎(chǔ)。 第三章Hsp20的神經(jīng)保護(hù)作用及其機(jī)制 目的：研究氧糖剝奪再灌注后Hsp20的神經(jīng)保護(hù)作用并探討其可能機(jī)制。 方法：將構(gòu)建成功的Hsp20野生型、Ser16磷酸化突變體Hsp20S16D及Serl6去磷酸化突變體Hsp20S16A的表達(dá)質(zhì)粒轉(zhuǎn)染小鼠腦神經(jīng)瘤N2a細(xì)胞,各組細(xì)胞經(jīng)歷氧糖剝奪再灌注后,采用MTT法、流式細(xì)胞技術(shù)及免疫熒光技術(shù),檢測細(xì)胞活力、凋亡率及線粒體的變化,并采用Western blot技術(shù)檢測Bax, Bcl-2及細(xì)胞色素C的釋放變化,探討Hsp20在保護(hù)神經(jīng)細(xì)胞線粒體凋亡通路中的作用。 結(jié)果：1.轉(zhuǎn)染Hsp20野生型及Ser16磷酸化突變體Hsp20s16D的細(xì)胞經(jīng)氧糖剝奪4小時并再灌注12及24小時后,與空載體相比,細(xì)胞活力增高(P0.05),細(xì)胞凋亡率下降(P0.01) 2.轉(zhuǎn)染Ser16去磷酸化突變體Hsp20s16A的細(xì)胞,經(jīng)氧糖剝奪再灌注后,其細(xì)胞活力及細(xì)胞凋亡率與空載體組相比沒有顯著性差異。 3.轉(zhuǎn)染Hsp20野生型及Ser16磷酸化突變體Hsp20s16D的細(xì)胞經(jīng)氧糖剝奪再灌注后,線粒體的碎裂程度明顯降低,而轉(zhuǎn)染Serl6去磷酸化突變體Hsp20s16A的細(xì)胞,其細(xì)胞內(nèi)線粒體碎裂程度較空載體組則并未見明顯區(qū)別。 4.轉(zhuǎn)染Hsp20野生型及Serl6磷酸化突變體Hsp20s16D的細(xì)胞經(jīng)氧糖剝奪再灌注后,Bcl-2的表達(dá)水平增高,Bax的表達(dá)水平下降,從線粒體釋放到胞漿中的細(xì)胞色素C減少(P0.05),而轉(zhuǎn)染Ser16去磷酸化突變體Hsp20S16A細(xì)胞中Bax,Bcl-2及細(xì)胞色素C的表達(dá),與空載體組相比則沒有顯著性差異。 結(jié)論：1.Hsp20在氧糖剝奪再灌注中具有神經(jīng)保護(hù)作用。 2. Hsp20在氧糖剝奪再灌注中的神經(jīng)保護(hù)作用與Serl6磷酸化有關(guān),阻斷Ser16磷酸化則其不再具有神經(jīng)保護(hù)作用。 3. Hsp20的神經(jīng)保護(hù)作用與保護(hù)線粒體結(jié)構(gòu)穩(wěn)定有關(guān)。 4. Hsp20的神經(jīng)保護(hù)作用與抑制線粒體凋亡通路有關(guān)。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 郭鵬;唐北沙;趙國華;劉小民;張付峰;張如旭;沈璐;江泓;梁靜慧;;腓骨肌萎縮癥的病理學(xué)特點(diǎn)和基因突變[J];中華醫(yī)學(xué)雜志;2005年34期

相關(guān)博士學(xué)位論文 前2條

1 李書劍;HSPB8蛋白與NEFL蛋白相互作用研究及HSPB8蛋白對細(xì)胞相對活力的影響[D];中南大學(xué);2007年

2 趙國華;CMT2L型致病基因的克隆與CMT的SIMPLE、RAB7、LMNA和MTMR2基因突變分析[D];中南大學(xué);2006年

相關(guān)碩士學(xué)位論文 前4條

1 陳蘭;沙鼠腦缺血再灌注損傷后Hsp22和HIF-1α表達(dá)的變化及神經(jīng)保護(hù)作用[D];中南大學(xué);2007年

2 郭曉倩;沙鼠腦缺血再灌注后Caspase-3和Hsp20的表達(dá)變化及西比靈干預(yù)的影響[D];中南大學(xué);2007年

3 曾六旺;沙鼠腦缺血再灌注和美滿霉素干預(yù)后高爾基體與TGF-β1的變化及TGF-β1在高爾基體定位的研究[D];中南大學(xué);2007年

4 傅敏;應(yīng)用變性高效液相色譜技術(shù)檢測MFN2基因突變[D];中南大學(xué);2007年

，

本文編號：1469823