【作者】 曾六旺；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 神经病学， 2011， 博士

【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(P<0.01)。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(P<0.05)。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(P<0.05)。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(P<0.05)。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(P<0.05),细胞凋亡率下降(P<0.01)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(P<0.05),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。更多还原

【Abstract】 Chapter I:Expression Pattern of Hsp20 in Mouse N2A Cells upon OGD/R treatmentObjective:To elucidate alteration of cellular viability and apoptosis, morphology of mitochondria and Golgi apparatus in mouse N2A cells upon oxygen-glucose deprivation followed by reperfusion (OGD/R),as well as expression pattern of Hsp20.Methods:We employed OGD/R model to mimic ischemic-like conditions in vitro. Cellular viability and apoptosis were measured using the MTT assay and flow cytometry. The morphologic change of mitochondria and Golgi apparatus was determined by immunofluorescence.GM130 and Hsp20 expression levels were determined by Western blot and quantitative Real-Time PCR.Results:1. Treatment with OGD/R markedly reduced cellular viability in mouse N2A cells at 12-hour and 24-hour time points recovery from 4-hour OGD (P<0.01), while increased apoptosis rate at 6-hour,12-hour and 24-hour time points recovery from 4-hour OGD (P<0.05)2. Expressions of Hsp20 were strongly downregulated in mouse N2A cells at 0-hour and 6-hour time points recovery from 4-hour OGD (P<0.05), both at mRNA and protein levels, and they returned to basal level 12 and 24 hours after OGD treatment.3. The protein levels of total and phosphorylated Hsp20 decreased to approximately the same extent at the 0-hour and 6-hour recovery time points following 4 hours of OGD. However, the ratio of phosphorylated to total Hsp20 protein was significantly higher than control 12 and 24 hours after OGD treatment (P<0.05)4. Upon OGD/R, compared to control, expressions of GM130 and the morphology of Golgi apparatus showed no significant differences. However, OGD/R induced mitochondria fragmentation. Many filamentous mitochondria converted small and round organelles following OGD/R.Conclusion:Cellular viability and apoptosis, as well as morphology of mitochondria damaged greatly in mouse N2A cells upon OGD/R treatment. Meanwhile, the expression pattern of Hsp20 was regulated upon OGD/R treatment. However, expressions of GM130 and the morphology of Golgi apparatus were not affected. ChapterⅡConstruction and Expression of Hsp20 and its mutantsObjective:To construct expression plasmids of Hsp20 and its constitutively dephosphorylated mutant Hsp20s16A, as well as the constitutively phosphorylated mutant Hsp20s16DMethods:Total RNA was isolated from N2a cell cultures and reverse transcription was then performed. Hsp20 was obtained by using primers with Restriction enzyme EcoR I and BamH I in 5’-Terminal. The PCR products were cloned to pEGFPNl vector using EcoR I and BamH I. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmids were transfected into N2a cells. Hsp20 expression in the transfected cells was assayed by immunofluorescence and immunoblotting.Results:We obtained CDS sequences of Hsp20 successfully by PCR. Enzyme digestion and DNA sequencing showed that the CDS sequences of Hsp20 were correctly inserted into pEGFPN1 vector. Same expression and distribution of Hsp20 and its mutants in N2a cells were confirmed by immunofluorescence. Expressions of Hsp20 and its mutants were confirmed by immunoblotting and their molecular weights were correct.Conclusion:We successfully constructed expression plasmids of Hsp20 and its constitutively dephosphorylated mutant Hsp20s16A, as well as the constitutively phosphorylated mutant Hsp20S16D. ChapterⅢThe Neuroprotective Effects of Hsp20 and its Underlying MechanismObjective:To elucidate the neuroprotective effects of Hsp20 against oxygen-glucose deprivation followed by reperfusion (OGD/R) and its potential mechanism.Methods:Mouse N2A cells were transfected with pEGFP-Hsp20, or pEGFP-Hsp20 (S16D) or pEGFP-Hsp20(S16A) for 36 h, and then treated with 12-hour reperfusion following 4-hour OGD. Cellular viability and apoptosis were measured using the MTT assay and flow cytometry. The morphologic change of mitochondria was determined by immunofluorescence. Bax, Bcl-2 and cytochrome c expression levels were determined by Western blot.Results:1, Compared with the control, the cellular viability was significantly higher in N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D (p<0.05). N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D also displayed a significant decrease in the number of apoptotic cells upon OGD/R exposure (p<0.01).2. pEGFP-Hsp20 (S16A)-transfected cells exhibited no significant alteration in OGD/R-induced apoptosis and cellular viability, compared with the control.3. N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D displayed much less damage to the structure of mitochondria. However, pEGFP-Hsp20 (S16A)-transfected cells exhibited no significant difference compared with the control.4. N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D displayed a marked decrease in the expression of Bax and an increase expression of Bcl-2. N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D also inhibited cytochrome c released from mitochondria into cytosol upon OGD/R (p<0.05). However, pEGFP-Hsp20 (S16A)-transfected cells exhibited no significant alteration in OGD/R-induced expression of Bax and Bcl-2 and failed to inhibit OGD-induced cytochrome c release. Conclusions:1. Hsp20 displayed neuroprotective effects against oxygen-glucose deprivation followed by reperfusion.2. Phosphorylation of Ser16 played an important role in the neuroprotective effect of Hsp20. Otherwise, blockade of Hsp20 phosphorylation abrogated its neuroprotective effect.3. Neuroprotective effects of Hsp20 were mediated by recovering mitochondria from the OGD/R damage.4. Neuroprotective effects of Hsp20 on OGD/R were also mediated through inhibition of mitochondrial apoptotic signaling pathways.更多还原