【作者】 张新民；

【导师】 戴翠莲；

【作者基本信息】 遵义医学院 ， 内科学， 2010， 硕士

【摘要】 1.目的和意义：泛素-蛋白酶体系统(Ubiquitin Proteasome System, UPS)是细胞内蛋白质选择性降解的主要途径,参与了细胞内80%以上的蛋白质更新。热休克蛋白70(Heat Shock Protein 70, HSP70)为细胞内源性保护蛋白,是热休克蛋白系统的重要成员,在防止应激引起的细胞损害和修复损伤方面起着重要作用。近期发现,热休克蛋白70羧基端相互作用蛋白(Carboxyl terminus of the Hsc70-interacting protein, CHIP)即是热休克蛋白的辅助分子伴侣又是一个E3泛素连接酶,作为热休克蛋白与泛素-蛋白酶体系统之间的联接蛋白,在应激条件下发挥关键的双分子开关作用。本研究以大鼠心肌缺血再灌注模型作为研究对象,分别观察再灌注前、再灌注2h、24h、7d的CHIP及HSP70的表达及7d的心功能情况,然后通过使用蛋白酶体抑制剂MG-132进行干预,在相应时间点观察上述指标变化,旨在探讨CHIP及HSP70对缺血再灌注心肌的影响。2.研究方法：结扎Sprague-Dawley大鼠左冠状动脉前降支(Left Anterior Descending, LAD)形成心肌缺血,30min后松开LAD达到再灌注,制作心肌缺血／再灌注(I／R)模型,54只大鼠分为以下三大组7个亚组：(1)缺血／再灌注对照(I／R)组：结扎大鼠LAD 30min后再灌注,于再灌注前5min股静脉注入3ml生理盐水,并根据取材时间点的不同,将该组分为I／R2h、I／R 24h、I／R 7d三个亚组,每个亚组8只大鼠。(2)缺血／再灌注+MG-132处理(I／R+T)组：缺血和再灌注时间以及分组情况完全同I／R组,于再灌注前5min股静脉注入0.75mg／kg(溶于3ml生理盐水中)的MG-132,每个亚组8只大鼠。(3)假手术(Sham)组：手术过程同I／R组,但只穿线不结扎LAD,穿线后25min股静脉注入3ml生理盐水,以穿线后开始观察2h；假手术组6只大鼠。连续监测每只大鼠手术前、结扎LAD即刻及再灌注2h的心电图,进行心律失常评分；计算各组存活率；相应时间点检测血流动力学各项指标,酶免法检测血清脑钠肽水平；电镜观察心肌超微结构变化；荧光定量PCR法检测CHIP、HSP70 mRNA表达,免疫组化法及蛋白质印迹法检测CHIP、HSP70的蛋白质水平。3.结果：(1)大鼠生存及死亡分析：8只大鼠于实验中死亡。Sham组大鼠均全部存活；I／R组存活19只,存活率为79%；I／R+T组存活21只,存活率为88%,I／R+T组较I／R组大鼠生存率显著增加(p<0.05),心律失常是死亡的主要原因。与I／R组相比,IR+T组首次发生心律失常的时间显著延迟(分别为51±22 s vs 129±43 s,p<0.01),室速、室颤的发生率降低(室速67% vs 40%,室颤19% vs 6%),持续时间缩短(室速27.1±10.2 s vs 15.8±8.1 s,p<0.05；室颤10.8±5.5 s vs 7.1±2.8 s,p<0.05),心律失常得分明显下降(分别为3.6±0.7分vs 2.4±0.4分,p<0.05)。(2)HE染色：光镜下,假手术组的心脏三层结构清晰,心肌纤维排列整齐。I／R 24h亚组心肌肌浆红染,梗死周围炎症反应明显,与其相比,I／R+T 24h亚组心肌肌浆红染、炎症反应减轻。I／R 7d亚组见大片心肌组织坏死溶解,被增生的肉芽组织所替代,其间可见散在分布呈岛状或细带状的存活心肌组织,横纹消失,肌丝断裂溶解,核固缩或溶解。I／R+T 7d亚组坏死心肌明显减少。(3)透射电镜：I／R 24h组电镜下可见肌丝排列紊乱、溶解或消失,线粒体肿胀,局部肌丝与闰盘分离,肌纤维明暗带模糊。I／R 7d组心肌细胞线粒体普遍损伤严重,嵴不清楚,细胞核浓缩,多切迹凹陷,染色质分布及排列异常。相应时间点的I／R+T组较I／R组超微结构损伤均有改善。(4)血流动力学变化：与Sham组比较,I／R 7d组大鼠心率增快,左室舒张末压升高,左室收缩压及左室最大收缩速率降低(p<0.05)。I／R 7d组和I／R+T 7d组左室收缩压分别为108±11mmHg和126±10mmHg,左室最大收缩速率分别为4660±480 mmHg/s和5355±554mmHg/s,与I／R组相比,I／R+T组的上述指标均有明显升高(p<0.05)；I／R 7d组和I／R+T 7d组左室舒张末压分别为19±4mmHg和15±3mmHg, I/R+T组有减低趋势。(5)血清脑钠肽(BNP)水平：与I／R 24h组相比,I／R+T 24h组的BNP水平有下降趋势(分别为528±116 pg/ml vs 452±88 pg/ml,p=0.07); I/R+T 7d组的BNP水平较I／R 7d组下降41%(分别为1486±142 pg/ml vs 883±104 pg/ml,p<0.01)。(6)心肌HSP70及CHIP mRNA水平：心肌HSP70 mRNA水平：与Sham组相比,I／R组和I／R+T组各亚组的HSP70mRNA表达显著升高(p<0.01)；I／R+T 2h组较I／R 2h组升高56%(分别为0.87±0.10 vs 0.50±0.06,p<0.01)；I／R+T 24h组、7d组比I／R相应亚组分别增加45%和48%,p值均小于0.01(I／R 24h组及I／R+T 24h组分别为0.33±0.04 vs0.57±0.07；I／R 7d组及I／R+T 7d组分别为0.19±0.03 vs 0.47±0.06)；I／R组与I／R+T组组内比较,均以2h亚组表达最多。心肌CHIP mRNA水平：I／R 2h组CHIP mRNA相对表达量为0.57±0.07,I／R+T2h组是其2.02倍(1.15±0.12),两组比较差异有显著性(p<O.01)；I／R 24h组和I／R+T 24h组CHIP mRNA水平分别为0.44±0.06和0.81±0.09,二者相比差异有显著性(p<0.01)；与I／R 7d组相比,I／R+T 7d组升高19%(分别为0.42±0.04 vs0.50±0.07)；I／R与I／R+T各组内比较,均以2h组表达最多。HSP70和CHIP mRNA相关性分析：以各只大鼠心肌组织的HSP70 mRNA表达量为因变量,CHIP mRNA表达量为自变量,采用直线相关分析法进行分析,结果显示：各组大鼠心肌组织中的HSP70 mRNA表达量与心肌组织中的CHIP mRNA表达量呈显著正相关(p<0.01)。所得的直线方程为Y=0.98X-0.12(R2=0.94)。(7)免疫组化检测蛋白质表达：I／R各组的HSP70积分光密度值与Sham组(96±10)相比有显著增加(p<0.01),其中I／R 2h组最高,为336±26,I／R 24h和7d组分别为266±14和228±13；I／R+T 2h、24h和7d组的HSP70分别为482±37、479±42和393±24,较I／R相应组增高44%、54%和72%,差异均有显著性(p<0.01)。Sham组CHIP积分光密度值为190+12,与其相比,I／R 2h组CHIP水平(278±13)明显升高(p<0.05),而I／R 24h和7d组较Sham组有所升高,但差异不显著(p>0.05)。MG-132处理增加了CHIP表达,I／R+T 2h、24h和7d组分别较I／R组升高30%、32%、19%(p<0.05)。(8) western-blot检测HSP70及CHIP蛋白质水平：以β-actin作为内对照,HSP70蛋白Sham组为0.21±0.02,I／R各组HSP70表达量均显著高于Sham组,其中I／R 2h组表达量为0.79±0.08,是Sham组的3.76倍；I／R 24h组和I／R 7d组表达量分别是Sham组的2.76倍和2.38倍(p<0.01)。MG-132显著刺激HSP70的表达,I／R+T 2h组、24h组和7d组的HSP70分别为1.28±0.16、0.88±0.12和0.99±0.17,较I／R相应各组升高了62%、52%、98%,差异均有显著性(p<0.01)。Sham组CHIP蛋白为0.68±0.13,I／R 2h、24h组分别为1.04±0.11和0.96±0.13,较Sham组升高53%和41%(p<0.01),I／R 7d组为0.80±0.10,仍较Sham组高。与I／R组相比,I／R+T 2h组的CHIP水平较I／R 2h组升高38%(p<0.01),I／R+T 24h组较I／R 24h组增加21%(p<0.05),I／R+T 7d组与I／R 7d组比较有升高趋势(10%)。HSP70与CHIP蛋白质表达相关性分析：以各只大鼠心肌组织HSP70蛋白水平为因变量,CHIP蛋白为自变量,采用直线相关分析法进行分析,结果显示：各组大鼠心肌组织中的HSP70与CHIP蛋白质水平呈显著正相关(p<0.05)。所得的直线方程为Y=1.22X-0.52(R2=0.89)。4.结论：(1)蛋白酶体抑制剂MG-132能够减少大鼠缺血再灌注早期心肌炎症细胞浸润、改善其超微结构变化,改善心功能,抑制脑钠肽水平,显著减少心肌缺血再灌注后室性心律失常的发生,降低死亡率,具有心肌保护作用。(2)蛋白酶体抑制剂MG-132通过诱导缺血再灌注大鼠心肌组织CHIP表达,上调心肌HSP70水平,从而促进损伤蛋白质的修复。更多还原

【Abstract】 Objective:Ubiquitin proteasome system (UPS), which is existed in all eukaryotic cells, is playing a central role in intracellular protein degradation, by which more than 80% intracellular proteins are degraded. It’s well known that heat shock protein 70 (HSP70), a endogenous protective protein, can prevent’ cells from stress-induced injury and also rescure many damaged proteins. Recent studies show that carboxyl terminus of the Hsc70-interacting protein (CHIP) is both a co-chaperone of HSPs and an E3 ubiquitin ligase. As a bridge protein between heat shock proteins and ubiquitin-proteasome system, CHIP acts as a double-switch in stress circumstances. The aim of this study is to investigate the changes of left ventricular structure, function, and the expression of CHIP and HSP70 in different phase of myocardial ischemia/reperfusion rats, and then explore whether proteasome inhibitor MG-132 could improve them.Methods:54 Adult Sprague-Dawley rats were randomly allocated into 3 groups:left anterior descending (LAD) coronary artery ligation for 30 minutes with subsequent 2 hours,24 hours and 7 days reperfusion (I/R groups), LAD ligation for 30 minutes with subsequent 2 hours,24 hours and 7 days reperfusion with treatment by MG-132 (I/R+T groups), and Sham group. After LAD was ligated for 25 min,5 min before reperfusion, MG-132 (0.75mg/kg) was given by vein injection in I/R+T groups, and other groups were given by the same capacity of sterile saline.Electrocardiogram was monitored continuously 2 hours after reperfusion. The incidence of malignant arrhythmia, hemodynamic parameters, the structure of myocardial tissue and the level of B-type natriuretic peptide were measured. Realtime-PCR was used to test the mRNA expression levels of Hsp70 and CHIP, and the protein levels of Hsp70 and CHIP were assayed by both immunohistochemistry and western-blot in myocardium.Results:(1) Survival ratial:8 rats died in this experiment including 5 rats of I/R group and 3 rats of I/R+T group due to several reasons and the predominant reason is malignant arrhythmias. The survival ratial is significant increased in I/R+T group compared with I/R group (88% vs 79%,p<0.05). Compared with I/R group, the initiate of ventricular arrhythmia was delayed (358±186s vs 102±78s,p<0.01), The duration and incidence of ventricular tachycardia and ventricular fibrillation, as well as arrhythmia scores in I/R+T group were pronounced decreased (p<0.05).(2) Myocardial histology:Myocardial tissue organization was well-aligned in Sham group. Inflammation reaction around the infarcted myocardium in I/R 24h subgroup can be found, and myocardium necrosis, fracture, disrupt and hyperplasia could be discovered in I/R 7d subgroup. Compared with these changes, MG-132 significant improved the myocardial structure in both I/R+T 24h subgroup and I/R+T 7d subgroup.(3) Myocardial ultramicrostructure:Transmission electron microscope of LV found that there were myofilament dissolution, mitochondrial swelling and vague light-shade zone fiber in I/R 24h subgroup, and acute injured mitochondria, condensed cell nucleus with multi-notch introcession, and abnormal arrayed chromatin in I/R 7d group. But administration of MG-132 could improve myocardial ultramicrostructure injury.(4) Measurement of haemodynamics:Compared with I/R 7d group, both the left ventricular systolic pressure (LVSP) and the maximum rate of left ventricular pressure (+dp/dt) were significantly increased in I/R+T group (126.45±13.41 vs 108.64±15.61, p<0.05 and 5355±754 vs 4860±680,p<0.05, respectively), and the left ventricular end diastolic presssure (LVEDP) in I/R 7d group as well as in I/R+T 7d group was increased, but when treated by MG-132 on rats, the level of LVEDP decreased.(5) The serum brain natriuretic peptide (BNP) level:The serum BNP levels of I/R and IR+T groups were significantly higher than Sham group (p<0.01). The level of serum BNP subgroup was more depressed in I/R+T 24h group (452±88 pg/ml) than in I/R 24 group (528±116 pg/ml). Compared with I/R 7d group, administration of MG-132 could further attenuate it in I/R+T 7d subgroup (1486±142 pg/ml vs 883±104 pg/ml,p<0.01)(6) Real-time PCR of CHIP and HSP70 mRNA:The level of myocardia HSP70 mRNA in I/R group and I/R+T group was obviously higher than Sham group (p<0.01). Compared to corresponding I/R subgroups, the level of HSP70 mRNA in I/R+T 2h, I/R+T 24h and I/R+T 7d subgroup had been risen 56%(0.87±0.10 vs 0.50±0.06, p<0.01),45%(0.33±0.04 vs 0.57±0.07,p<0.01) and 48%(0.19±0.03 vs 0.47±0.06, p<0.01) respectively, and the HSP70 level in 2h subgroup was the highest compared within each intragroup.The levels of myocardia CHIP mRNA in I/R and I/R+T groups were also significantly higher than Sham group (0.57±0.07 vs 1.15±0.12, p<0.01). The level of I/R+T 2h subgroup was 2 fold more than I/R 2h subgroup (p<0.01), I/R+T 24h subgroup was 84% higher than I/R 24h subgroup (0.44±0.06 vs 0.81±0.09,p<0.01), and I/R+T 7d subgroup was 19% higher than I/R 24h subgroup (0.42±0.04 vs 0.50±0.07,p=0.08). Similarly, it was the highest level in 2h subgroup compared within each intragroup.The correlation analysis between HSP70 and CHIP mRNA levels:We defined HSP70 mRNA level as the dependent variable and CHIP level as the independent variable. The result indicated that the mRNA expression between CHIP and HSP70 was positive correlation, and the linear equation was Y= 0.98X-0.12 (R2=0.94).(7) CHIP and HSP70 proteins expression tested by immunohistochemistry:The IOD level of Hsp70 protein in Sham group was 96±10. And it was 336±26,266±14 and 228±13 in I/R 2h,24h and 7d subgroup respectively, and they were higher than Sham group (p<0.01). Administration of MG-132 could induce the expression of HSP70 in I/R+T 2h,24h and 7d subgroup. Compared with each I/R subgroup, the HSP70 protein levels were increased 44%,54% and 72% than in the corresponding I/R+T subgroup. The integrated optical density (IOD) level of CHIP protein in Sham group was 190±12, compared with it, IOD in I/R 2h subgroup was significantly increased (278±13,p<0.01). They were also higher in I/R 24h subgroup and 7d subgroup than Sham group. MG-132 had stimulated the CHIP protein expression, and they were 30%, 32% and 19% higher than each I/R+T subgroup compared with I/R 2h,24h and 7d subgroups.(8) CHIP and HSP70 proteins expression assayed by western-bolt:The western-bolt method was also used to measure the myocardial expression levels of HSP70 and CHIP protein.HSP70 protein was low level in Sham group (0.21±0.02). It was significantly increased in I/R 2h,24h and 7d subgroups (0.79±0.08,0.58±0.12 and 0.50±0.09 respectively) compared with Sham group (p<0.01). Treated by MG-132, the HSP70 protein levels of I/R+T 2h,24h and 7d subgroups were 62%,52% and 98% higher than I/R 2h,24h and 7d subgroups (p<0.01).The CHIP protein level of Sham group was 0.68±0.13. Compared with Sham group, the levels of CHIP protein were raised 53% and 41% in I/R 2h and 24h subgroups (p<0.01), and 17% in I/R 7d subgroup (p=0.06). MG-132 could markedly increased the expression of CHIP protein in I/R+T 2h and 24h subgroups by 38% and 21% compared with their control subgroups (p<0.01), and raised I/R+T 7d subgroup by 10% in contrast to I/R 7d subgroup.We also analyze the correlation of HSP70 and CHIP protein levels and show that the linear equation is Y=1.22X-0.52 (R2=0.89), which demonstrated that they have a very strong relationship.Conclusion:(1) Administration of proteasome inhibitor MG-132 could attenuate myocardial inflammatory reaction and improve the myocardial structure and function in myocardial ischemia/reperfusion rats. It also decreased the BNP level as well as the appearance of ventricular arrhythmias. These findings demonstrate that MG-132 plays a very important role in cardioprotective effect in ischemia/reperfusion injured rats. (2) Proteasome inhibitor MG-132 could stimulate the expression of CHIP in myocardial ischemia/reperfusion rats. Consequently, the HSP70 level could be up-regulated by CHIP, which facilitated the damaged proteins to be repaired.更多还原