【作者】 邱原刚；

【导师】 朱建华；

【作者基本信息】 浙江大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 第一部分环磷酰胺保护心脏缺血/再灌注损伤的机制研究目的与背景：炎症在心肌缺血/再灌注损伤的发生发展过程中起着重要的作用,但目前尚不清楚抗炎治疗对心肌缺血/再灌注损伤的疗效。环磷酰胺是一种免疫抑制剂,它具有广泛的抗炎症作用,多年以来被广泛应用于风湿病及其他与自身免疫相关疾病的治疗,包括系统性红斑狼疮、慢性肾脏疾病、以及类风湿性关节炎等。我们推测应用环磷酰胺进行抗炎治疗可能有益于减轻心肌缺血/再灌注损伤。方法：在大鼠原位心脏缺血/再灌注模型上,探讨小剂量环磷酰胺对心脏缺血/再灌的保护作用及可能机制。先缺血30分钟,继以再灌注3,12和24小时。术前于腹腔内注射环磷酰胺(0.75g/m2,干预组)或2ml生理盐水(对照组)。实验分为3组：假手术组,缺血/再灌注损伤组和环磷酰胺干预组。假手术组在术中不结扎冠状动脉,故实验过程中无心脏缺血/再灌注损伤过程。测量血流动力学指标、心肌梗死面积、心肌病理损伤程度、外周血高敏C-反应蛋白水平、肿瘤坏死因子α浓度,并用免疫组化法检测心肌肿瘤坏死因子α水平。结果应用方差分析进行统计学处理。结果：与缺血/再灌注损伤组比较,环磷酰胺干预组明显改善左心室收缩压(LVSP)。在再灌注3小时为88.3±3.8mmHg,但缺血/再灌注损伤组为68.6±3.8mmHg,P<0.01；再灌注12小时为92.0±3.8vs63.7±4.9mmHg, P<0.01;再灌注24小时为90.4±4.0vs64.2±4.9mmHg, P<0.01。环磷酰胺干预组左室舒张末压(LVEDP)及左室压力上升变化最大速率(+dp/dtmax)也有所改善。在再灌注3小时,左室舒张末压在环磷酰胺干预组为14.57±0.94mmHg,在缺血/再灌注损伤组为14.45±0.48mmHg, P>0.05;再灌注12小时为13.00±0.88vs15.13±0.88mmHg, P<0.05;再灌注24小时为13.24±0.79vs15.50±0.81mmHg, P<0.05。在再灌注3小时,+dp/dtmax在环磷酰胺干预组为4675.8±342.4,而在缺血/再灌注损伤组为3477.0±342.4mmHg/s,P<0.05；再灌注12小时为5094.3±342.4vs5182.7±442.0mmHg/s,P>0.05；再灌注24小时为4992.6±300.3vs3608.7±326.5mmHg/s, P<0.05。但环磷酰胺治疗不影响左室压力下降变化最大速率(-dp/dtmax)的数值。应用环磷酰胺后心肌梗死面积也显著减少。在再灌注3小时,在环磷酰胺干预组为26.1±0.4%,而在缺血/再灌注损伤组为40.4±0.4%,P<0.01；再灌注12小时为21.6±0.4%vs49.9±0.4%,P<0.01；再灌注24小时为21.6±0.4%vs40.0±0.4%,P<0.01。心肌病理损伤评分在环磷酰胺干预组也显著降低。在再灌注3小时,在环磷酰胺干预组为1.83±0.14,而在缺血/再灌注损伤组为2.17±0.14,P<0.01；在再灌注12小时为2.33±0.14vs3.17±0.14,P<0.01；再灌注24小时为2.83±0.14vs3.83±0.14,P<0.01。环磷酰胺干预组心肌中性粒细胞计数显著减少,在再灌注3小时,环磷酰胺干预组为6.9±2.7/HPF,而在缺血/再灌注损伤组为20.4±1.5/HPF,P<0.01；再灌注12小时为18.6±2.8vs37.5±3.6/HPF,P<0.01；再灌注24小时为16.8±2.5vs58.8±5.0/HPF,P<0.01。血清高敏C-反应蛋白在环磷酰胺干预组也明显降低。再灌注3小时,环磷酰胺干预组为29.3±0.5,而缺血/再灌注损伤组为32.3±0.5ng/ml, P<0.01;再灌注12小时为29.1±0.5vs31.8±0.5ng/ml, P<0.01;再灌注24小时为28.6±0.5vs31.9±0.5ng/ml, P<0.01。环磷酰胺干预组血清肿瘤坏死因子α(TNF-α)表达也减少。再灌注3小时,环磷酰胺干预组血清肿瘤坏死因子α为13.3±2.6pg/mL,而缺血/再灌注损伤组为14.1±6.0pg/mL, P<0.05;再灌注12小时为10.1±2.7vs12.5±5.0pg/mL,P<0.05;再灌注24小时为10.4±5.6vs13.0±3.6pg/mL, P<0.05。免疫组化检测心肌TNF-a,其染色密度分别为,再灌注3小时,环磷酰胺干预组为2.42±0.38,而缺血/再灌注损伤组为3.33±0.36,P<0.01；再灌注12小时为3.75±0.52vs5.08±0.58,P<0.01；再灌注24小时为6.50±0.45vs8.08±0.80,P<0.01。结论：环磷酰胺对大鼠原位心脏缺血/再灌注损伤有确切的保护作用,同时还带来心肌肿瘤坏死因子α及中性粒细胞浸润的减少,以及血清炎症指标高敏C-反应蛋白及肿瘤坏死因子α明显下降。我们认为环磷酰胺的心肌保护作用至少部分与其抗炎症作用有关。第二部分内质网应激-未折叠蛋白反应在心脏缺血/再灌注损伤时的作用目的与背景：在心肌缺血/再灌注时,存在内质网应激和炎症反应,其中间过程及信号传导通路尚不清楚。其他领域的研究提示,各种相应的刺激引起内质网应激后,经两个参与炎症反应的信号分子NF-κb和JNK介导,进而诱导炎症转录因子及各种炎症因子的表达,启动炎症反应,进而使细胞损伤加重。我们推测在心肌缺血/再灌注导致内质网应激-炎症反应的过程中,NF-κb起着重要的作用方法：在心肌细胞缺氧/复氧模型上,观察心肌缺氧/复氧所引起内质网应激-炎症反应的过程中,NF-Kb的作用。将原代培养的乳鼠心肌细胞,进行缺氧12小时和复氧12小时处理,实验分五组,(1)空白对照组(Control组)；(2)衣霉素组(Tm组,即阳性对照组,含衣霉素1mg/L);(3)缺氧/复氧组(H/R组,缺氧12h,再复氧12h)；(4)衣霉素+SN50组(Tm+SN50组,含衣霉素lmg/L, SN5050μg/ml);(5)缺氧/复氧+SN50组(H/R+SN50组,含SN5050μg/ml)。采用Western blot法测定GRP78蛋白含量,实时荧光定量PCR检测GRP78mRNA表达情况,CCK-8(Cell Counting Kit-8)法检测心肌细胞增殖及活性水平,流式细胞仪测量肿瘤坏死因子α浓度,倒置相差显微镜观察细胞形态及搏动情况,苏木素伊红(HE)染色后观察心肌细胞形态学变化。结果应用方差分析进行统计学处理。结果：心肌缺氧/复氧后,内质网应激标志蛋白GRP78有升高,在使用NF-κb抑制剂SN50组,GRP78下降。以GRP78/GAPDH代表内质网应激蛋白GRP78水平。蛋白印迹实验发现,在空白对照组(Control组)GRP78/GAPDH为0.80±0.02,衣霉素组(Tm组)为1.84±0.11,缺氧/复氧组(H/R组)1.44±0.02,衣霉素+SN50组(Tm+SN50组)为1.12±0.07,缺氧/复氧+SN50组(H/R+SN50组)为1.16±0.12。其余各组与对照组相比,p<0.01；衣霉素+SN50组与衣霉素相比,p<0.05；缺氧/复氧+SN50组与缺氧/复氧组相比,p<0.05。心肌缺氧/复氧后,内质网应激标志蛋白GRP78mRNA升高,在使用NF-κb抑制剂SN50组,GRP78mRNA下降。RT-PCR实验发现,在空白对照组(Control组)内质网应激蛋白GRP78mRNA为0.28±0.06,衣霉素组(Tm组)为12.91±2.08,缺氧/复氧组(H/R组)6.91±0.81,衣霉素+SN50组(Tm+SN50组)为4.66±0.38,缺氧/复氧+SN50组(H/R+SN50组)为1.95±0.86。Tm组最高,H/R组次之,加用SN50后均显著降低。其余各组与对照组相比,p<0.01；衣霉素±SN50组与衣霉素相比,p<0.05；缺氧/复氧+SN50组与缺氧/复氧组相比,p<0.05。心肌缺氧/复氧后,炎症因子TNF-α有升高,在使用NF-κb抑制剂SN50组,TNF-α下降。在空白对照组(Control组)TNF-a为2.10±0.54,衣霉素组(Tm组)为10.50±0.54pg/ml,缺氧/复氧组(H/R组)11.70±1.31pg/ml,缺氧/复氧+SN50组(H/R+SN50组)为8.10±1.12pg/ml。其余各组与对照组相比,p<0.01；缺氧/复氧+SN50组与缺氧/复氧组相比,p<0.05。心肌缺氧/复氧对心肌损害程度的评估及SN50的作用。我们采用二个指标评估心肌损害的程度,即测定CCK-8及心率计数。CCK-8测定及心率计数发现,心肌缺氧/复氧后存在心肌损害,SN50使用后可减轻心肌损害。在空白对照组(Control组),CCK8为1.61±0.03,衣霉素组(Tm组)为0.74±0.08,缺氧/复氧组(H/R组)为0.81±0.06,衣霉素+SN50组(Tm+SN50组)为0.95±0.06,缺氧/复氧+SN50组(H/R+SN50组)为1.21±0.05。其余各组与对照组相比,p<0.01；衣霉素+SN50组与衣霉素相比,p<0.05；缺氧/复氧+SN50组与缺氧/复氧组相比,p<0.01。CCK-8研究发现,心肌缺氧/复氧后存在心肌损害,SN50使用后心肌损害减轻。在空白对照组(Control组)心率(HR,次/分)为125.3±2.5,衣霉素组(Tm组)为32.7±2.5,缺氧/复氧组(H/R组)58.7±5.5,衣霉素+SN50组(Tm+SN50组)为65.7±10.0,缺氧/复氧+SN50组(H/R+SN50组)为97.3±1.2。其余各组与对照组相比,p<0.01；衣霉素+SN50组与衣霉素相比,p<0.01；缺氧/复氧+SN50组与缺氧/复氧组相比,p<0.01。病理研究发现,心肌缺氧/复氧后存在心肌损害,SN50使用后可减轻心肌损害。在空白对照组(Control组)病理损伤评分为3.67±0.58,衣霉素组(Tm组)为1.67±0.58,缺氧/复氧组为(H/R组)1.33±0.58,衣霉素+SN50组(Tm+SN50组)为3.33±0.58,缺氧/复氧+SN50组(H/R+SN50组)为1.66±0.58。其余各组与对照组相比,p<0.01；衣霉素+SN50组与衣霉素相比,p<0.05；缺氧/复氧+SN50组与缺氧/复氧组相比,p<0.01。结论：心肌缺氧/复氧后,产生内质网应激-炎症反应,在此过程中NF-κb起着重要的桥梁作用,我们的这一结果有助于对心肌缺血/再灌注损伤发病机制的理解。更多还原

【Abstract】 Part1:The mechanism of cyclophosphamide protects myocardial ischemia-reperfusion injuryObject:Cyclophosphamide has been used in the treatment of autoimmune disorders, including Systemic Lupus Erythematosus (SLE), Chronic Glomerulonephritis, Rheumatoid Arthritis, et al. There is inflammation in myocardial ischemia-reperfusion injury similar to that in autoimmune disorders. We hypothesized that myocardial ischemia-reperfusion injury could be partially ameliorated with cyclophosphamide treatment.Methods:Open chest rats were submitted to30min of ischemia followed by3h,12h or24h of reperfusion. Rats were divided into sham group, I/R group and cyclophosphamide group, and each group included3time-point subgroups (3h,12h and24h; n=8for each subgroup).750mg/m2cyclophosphamide or saline was intraperitoneally administrated in cyclophosphamide group or I/R group. A polyethylene tube was inserted into the left ventricular cavity to detect left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and±dp/dt max. At the end of experiment, hearts were harvested for histopathological assessment and infarct size determination and blood was collected for hs-CRP and TNF-adetection. Serum TNF-a was measured by cytometric bead array (CBA) and immunohistochemistry was used to detect TNF-a in myocardium.Results:Compared with I/R group, rats treated with low dose cyclophosphamide showed a significant recovery in myocardial function with improved left ventricular systolic pressure (LVSP)(88.3±3.8vs.68.6±3.8mmHg of3h reperfusion;92.0±3.8vs.63.7±4.9mmHg of12h;90.4±4.0vs.64.2±4.9mmHg of24h; P<0.01respectively). The left ventricular end diastolic pressure (LVEDP) and±dp/dt max also had the similar trends.(for LVEDP,14.57±0.94vs14.45±0.48mmHg of3h reperfusion, P>0.05;13.00±0.88vs15.13±0.88mmHg of12h, P<0.05;13.24±0.79vs15.50±0.81mmHg of24h, P<0.05). The myocardial infarct size was reduced after low dose cyclophosphamide treatment compared with I/R group (26.1±0.4%vs40.4±0.4%of3h reperfusion;21.6±0.4%vs49.9±0.4%of12h;21.6±0.4%vs40.0±0.4%of24h, P<0.01respectively.); Histopathological damage score was attenuated (1.83±0.14vs.2.17±0.14of3h reperfusion;2.33±0.14vs.3.17±0.14of12h;2.83±0.14vs.3.83±0.14of24h; P<0.01respectively); Polymorph nuclear leukocytes (PMNs) infiltration in myocardium also decreased after low dose cyclophosphamide treatment (6.9±2.7vs20.4±1.5/HPF of3h reperfusion;18.6±2.8vs37.5±3.6/HPF of12h;16.8±2.5vs58.8±5.0/HPF of24h, P<0.01respectively.).High sensitive C-reactive protein (hs-CRP) was elevated pronouncedly in I/R group at3h,12h and24h after reperfusion. The increase of hs-CRP was prevented after cyclophosphamide administration at three time-points (29.3±0.5vs32.3±0.5ng/ml of3h reperfusion;29.1±0.5vs31.8±0.5ng/ml of12h;28.6±0.5vs31.9±0.5ng/ml of24h, P<0.01respectively). Compared with I/R group, rats treated with cyclophosphamide showed a significant difference with decreased serum TNF-α (13.3±2.6vs14.1±6.0pg/mL at3h reperfusion,10.1±2.7vs12.5±5.0pg/mL at12h,10.4±5.6vs13.0±3.6pg/mL at24h reperfusion, P<0.05respectively). And also the immunostaining was less intense with cyclophosphamide injection at each reperfusion time. The score of the intensity of myocardial TNF-α staining was down regulated with cyclophosphamide injection in each time-point between cyclophosphamide and I/R group (2.42±0.38vs3.33±0.36at3h reperfusion,3.75±0.52vs5.08±0.58at12h reperfusion,6.50±0.45vs8.08±0.8at24h reperfusion, P<0.01respectively).Conclusions:Low dose cyclophosphamide might improve cardiac function and alleviate histopathological damage in myocardial ischemia-reperfusion rats. Cyclophosphamide was a feasible strategy for anti-inflammation to protect myocardial ischemia-reperfusion injury.Part2:The study of endoplasmic reticulum stress-inflammation response of myocardial ischemia/reperfusion injuryObject:Myocardial ischemia/reperfusion activates endoplasmic reticulum stress and inflammatory responses. The stress-response pathways, however, are still unclear. Studies in other areas have suggested that endoplasmic reticulum stress induced by a variety of appropriate stimulations, mediated by two signaling molecule NF-Kb and JNK, cause the expression of inflammatory transcription factors and a variety of inflammatory cytokines, start the inflammatory response, thereby increase cell damage. We speculate that NF-Kb plays an important role in the process of endoplasmic reticulum stress-inflammation responses induced by myocardial ischemia/reperfusion.Methods:Primary cultured neonatal rat cardiomyocytes were exposed to hypoxia for12hours and subsequently reoxygenation for12hours. Five groups were studied,(1) Control group;(2) Tunicamycin group (Tm group, containing tunicamycin lmg/L) was the positive control of this study;(3) hypoxia/reoxygenation group (H/R group,12h hypoxia followed by12h reoxygenation);(4) tunicamycin+SN50group (Tm+SN50group, containing tunicamycin1mg/L and SN5050μg/ml);(5) hypoxia/reoxygenation+SN50group (H/R+SN50group, containing SN5050μg/ml). Western blot and RT-PCR were applied to monitor the expression of GRP78(glucose regulated protein78) and GRP78mRNA. TNF-a was measured by cytometric bead array (CBA). The cell viability of primary cultured neonatal rat cardiomyocytes was explored by CCK-8(Cell Counting Kit-8) assay. Myocardial activity was observed by inverted phase contrast microscope. Hematoxylin-eosin (HE) staining was appiled to observe myocardial cell morphology and pulsation.Results:Compared with the control group, the hypoxia/reoxygenation group significantly increase the expression of GRP78and GRP78mRNA. The concentration of TNF-a is increased significantly, too. CCK-8and HR are decreased. Histopathological damage score shows myocardial injury is more serious. SN50, however, significantly decrease the changes which induced by Hypoxia/reoxygenation. Compared with the H/R group, the H/R+SN50group significantly decrease the expression of GRP78(indicated by GRP78/GAPDH,1.16±0.12vs1.44±0.02) and GRP78mRNA(1.95±0.86vs6.91±0.81), the concentration of TNF-a is decreased (8.10±1.12vs11.70±1.31pg/ml), too. CCK-8(1.21±0.05vs0.81±0.06) and HR (97.3±1.2vs58.7±5.5) is also increased. And histopathological damage score is decreased (1.66±0.58vs1.33±0.58).Conclusions:These findings suggest that hypoxia/reoxygenation can induce endoplasmic reticulum stress and inflammation. And more importently, our results show NF-κb plays an important role in ERS-imfammation pathway.更多还原