【作者】 王敏；

【导师】 柴颖儒； 肖传实；

【作者基本信息】 山西医科大学 ， 心血管内科， 2012， 硕士

【摘要】 第一部分脂联素受体在正常SD大鼠乳鼠心肌细胞的分布和表达目的：研究脂联素受体1（AdipoR1）和脂联素受体2（AdipoR2）及T-钙黏蛋白（T-cadherin，T-cad），三种脂联素受体在大鼠乳鼠心肌细胞上的分布和表达。方法：1.利用酶消化法、差速贴壁法、化学试剂(5-brdu)抑制法培养原代的乳鼠心肌细胞，采用α-肌动蛋白细胞免疫荧光法对培养的心肌细胞进行鉴定，然后在倒置相差显微镜下观察细胞生长状态、细胞形态和其自发搏动。2.选用原代培养72~96h的单层原代心肌细胞进行实验，采用RT-PCR和细胞免疫组织化学法检测AdipoR1、AdipoR2和T-cadherin的mRNA和蛋白的表达及分布。结果：1.经细胞免疫组织化学法鉴定培养的原代细胞为心肌细胞，且其纯度可达95%以上；2.在SD大鼠乳鼠心肌细胞上均检测到了AdipoR1、AdipoR2、T-cadherin的mRNA和蛋白表达，其中以AdipoR1和T-cadherin的mRNA和蛋白表达量高，与AdipoR2的表达量相比差异显著（P<0.01）。结论：1.脂联素受体1、脂联素受体2和T-钙黏蛋白，三种脂联素受体在SD大鼠乳鼠心肌细胞上均有表达。2.心肌细胞上以脂联素受体1和T-钙黏蛋白的表达为主，脂联素受体2表达量较少。第二部分脂联素对心肌细胞缺氧/复氧损伤后AdipoR1、AdipoR2及T-cadherin表达的影响目的：通过原代培养SD大鼠乳鼠心肌细胞并建立缺氧/复氧（H/R）模型以模拟在体缺血/再灌注，观察不同浓度脂联素（APN）对心肌细胞H/R损伤后脂联素受体1（AdipoR1）和脂联素受体2（AdipoR2）及T-钙黏蛋白（T-cadherin，T-cad）表达的影响，为心脏缺血/再灌注损伤的防治提供相应的理论依据。方法：1.利用酶消化法、差速贴壁法、化学试剂（5-brdu）抑制法培养原代的乳鼠心肌细胞，采过α-肌动蛋白免疫荧光法对培养的原代心肌细胞进行鉴定，在倒置相差显微镜下观察细胞生长状态、细胞形态和其自发搏动。2.随机分组：1）正常对照组、2）单纯H/R组、3）H/R+3 ug/ml脂联素组、4）H/R+10 ug/ml脂联素组、5）H/R+20 ug/ml脂联素组、6）H/R+30 ug/ml脂联素组。3.在倒置相差显微镜下观察原代心肌细胞形态变化，化学比色法测定乳酸脱氢酶（LDH）的释放，流式细胞术、脱氧核糖核苷酸缺口末端标记（TUNEL）法来检测心肌细胞的凋亡。4.用RT-PCR和Western blotting方法检测AdipoR1、AdipoR2、T-cadherin的表达。结果：1.经各项鉴定培养原代细胞为心肌细胞，且纯度可达95%以上。2.与正常对照组相比，H/R组，原代心肌细胞生长状态较差，原代心肌细胞伪足缩短或消失，搏动减弱甚至消失，折光性下降。细胞凋亡率显著增加，TUNEL阳性信号绿色荧光明显增多，上清液中LDH的释放量增加，经过不同浓度APN预处理后再进行H/R，可较大程度的逆转各指标变化。3.与正常对照组相比，单纯H/R后，T-cad在mRNA和蛋白水平表达明显下降，经过不同浓度APN预处理后再进行H/R，可较大程度地逆转各指标变化，并且与单纯H/R组相比均有显著差异（P<0.01），并且呈现一定的浓度依赖性。4.与正常对照组相比，单纯H/R后，AdipoR1在mRNA和蛋白水平表达明显下降，经过不同浓度APN预处理后进行H/R，可逆转各指标变化，与单纯H/R组相比均有显著差异（P<0.01），但各APN处理组不依赖APN浓度变化而变化。5.与正常对照组相比，单纯H/R后，AdipoR2在mRNA和蛋白水平表达稍下降，不同浓度APN预处理后进行H/R，AdipoR2在mRNA和蛋白水平表达无明显变化，与H/R组相比无显著差异（P＞0.01），且不依赖APN浓度变换而变化。结论：1.缺氧/复氧可以诱导心肌细胞损伤；2.脂联素对缺氧/复氧后心肌细胞的损伤具有保护作用。3.脂联素可能通过上调T-cad的表达逆转H/R所致的心肌细胞损伤及凋亡，并且脂联素对心肌细胞的保护作用可能主要通过结合其受体T-cad而起作用。4.脂联素受体1和脂联素受体2可能是脂联素对心肌细胞保护的次要结合受体，并且两独立于APN而存在，不随APN浓度变化而变化，且AdipoR2在脂联素发挥心肌细胞保护作用中可能所起作用较弱。更多还原

【Abstract】 PartⅠDistribution and expression of adiponectin receptors in SD ratcardiomyocytesObjective To investigate the distribution and expression of adiponectin receptors in SD ratcardiomyocytes.Methods1. Primary myocardial cells were obtained from neonatal rats by enzymatic digestionmethod.The molecular markerα-actin of the cardiomyocytes was observed byimmunocytochemistry.The morphology of neonatal rat cardiomyocytes was studied by phasecontramicroscope.2. The cells cultured for 72~96 h were used in the experiments, The expressions of AdipoR1、AdipoR2、T-cadherin were detected by RT-PCR and immunocytochemistry.Results1. Cardiomyocytes were confirmed and the purity was more than 95%.2. The mRNA and proteins of AdipoR1、AdipoR2 and T-cadherin were all observed in SD ratcardiomyocytes, while the expression of AdipoR1 and T-cadherin were much higher than theexpression of AdipoR2 in cardiomyocytes(P<0.01).Conclusions1. AdipoR1、AdipoR2 and T-cadherin were all distributed in SD rat cardiomyocytes.2. AdipoR1、AdipoR2 and T-cadherin were all distributed in SD rat cardiomyocytes, especiallyAdipoR1 and T-cadherin, while AdipoR2 expressed lower.PartⅡEffects of adiponectin on the expressions of AdipoR1、AdipoR2 andT-cadherin in cardiomyocytes injure after hypoxia-reoxygenationObjective To investigate the effects of adiponectin on the expression of AdipoR1、AdipoR2and T-cadherin on cultured SD rat cardiomyocytes by hypoxia-reoxygenation.Methods1. Primary myocardial cells were obtained from neonatal rats by enzymatic digestion method.The molecular markerα-actin of the cardiomyocytes was observed by immunocytochemistry.Themorphology of neonatal rat cardiomyocytes was studied by phase contramicroscope.2. The cells cultured for 96 h were used in the experiments, and they were divided into groupsrandomly: control group, H/R group, H/R+3 ug/ml APN group, H/R+10 ug/ml APN group,H/R+20 ug/ml APN group, H/R+30 ug/ml APN group. 3. The changes of morphology of cardiomyocytes were observed by phase contractedmicroscope.The content of LDH was detected by chemistry chromatometry; The ratescardiomycocytes apotosis was detected by flowcytometry and terminal deoxynucleotidyltransferrase-mediated dUTP nick end labeling (TUNEL).4. The expression of AdipoR1,AdipoR2,T-cadherin were detected by RT-PCR and Westernblotting.Results1. Cardiomyocytes were confirmed and the purity was more than 95%.2. Compared with control group,the rate of apoptosis in group H/R was significantly increased,the TUNEL-positive cells(green) increased significantly,the release of LDH wasincreased,Pretreatment with APN, the above changes were significantly reversed.3. Compared with control group, the expresstion of T-cad mRNA and the protein was decreasedin group H/R. Pretreatment with APN, the above changes were significantly reversed. Comparedwith group H/R,the indexes improved obviously(P<0.01) in a dose-dependent manner.4. Compared with control group, the expresstion of AdipoR1 mRNA and the protein wasdecreased in group H/R. Pretreatment with APN, the above changes were significantly reversed.Compared with group H/R,the indexes improved obviously(P<0.01), but each APN treatmentgroup do not depend on the APN concentration changes.5. Compared with control group, the expresstion of AdipoR2 mRNA and the protein was in alittle down in group H/R. Pretreatment with APN, the levels of AdipoR2 mRNA and protein wasno obvious change(P＞0.01). Compared with group H/R,there was also no obvious change(P＞0.01), and levels were independent of APN concentration changes.Conclusions1. Hypoxia-reoxygenation can induce injury in rat Cardiomyocytes.2. APN can protect cardiomyocytes from the injury induced by hypoxia-reoxygenation .3. APN may be protect cardiomyocytes from hypoxia-reoxygenation injury by upregulating theT-cadherin expression. The protection may be mainly through combined with its receptor T-cad.4. AdipoR1 and AdipoR2 may be the adiponectin secondary receptors to the protection of thecardiomyocytes, and both were independent of APN concentration changes,and AdipoR2protection against damage effect of cardiomyocytes may be relatively weak.更多还原