【作者】 王裕勤；

【导师】 冯义柏；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 第一部分粉防己碱对缺氧/复氧诱导乳鼠心肌细胞损伤及前炎症因子的影响实验一新生大鼠心肌细胞培养及缺氧/复氧模型建立目的:建立新生大鼠心肌细胞培养及缺氧/复氧模型以及研究粉防己碱对离体培养的大鼠心肌细胞在缺氧/复氧条下对心肌酶:乳酸脱氢酶(LDH)、肌酸激酶(CK)的影响。方法:利用原代培养的SD大鼠乳鼠心肌细胞建立心肌缺氧/复氧(A/R)模型,模拟心肌缺血/再灌注损伤,为研究粉防己碱对大鼠心肌缺血/再灌注损伤影响提供研究平台。心肌细胞的鉴定采用免疫组织化学方法。实验分为正常对照组(CON)和缺氧/复氧组(A/R)。CON组:在培养的心肌细胞中加入适量0.9%盐水,放入培养箱培养26h。A/R组:用氩气饱和后更换使用无糖无血清培养基再灌入高纯度氩气封口,在培养箱中孵育2h,再拆开封口在培养箱中孵育24h。检测心肌细胞在缺氧/复氧这一病理生理过程当中心肌酶LDH、CK的变化。结果:1.新生大鼠心肌细胞体外培养结果:心肌细胞分离后原代培养2小时绝大多数非心肌细胞(主要为成纤维细胞)已经贴壁,而心肌细胞仍然悬浮,呈圆形或椭圆形。新生大鼠心肌细胞在种植后第2天,可在倒置显微镜下见散在的具有搏动能力的心肌细胞,第3-4天可见散在的心肌细胞相互间连接成合胞体。抗a-Sacromeric actin免疫细胞化学细胞染色显示96%以上的细胞呈现强阳性。心肌细胞体外培养成功。2.缺氧导致培养的心肌细胞中LDH、CK轻度增高:对照组(CON)组与A/R组在缺氧前LDH和CK值未见显著性差异(74.67±3.51 VS 73.08±2.08,P>0.05),表明两组具有可比性。A/R组缺氧2h后LDH和CK分别较缺氧前增加3.50倍(260.99±6.73 VS 74.67±3.51)和2.68倍(4.74±0.45 VS 1.77±0.40);而CON组中在培养2h后,与培养前相比LDH和CK均未见明显变化。3.复氧导致培养的心肌细胞中LDH、CK进一步增高:A/R组缺氧2h和复氧24h后,检测培养心肌细胞中LDH、CK,显示LDH、CK在复氧24h分别较缺氧2h时增加1.93倍(503.72±6.26 VS 260.99±6.73)和1.59倍(7.54±0.66 VS 4.74±0.45);而对照组在培养24h后较培养2h无明显变化。实验二粉防己碱对缺氧/复氧乳鼠心肌细胞前炎症因子的影响目的:研究粉防己碱对离体培养的大鼠心肌细胞在缺氧/复氧条下对心肌酶LDH、CK和前炎症因子:肿瘤坏死因子(TNF-α)、白细胞介素-1(IL-1β)、白细胞介素-6(IL-6)的影响。方法:乳鼠心肌细胞体外培养心肌细胞成功后,随机分为四组:CON组、A/R组、粉防己碱组(Tet)、辛伐他汀组(Sim)。CON组:不进行A/R处理,正常情况下连续培养26h;A/R组:在高纯度氩气,无糖无血清培养基条件下培养2h,复氧开始时加入0.9%盐水,再复氧培养24h;Tet组:预先给予终浓度为30μmol/l的Tet孵育60min,再在无血清低糖DMEM培养基缺氧孵育2h,然后继续复氧培养24h;Sim组:预先给予终浓度为10μmol/l的Sim孵育60min,再在无血清低糖DMEM培养基缺氧孵育2h,然后继续复氧培养24h;检测各组在复氧24h后的LDH、CK、TNF-α、IL-1β、IL-6的水平。结果:1.缺氧/复氧24h后A/R组、Tet组、Sim组心肌酶LDH、CK均较CON组显著升高(p<0.01),药物预处理组Tet组和Sim组心肌酶LDH、CK显著低于A/R组(p<0.01)。2.缺氧/复氧24h后A/R组、Tet组、Sim组前炎症因子TNF-α、IL-1β、IL-6均较CON组显著升高(p<0.01),药物预处理组Tet组和Sim组前炎症因子TNF-α、IL-1β、IL-6显著低于A/R组(p<0.01)。3.缺氧/复氧24h后的LDH、CK、TNF-α、IL-1β、IL-6的水平,Tet组和Sim组之间均无显著性差异(p>0.05)。第二部分粉防己碱调控前炎症因子减轻大鼠心肌缺血/再灌注损伤目的:研究粉防己碱通过对心肌缺血/再灌注过程中前炎症因子TNF-α、IL-1β、IL-6的调控减轻心肌缺血/再灌注损伤。方法:80只健康健康雄性SD大鼠随机分为4组:缺血/再灌注损伤组(I/R)、假手术组(Sham)、粉防己碱治疗组(Tet),辛伐他汀治疗组(Sim)。I/R组结扎大鼠冠状动脉左前降支造成心肌缺血30分钟后再灌注24小时后处死大鼠;Sham组只穿针不结扎,余步骤同I/R组; Tet组在缺血前20分钟腹腔注射Tet,辛伐他汀组(Sim)结扎手术前予以辛伐他汀药物2mg/kg连续灌胃14天,余步骤同I/R组。抽血检测血清中肌酸磷酸激酶(CK),乳酸脱氢酶(LDH)。二维超声评价心脏功能:检测左心室舒张和收缩末期内径LVDd、LVDs、左心室短轴缩短率%FS=〔[(LVDd-LVDs)/LVDd〕×100],射血分数(EF)。每个指标连续测量三个心动周期,取其平均值。24小时后取心肌标本,用酶联免疫吸附法(ELISA)检测血清和心肌组织中IL-1β、IL-6、TNF-α表达水平。检测髓过氧化物酶(MPO)了解心肌组织中性粒细胞的浸润程度。伊文氏兰/氯化四唑(EB/TTC)双染色法测量心肌梗塞面积(MIS)。结果:1.各组血清心肌酶水平比较:I/R组、Tet组、Sim组水平显著高于Sham组(p<0.01),Tet组与Sim组与I/R组相比显著降低(p<0.01),两药物处理组之间无显著性差异(p>0.05)。2.各组心肌梗死面积比较:心肌梗死面积、梗死面积/左室面积%、梗死面积/缺血面积%三项指标数据显示:Tet组与Sim组显著低于I/R组,而Tet组与Sim组之间无显著性差异(p>0.05)。3.心肌酶CK、LDH与心梗面积/缺血面积%相关性分析:心肌酶CK、LDH与心梗面积/缺血面积%呈密切正相关(r=0.922,0.975, p<0.01)。4.各组再灌注24h后超声检测心功能比较:以假手术组术前心功能数据为正常公共对照组数据。FS%、EF值在I/R组、Tet组、Sim组均较正常组显著减低(p<0.01)。药物预处理的Tet组与Sim组FS%、EF值显著高于I/R组(p<0.01)。E/A比值在I/R组、Tet组、Sim组均较正常组显著减低(p<0.01)。药物预处理的Tet组与Sim组E/A比值显著高于I/R组(p<0.01)。5.各组缺血心肌局部MPO酶活性比较:I/R组、Tet组、Sim组各组显著高于Sham组(p<0.01),药物预处理的Tet组与Sim组E/A比值显著高于I/R组(p<0.01)。Tet组与Sim组之间无显著性差异(p>0.05)。6.各组MPO活性与组织TNF-α表达水平相关性分析:各组MPO活性与TNF-α呈密切正相关关系(r=0.936,p<0.01)。7.各组大鼠血清及心肌组织局部前炎症因子TNF-α、IL-1β、IL-6表达水平比较:I/R组、Tet组、Sim组各组显著高于Sham组(p<0.01),药物预处理的Tet组与Sim组E/A比值显著高于I/R组(p<0.01)。Tet组与Sim组之间无显著性差异(p>0.05)。8.各组心肌组织局部TNF-α表达水平与IL-1β、IL-6表达水平线性相关分析:相关分析结果显示IL-1β、IL-6表达水平与TNF-α表达水平成密切正相关(r=0.928, 0.948; p<0.01)。第三部分粉防己碱调控前炎症因子减轻大鼠心肌缺血/再灌注损伤的分子机制目的:通过建立大鼠缺血/再灌注模型,观察细胞内前炎症因子TNF-α、IL-1β、IL-6以及相关调控蛋白I-κBα、NF-κB的变化揭示其抗再灌注损伤的细胞分子机制。方法:80只健康雄性SD大鼠随机分为4组:缺血/再灌注损伤组(I/R)、假手术组(Sham)、粉防己碱治疗组(Tet),辛伐他汀治疗组(Sim)。I/R组结扎大鼠冠状动脉左前降支造成心肌缺血30分钟后再灌注24小时随后处死大鼠;Sham组只穿针不结扎,余步骤同I/R组; Tet组在缺血前20分钟腹腔注射Tet,辛伐他汀组(Sim)结扎手术前予以辛伐他汀药物2mg/kg连续灌胃14天。余步骤同I/R组。24小时后取心肌标本,实时RT-PCR(Real Time RT-PCR )检测TNF-α、IL-1β、IL-6 mRNA表达水平。应用免疫组织化学和蛋白电泳(Western-Blot)方法检测磷酸化IκB-α(P-IκB-α)在心肌细胞胞浆中的表达,凝胶电泳迁移率(EMSA)变化检测NF-κB的活性。结果:①实时RT-PCR实验结果显示:手术各组的前炎症因子在经历再灌注后均由显著升高,各组的mRNA转录水平与假手术组比较均显著升高(p<0.01)。粉防己碱组、辛伐他汀组的前炎症因子mRNA拷贝相对值较缺血/再灌注组显著降低(p<0.01)。②免疫组化和蛋白电泳(Western-Blot)实验结果均显示各实验组P-IκBα表达水平与假手术组相比显著增加(p<0.01),粉防己碱组、辛伐他汀预处理组P-IκB-α表达水平显著低于缺血/再灌注组(p<0.01)。③EMSA蛋白凝胶电泳结果:可见缺血/再灌注组心肌细胞核内有核因子NF-κB蛋白表达,表达水平与假手术组、粉防己碱组、辛伐他汀组相比较均显著升高(p<0.01)。而经过药物预处理的粉防己碱组、辛伐他汀组的NF-κB蛋白表达水平在经过再灌注24h后显著低于缺血/再灌注组(p<0.01),并且与假手术组之间无显著性差异(p>0.05)。两药物预处理组之间的NF-κB蛋白表达水平也无显著性差异(p>0.05)。结论:1.体外分离、培养乳鼠心肌细胞,通过对培养心肌细胞施行缺氧/复氧可成功建立模拟心肌细胞缺血/再灌注损伤模型。2.缺血/再灌注可造成心肌细胞损伤;3.缺血/再灌注后心肌细胞发生炎症反应和前炎症因子引发的细胞因子级联反应;4.前炎症因子参与了心肌缺血/再灌注损伤;5.粉防己碱可减少缺血/再灌注损伤后心肌酶的释放,保护膜结构;减少局部中性粒细胞浸润;改善心功能;6.粉防己碱可以减少前炎症因子的生成;7.粉防己碱可以减少前炎症因子mRNA的生成;8.粉防己碱抑制缺血/再灌注局部心肌细胞胞浆内I-κBα的磷酸化;9.粉防己碱抑制缺血/再灌注局部心肌细胞胞浆内NF-κB的活化转位进入细胞核;10.粉防己碱减轻缺血/再灌注损伤,抑制炎症反应以及细胞因子级联反应,改善心功能,部分机制与其抑制前炎症因子生成,阻断再灌注损伤关键始动因素,打断恶性循环有关,另外与其抑制中心粒细胞浸润、抑制I-κBα的磷酸化、以及其钙通道阻滞的多靶点作用相关。更多还原

【Abstract】 PARTⅠThe effect of tetrandrine on anoxia/reoxygenation-induced injury and the release of proinflammatory factors in cultured cardiomyocyte of neonate ratsExperimentⅠCulturing Neonate Rat Cardiocyte and Establishing Anoxia/Reoxygenation ModelObjective:To establish an anoxia/reoxygenation model of cultured cardiocyte using neonate rat and investigate the level of myocardial enzyme LDH、CK under anoxia/reoxygenation conditions.Methods:Using primary cultured SD neonate rat’s myocardial cell establishes an anoxia/ reoxygenation model to simulate myocardial ischemia/reperfusion injury. Take this model as a platform to investigate the effect of tatrandrine on rat’s myocardial ischemia/ reperfusion injury. Cardiomyocyte was identified by using immunohistochemistry method. Cultured cardiocytes were divided into 2 groups: control group (CON) and anoxia/ reoxygenation group (A/R). Each group was treated as follow: CON group-0.9% saline was added into culture fluid, and cardiocytes were cultured in normal circumstance 26h. A/R group-O2 and CO2 in the culture plate which carried cultured cardiocytes were blowed out by argon gas, and nutritive medium was replaced by non- saccharide non- serum culture medium, followed 95% argon gases were infused. The culture plated was sealed and cultured in incubator for 2h under anoxia, after that the seal was opened it was incubated in incubator for the next 24h reoxygenation. Lactate dehydrogenase (LDH) and creatine kinase (CK) were detected and compared during the process of cardiocyte anoxia/reoxygenation.Result:1. The result of cutured neonate rat in vitro: myocardial cell was separated and primary cultured 2h, most non-cardiocytes (mainly mechanocyte) were adherence and cardiocytes still floating in cultured flui. The shape of cultured cardiocyte is round or ellipse. At the day after cardiocyte was implanted 2d, some cardiocytes possessing beating ability were seen under inverted microscope. At the day after cardiocyte was implanted 3-4d, some poradic cardiocytes were connected each other to symplasm. These cultured cardioctes were stained by using antiα-Sacromeric immunohistochemistry method; more over 96% cardiocytes were present strong positive result. This result identified this model was established successful. 2. Anoxia lead LDH and CK increased lightly: there were no significant difference between CON group and A/R group before anoxia beginning (74.67±3.51 VS 73.08±2.08, p>0.05).This phenomenon indicates the two groups have comparability. The numerus of LDH and CK in A/R group were increased 3.5 (260.99±6.73 VS 74.67±3.51, p<0.01) and 2.68 (4.74±0.45 VS 1.77±0.40, p<0.01) times respectively after experienced 2h anoxia culture compared with their numerus before anoxia;There was no significant difference on the numerus of LDH and CK in CON group before and after cultured 2h (72.24±2.25 VS 73.08±2.08, p>0.05) (1.78±0.28 VS 1.73±0.12, p>0.05). 3. Reoxygenation lead to the LDH and CK in cultured cardiocyte increased higher: there were obviously increased on LDH and CK compared between anoxia 2h and reoxygenation 24h in A/R group, they were elevated 1.93 (503.72±6.26 VS 260.99±6.73, p<0.01) and 1.59 (7.54±0.66 VS 4.74±0.45, p<0.01) times respectively; but there were no significant difference found in CON group (74.32±2.78 VS 72.24±2.25, p>0.05) (1.93±0.21 VS 1.78±0.28, p>0.05). ExperimentⅡEffect of tetrandrine on anoxia/reoxygenation-induced release of proinflammatory factors in cultured cardiocyte of neonate ratsObjective:To investigate the effect of tetrandrine on anoxia/reoxygenation-induced the release of myocardial enzyme LDH, CK and proinflammatory factors: TNF-α, IL-1β, IL-6 in cultured cardiocytes of neonate rates.Mthods:After cardiocytes were cultured in vitro successfully, it were divided into 4 group: control group (CON), anoxia/reoxygenation group (A/R), tetrandrine group (Tet), simvastatin (Sim) in random. Each group was treated as follow: CON group-not treated anoxia/reoxygenation, continuous incubated 26h under normal circumstance. A/R group- first anoxia incubate carried, cells were incubated on the non- saccharide non- serum culture medium, which saturate by 95% argon gases 2h, reoxygenation incubate followed, cells were incubated in normal circumstance 24h.0.9% saline were added into culture fluid before the beginning of reoxygenation. Tet group and Sim group–the procedure of anoxia/reoxygenation was same to A/R group, the difference of these two groups was they added Tet (30μmol/L) or Sim (10μmol/L) respectively into culture fluid and incubated 60min before anoxia beginning. LDH, CK, TNF-α, IL-1β, IL-6 were detected after reoxygenation 24h.Conclusion:The LDH and CK were increased significantly in A/R, Tet, and Sim groups compared with CON group (p<0.01). The LDH and CK in Tet and Sim group were lower significant than A/R group (p<0.01). 2. The proinflammatory factors TNF-α, IL-1βand IL-6 were increased significantly in A/R, Tet, and Sim groups compared with CON group (p<0.01). And it were lower significant than A/R group (p<0.01). 3. The level of LDH, CK, TNF-α, IL-1β, IL-6 were no significant difference between Tet group and Sim group (p>0.05). PARTⅡTetrandrine regulate proinflammatory factors to attenuate rat myocardial ischemia/reperfusion injuryObjective:To investigate how tetrandrine through regulate the pro-inflammation factors TNF-α、IL-1β、IL-6 to attenuate rat ischemic/reperfusion injury.Methods:80 male Sprague-Dawley(SD) rats were randomly divided into 4 group: Sham group, ischemia/reperfusion (I/R) group, Tetrandrine group (Tet) and simvastatin group (Sim).The SD rat underwent 30 min of left anterior descending (LAD) coronary occlusion and 24h reperfusion to make ischemia/reperfusion (I/R) injury model in vivo. Sham group were not subjected to occlusion of artery. Tet group were injected tetrandrine to abdominal cavity 20min before ischemia starting. The rat in Sim group was administrated simvastatin 2mgkg/L intragastricly every day, administrating drugs lasted 14 days. The other procedures were same to the I/R group. Samples were collected after 24h reperfusion. The expression level of TNF-α、IL-1β、IL-6 protein in serum and myocardial tissue was detected by ELISA. LDH and CK were detected too. The neutrophil infiltration degree in myocardium was determined by using measuring the activity of myeloperoxidase (MPO) method. Cardiac function which includes FS%, EF and E/A was measured by using ultrasound. EB/TTC (Azovan Blue/2,3,5-Tripheny-2H-Tetrazoliam Chloride) dyeing method was used to measure the infraction size.Result:1. The effect of Tet on the release of myocardial enzyme during ischema/reperfusion: the LDH and CK were significantly higher in I/R, Tet and Sim groups compared with Sham group (p<0.01), but it were much lower in Tet and Sim groups compared with I/R group. 2. The effect of Tet on the cardiac function and infraction size: the cardiac function of systolic and dilator in experimental group was decreased significantly compared with normal heart’s function. In Tet and Sim group, which was experienced pharmacological preconditioning their cardiac function were significant higher than I./Rgroup(p<0.01),but no significant difference between Tet and Sim on EF and E/A. intense There were intense liner correlation between myocardium(CK,LDH) and tumor necrosis factor alpha(TNF-α) (r=0.922,0.975, p<0.01). 3.The effect of Tet on the neutrophil infiltration: the activity of MPO was significantly increased after reperfusion, its activity in experimental groups were much higher than Sham group(p<0.01), notwithstanding its activity in Tet nad Sim groups were significantly lower than I/R group(p<0.01). No significant difference was found between Tet and Sim group. There were intense liner correlation between MPO and TNF-α(r=0.936, p<0.01) 4. The effect of Tet on the release of proinflammatory factors(TNF-α、IL-1β、IL-6): The similar results were obtained after measuring the serum and tissue concentration of TNF-α、IL-1βand IL-6,that was in Tet and Sim group the expression of proinflammatory factors(TNF-α、IL-1β、IL-6) were significant lower compared with I/R group(p<0.01) and significant higher than shame group(P<0.01). The expression level of IL-1βand IL-6 has intense liner correlation with TNF-α(r=0.928, 0.948, p<0.01).PARTⅢThe molecule mechanism of tetrandrine attenuate myocardial ischemia/reperfusion injuryObjective:Observing the mRNA change of proinflammatory factors(TNF-α,IL-1β,IL-6) and modulating protein I-κBα,NF-κB during ischemia/reperfusion to reveal the molecular mechanism of tetrandrine attenuating myocardial ischemia/reperfusion injury.Methods:80 male SD rats were randomly divided into 4 groups: Sham group, ischemia/reperfusion (I/R) group, Tet group and Sim group. The SD rats underwent 30 min of left anterior descending (LAD) coronary occlusion and 24 hours reperfusion to make ischemia/ reperfusion (I/R) injury model in vivo. Sham group were not subjected to occlusion of artery. Tet group were injected tetrandrine to abdominal cavity 20 minute before ischemia starting. The rat in Sim group was administrated simvastatin 2mg/kg intragastricly every day, administrating drugs lasted 14 days. The other procedures were same to the I/R group. Samples were collected after 24h reperfusion. TNF-αmRNA, IL-1βmRNA, IL-6mRNA was detected by real time RT-PCR. Western-Blot and immunohistochemistry were used to detect the expression of phosphorylation I-κBα(P- I-κBα) in the kytoplasm of cardiocyte. Electrophoretic mobility shift assay (EMSA) was used to detect the expression NF-κB in the nuclear of cardiocyte.Recult:1. The result of real time of RT-PCR: after reperfusion the mRNA expression of proinflammatory factors increased significantly in experimental groups (p<0.01). The copies of mRNA in Tet and Sim group was decreased significantly compared with I/R group (p<0.01). 2. The result of Western-Blot and immunohistochemistry were proved that the expression of P- I-κBαincreased significantly in experimental groups (p<0.01) and the expression of P- I-κBαin Tet and Sim group was much lower than I/R group (p<0.01).3. There was the expression of NF-κB in I/R group and it was much higher compared with Sham, Tet, Sim group (p<0.01). Comparing the expression of NF-κB among Sham, Tet, Sim groups, there was no significant difference found (p>0.05). 3. The expression of P- I-κBαin cytoplasm had intense liner correlation with NF-κB(r=0.751, p<0.01).Conclusion:1.Neonate rat cardiocytes were separated and cultured in vitro, through the procduce of anoxia/reoxygenation establishing myocardial cell ischemia/reperfusion injury model.2. Ischemia/reperfusion can cause to cardiocyte damage.3. Inflammation raction of cardiocyte and cascade of cytokine induced by proinflammatory factors were emerge after ischemia/reperfusion.4. Proinflammatory factors participate ischemia/reperfusion injury.5. Tetrandrine can decrease the release of myocardial enzyme induced by ischemia/ reperfusion injuryr, diminish the infiliation of neutrophil; improve cardiac founction.6. Tetrandrine could rduce the production of proinflammatory factors.7. Tetrandrine can rduce the mRNA production of proinflammatory factors.8. Tetrandrine inhibite the phosphorylation of I-κBαin cytoplasm at local myocardium.9. Tetrandrine inhibite the activation of NF-κB, and inhibite its translocation into nuclear.10. The mechanism of tetrandrine inhibiting inflammation reaction, inturpting the cascade of cytokine, improving cardiac founction part releated to the founction of inhibiting the priduction of proinflammatory factors and blocking the key point of initiaiting agents inturping the vicious circule part releated to its multitarget founction such as: reducing the infilation of leukocytes, inhibiting the phosphorylation, blocking calcium channel.更多还原