缺氧/氧再灌注诱导心肌细胞衰老的机制及其干预研究

【作者】 张凤祥；

【导师】 曹克将；

【作者基本信息】 南京医科大学 ， 内科学， 2007， 博士

【摘要】 研究背景衰老是细胞脱离细胞周期并不可逆地丧失增殖能力后进入一种相对稳定的状态。自Hayflick等首次从人类纤维原细胞发现衰老现象后，人们逐渐发现人类其它各类细胞和其它物种的纤维细胞也存在着衰老。细胞发生衰老以后主要表现在细胞周期的改变、β-半乳糖苷酶活性的变化、线立体的皱缩与衰老基因的表达增加等。细胞衰老可以促进生物衰老，不仅与肿瘤发生有关，而且与心力衰竭、动脉粥样硬化、心律失常等心血管疾病发生有着密切的关系。因此，深入研究衰老的机制有着重要意义。缺氧／氧再灌注是细胞、组织与器官最常见的损伤因素，也是诱导细胞发生衰老的常见方法。最近国外有研究显示缺氧与氧再灌可以使骨髓造血细胞发生衰老。他汀类药物通过抑制3-羟基-3-甲基戊二酰辅酶的活性而抑制胆固醇的合成，常用来治疗高胆固醇血症，他汀类药物还可降低冠心病患者的死亡率、提高心脏移植患者的生存率、改善心肌重构等作用。除了调节胆固醇代谢外，最近Assmus等报道他汀类药物可通过上调细胞周期蛋白，下调细胞周期抑制因子p27^Kipl，抑制内皮祖细胞的衰老。Pravastatin除了心血管保护作用给患者带来的益处外，是否同时具有抑制缺氧／氧再灌注诱导心肌细胞衰老，对心脏产生保护作用呢?在本研究中，我们将了解缺氧／氧再灌注是否能诱导培养SD乳大鼠心肌细胞发生衰老；研究缺氧／氧再灌注诱导培养SD乳大鼠心肌细胞发生衰老的机制；并拟采用pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞的衰老进行干预，观察细胞周期与β-半乳糖苷酶活性等经典的细胞衰老指标的变化，以探讨pravastatin是否对心肌细胞衰老有抑制作用及可能机制。第一部分SD乳大鼠心肌细胞的培养、鉴定与缺氧／氧再灌注诱导建立SD乳大鼠心肌细胞衰老模型目的在成功分离、培养SD乳大鼠心肌细胞的基础上，探讨获得较纯心肌细胞的方法以及采用缺氧／氧再灌注诱导建立SD乳大鼠心肌细胞衰老模型的可行性。方法采用胰蛋白酶消化心肌组织分离单个心肌细胞，联合差速贴壁与培养基中加入BrdU杀死心肌成纤维细胞。心肌细胞在含1％O₂与99％N₂的孵育箱中缺氧培养6h，在21％O₂与5％CO₂孵育箱中进行氧再灌注培养24和48h，建立心肌细胞衰老模型。采用免疫组化检测培养细胞α-actin表达情况；透射电镜观察心肌细胞超微结构改变；BrdU掺入试验了解心肌细胞增殖情况；流式细胞仪检测细胞周期；β-半乳糖苷酶试剂盒检测β-半乳糖苷酶活性。结果经α-actin抗体鉴定显示分离培养的细胞中95％以上是心肌细胞。心肌细胞经缺氧／氧再灌注诱导后，细胞体积变大、线立体明显皱缩；细胞增殖结果显示缺氧／氧再灌注组心肌细胞BrdU阳性率较对照组有显著下降（P＜0.01）；细胞周期检测结果显示缺氧／氧再灌注组心肌细胞G0／G1期细胞较对照组有显著增多（P＜0.01），S期细胞较对照组有显著减少（P＜0.01）；β-半乳糖苷酶活性检测结果显示氧再灌注组心肌细胞β-半乳糖苷酶活性较对照组与缺氧6 h有显著提高（P＜0.01）。结论1．采用胰蛋白酶消化心肌组织分离心肌细胞，联合差速贴壁与培养基中加入BrdU杀死心肌成纤维细胞，可以获得较纯的心肌细胞，经α-actin抗体鉴定显示分离培养的细胞中95％以上是心肌细胞。2．我们首次采用缺氧／氧再灌注，成功诱导建立了SD乳大鼠心肌细胞衰老模型，为心肌细胞衰老机制的研究提供了稳定可靠的模型。第二部分缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的机制研究目的从细胞周期调控方面探讨缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的分子机制方法采用real-time quantitative PCR与western blot方法检测培养SD乳大鼠心肌细胞在对照组、缺氧6h、氧再灌注24和48h，心肌细胞周期相关蛋白（D1），细胞周期依赖激酶（CDK4），细胞周期蛋白和细胞周期依赖激酶复合物抑制剂（p21WAF1，p16IN4Ka），Rb蛋白，p53等基因的mRNA与蛋白水平的表达变化。结果（1） Cyclin D1与β-actin基因mRNA水平的比值在缺氧6h、氧再灌注24和48h分别为0.024，0.082，0.092，与对照组0.005相比有显著增高（P＜0.05或P＜0.01）。Cyclin D1与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h分别为0.735，0.993，1.181，与对照组0.437相比升高明显（P＜0.05或P＜0.01）。（2） CDK4与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.008，0.005，0.005与对照组0.011相比有显著降低（P＜0.05或P＜0.01）。CDK4与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.854，0.432，0.171与对照组1.102相比有明显减少（P＜0.05或P＜0.01）。p21WAF1是通过降低CDK4的活性导致细胞分裂停止，p21WAF1与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.009，0.009，0.012与对照组0.006相比有显著增高（P＜0.05或P＜0.01）。p21WAF1与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.831，1.978，1.736与对照组0.456相比升高明显（P＜0.05或P＜0.01）。p53是通过其靶蛋白p21WAF1起作用与p21WAF1的变化相平行。p53与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.002，0.003，0.004与对照组0.001相比有显著增高（P＜0.05或P＜0.01）。p53与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.678，0.978，1.236与对照组0.213相比升高明显（P＜0.01）。（3） Rb与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.003，0.004，0.003，与对照组0.002相比有显著增高（P＜0.05）。但pRb与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.926，1.231，0.351，与对照组2.502相比有显著降低（P＜0.01），p16IN4Ka能阻止CDK4／6与cyclinD1的结合对Rb的磷酸化。p16IN4Ka与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.007，0.022，0.022与对照组0.001相比有显著增高（P＜0.05或P＜0.01）。p16IN4Ka与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.078，0.082，0.109与对照组0.001相比升高明显（P＜0.01）。结论缺氧／氧再灌注通过调节细胞周期相关蛋白（cyclin D1、CDK4、p16IN4Ka、p21WAF1、p53等）而抑制心肌细胞增殖，促进细胞衰老。第三部分Pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的干预研究目的了解pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老是否有抑制作用及其机制。方法Pravastatin干预浓度为10^-7-10^-5mol／l，方法参考第一部分方法。结果Pravastatin干预后对照组、缺氧6h、氧再灌注24和48h的G0／G1期细胞分别为71.98％，86.47％，84.98％，88.78％较实验组72.71％，87.45％，87.97％，89.78％没有明显减少（P＞0.05），同样对照组、缺氧6h、氧再灌注24和48h的S期细胞分别为20.34％，9.02％，6.06％，5.79％较实验组20.03％，9.01％，5.06％，4.93％没有明显增多（P＞0.05）。Pravastatin干预后，对照组、缺氧6h、氧再灌注24和48h的半乳糖苷酶活性阳性细胞分别为9.13％，12.55％，42.15％，53.48％较实验组8.56％，12.37％，42.34％，58.28％没有明显减少（P＞0.05），不同浓度pravastatin的干预，结果相似。结论Pravastatin不能抑制缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老。更多还原

【Abstract】 BACKGROUNDCellular senescence has been defined by Hayflick as the ultimate andirreversible loss of replicative capacity occurring in primary somatic cellculture. After Hayflick identifying senescence in human fibroblast firstly,cellular senescence was identified in diverse type cells in human orfibroblasts in other species. Different markers of the senescent phenotypeas, for instance, G0/G1 specificity of cell cycle arrest,senescence-associatedβ-galactosidase activity, mitochondrialdehydration, or senescence-associated gene expression. Cellularsenescence may not only result in the aging of organism, but induceoccurrence of tumors, heart failure, atherosclerosis or arrhythmia.Therefor, it is very important to study the mechanism of cellularsenescence.Hypoxia/reoxygenation （H/R） was the most common injury factorfor cell, tissue or organism. It was also an effective way to induce cellularsenescence. Recetenly, a report has shown that premature senescencecould be induced by H/R in FA bone marrow hematopoietic cells.HMG-CoA reductase inhibitors, i.e. statins, were reversibleinhibitors of the rate-limiting step in cholesterol biosynthesis and weregenerally used for the treatment of hypercholesterolemia. Statins also could reduce mortality for patients with coronary heart disease, increasesurvival for patients with heart transplantation, and improve ventricularremodeling. Besides regulating lipid metabolism, recently, Assmusreported that atorvastatin could reduce senescence and increaseproliferation of endothelial progenitor cells via regulation of modulationexpression of cell cycle genes including upregulation of cyclins anddownregulation of the cell cycle inhibitor p27Kipl. Apart from statinsplay very important role in protection for patients with cardiovasculardiseases, whether pravastatin could inhibit premature senescence ofneonatal SD rat cardiomyocytes induced by H/R or not?In this study, we would investigate whether premature senescencecould be induced by H/R in neonatal SD rat cardiomyocytes. To explorecellular and molecular mechanisms of premature senescence in neonatalrat cardiomyocytes exposed to H/R. To investigate whether pravastatincould inhibit premature senescence of neonatal SD rat cardiomyocytesinduced by H/R and assosiated mechanisms.PartⅠIsolation, culture, purification and identification ofneonatal cardiomyocytes from SD rat heart. Establishmentthe premature senescence model of cardiomyocytes inducedby H/R AIMOn the base of succeeding in isolation, culture neonatalcardiomyocytes from SD rat heart, to explore how to get morecardiomyocytes. To investigate whether premature senescence could beinduced by H/R in neonatal SD rat cardiomyocytes.METHODSTrypsin was used to cut pieces of heart tissue into single cells.Combination diferent time for cells to stain wall and mediumsupplemented with BrdU to remove cardiac fibroblasts.Cardiomyocytes were isolated from neonatal SD rat heart andidentified by a-actin immunohistochemistry. The control cultures wereincubated at 37℃in humidified atmosphere of 5％CO₂, 95％air. Thehypoxic cultures were （within a modular incubator chamber filled with 1％O2, 5％CO2, and balance N2） for 6 h. The reoxygenated cultures weresubjected to 1％O2, 5％CO2 for 6 h then 21％oxygen for 24 and 48 h,respectively. Cell proliferation was determined using BrdU labelling.Ultrastructure of cardiomyocytes was observed by using electronmicroscope. Flow cytometry were used to investigate alteration of cellcycle.β-galactosidase activity was determined by using Senescenceβ-galactosidase Staining Kit.RESULTSMost cells （＞95%） in the isolated cultures were positive forα-actin antibody. The percentage of BrdU positive cells reduced significantly inH/R treated group （P＜0.01）. Under the condition of H/R, mitochondrialdehydration appeared. Most cardiomyocytes resided in G0 and G1 phase inthe group of hypoxia 6 h, reoxygenation 24 and 48 h compared withcontrol one （P＜0.01）. Cardiomyocytes did not stain forβ-galactosidasein the group of control, hypoxia 6 h, but did intensely for it inreoxygenation group of 24,48 h, compared with control one （P＜0.01）.CONCLUSIONS1. Combination diferent time for cells to stain wall and mediumssupplemented with BrdU to remove cardiac fibroblasts are a good wayto purify cardiomyocytes.2. Premature senescence could be induced in neonatal SD ratcardiomyocytes exposed to H/R.PartⅡA study on mechanism of premature senescence ofcardiomyocytes induced by H/RAIMTo explore cellular and molecular mechanisms of prematuresenescence in neonatal rat cardiomyocytes exposed to H/R. METHODSReal-time quantitative PCR and western blot were used to analyzemRNA and protein level of cyclin D1, cyclin dependent kinases （CDK4）,inhibitors of the cyclin-CDK complexes （p16IN4Ka, p21WAF1）, pRb,p53 in cardiomyocytes of neonatal SD rat in the group of control, hypoxia6h, reoxygenation 24 and 48 h.RESULTSCyclin D1 mRNA and protein levels significantly increased in H/Rtreated group compared with control one （P＜0.05 or P＜0.01,respectively）.CDK4 mRNA and protein levels significantly decreased in H/Rtreated group compared with control one （P＜0.05 or P＜0.01,respectively）. p21WAF1 inhibited cell cycle through reducing expressionof CDK4. p21WAF1 mRNA and protein levels increased significantly inH/R treated group compared with control one （P＜0.05 or P＜0.01,respectively）. Paralleled to the change of p21WAF1, the mRNA andprotein levels of p53 were rosen significantly in H/R treated groupcompared with control one （P＜0.05 or P＜0.01, respectively）.Rb mRNA level increased significantly in H/R treated groupcompared with control one （P＜0.05, respectively）. But pRb leveldecreased significantly in H/R treated group compared with control one（P＜0.01, respectively）, p16IN4Ka mRNA and protein levels, which prevented CDK4/6 to catalyze phosphorylation of Rb, were rosensignificantly in H/R treated group compared with control one （P＜0.01,respectively）.CONCLUSIOSH/R inhibited cell proliferation and accelerated prematuresenescence through regulating protein related to cell cycle （cyclin D1,CDK4, p16IN4Ka, p21WAF1, p53）.PartⅢA study about the effect of pravastatin onpremature senescence of neonatal SD rat cardiomyocytesinduced by H/RAIMTo investigate whether pravastatin could inhibit prematuresenescence of neonatal SD rat cardiomyocytes induced by H/R.METHODSIntervention concentration of pravastatin was 10^-7-10^-5 Mol/L, othermethods were as same as partⅠRESULTSCompared with cells resided in G0/G1 phase in experimental group （72.71％, 87.45％, 87.97％, 89.78％in control group, hypoxia 6 h,reoxygenation 24 h and 48 h, respectively）, pravastatin could not reversecells resided in G0/G1 phase （71.98％, 86.47％, 84.98％, 88.78％incontrol group, hypoxia 6 h, reoxygenation 24 h and 48 h, respectively） （P＞0.05）. Pravastatin also could not reverse cells disappeared in S phase （P＞0.05）. Compared with the percentage ofβ-galactosidase positivestaining cells in experimental group （9.13％, 12.55％, 42.15％, 53.48％in control group, hypoxia 6h, reoxygenation 24h and 48h, respectively）pravastatin could not reduceβ-galactosidase activity, the percentage ofβ-galactosidase positive staining cells is 8.56％, 12.37％, 42.34％,58.28％, in control group, hypoxia 6 h, reoxygenation 24 h and 48 h,respectively （P＞0.05）, different intervetion concertration of pravastatin,with similar outcome.CONCLUSIONSPravastatin could not inhibit premature senescence of neonatal SDrat cardiomyocytes induced by H/R.更多还原