【作者】 李昕；

【导师】 郑建全； 张永祥；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2008， 博士

【摘要】 目的和意义:心肌肥厚是众多心血管疾病共有的病理改变和独立的危险因子,如高血压、缺血性心脏病、心率失常和心瓣膜病等。持续性的心肌肥厚最终可导致心衰和死亡。心肌肥厚和肺阻力血管的结构重构是缺氧所致的肺动脉高压形成过程中的相关效应,是肺动脉高压维持和发展的重要病理基础。对心肌肥厚和血管重构的逆转作为心血管疾病临床治疗的靶向之一,也日益受到重视,但发生机制目前还不十分清楚。已知内皮素（endothelin,ET）-1在心肌肥厚和血管重构的发生发展中起重要作用。最新研究发现,心肌过表达组织型转谷氨酰胺酶（tissuetransglutaminase,tTG）的转基因小鼠可发生独特的心肌肥厚表型;小动脉重构可能依赖于tTG的参与。提示:心肌肥厚的发生及小动脉重构可能与tTG相关。综上所述,本研究将重点探讨tTG在ET-1诱导的心肌细胞肥大和缺氧所致的大鼠心肌肥厚与肺小动脉重构中的作用,为最终阐明心肌肥厚和小动脉重构的发生机制提供新的思路和实验依据。方法和结果:本研究在ET-1诱导的新生乳鼠心肌细胞肥大实验模型上观察了ET-1对tTG mRNA和蛋白的表达及其多功能酶活性的影响;在慢性缺氧诱导的大鼠右心室肥厚及肺小动脉重构实验模型上观察了tTG mRNA和蛋白表达的变化;此外,在细胞和整体实验模型上还观察了tTG竞争性抑制剂对这些病理过程的作用,具体方法和结果如下:根据Simpson and Savion建立的方法进行心肌细胞的原代培养:心肌细胞培养4天后,分别用0.1%DMSO、ET-1（10 nM）处理24 h或48 h,然后在光学显微镜下进行心肌细胞形态的观察,并用计算机辅助软件对细胞表面积进行测量;采用[³H]-leucine掺入细胞法测定心肌细胞蛋白质合成速度;若丹明标记鬼笔环肽对细胞进行染色,激光扫描共聚焦显微镜下对心肌细胞肌节重组进行鉴定;用RT-PCR方法检测心肌细胞肥厚基因的重新表达。结果显示:ET-1 10 nM处理心肌细胞24 h可使心肌细胞表面积和蛋白质合成速度较对照组相比明显增大;使心肌细胞的肌节重新组装及诱导ANF和MLC-2 mRNA的重新表达。在ET-1诱导的心肌细胞肥大模型上,采用RT-PCR和Western blot方法观察了ET-1对心肌细胞tTG mRNA和蛋白表达水平的影响。心肌细胞用0.1%DMSO或ET-1（0.1-100 nM）处理24 h或单一浓度的ET-1（10 nM）处理6-48 h后,tTGmRNA和蛋白的表达水平较对照组相比显著升高,并呈浓度和时间依赖性。提示:tTG可能参与了心肌细胞肥大的形成。在上述实验模型上,为了观察tTG抑制剂对ET-1诱导的心肌细胞肥大的作用,心肌细胞用tTG竞争性抑制剂单丹磺酰尸胺（Monodansyl cadaverine,MDC）（100μM）和ET-1（10 nM）联合处理24 h,ET-1诱导的心肌细胞表面积的增大可被MDC所抑制,抑制百分率达61.9%;此外,MDC在0.1-100μM浓度范围内可阻断ET-1诱导的心肌细胞蛋白质合成速度的加快,抑制百分率分别为33%、59%、63%和76%;MDC（100μM）还可抑制ET-1（10 nM）诱导的心肌细胞肌原纤维节的重新组装和胚胎基因的重新表达;而MDC（100μM）单独处理心肌细胞24 h对对照组细胞上述参数无显著影响。这些结果提示:tTG以正性调控作用参与了ET-1诱导的心肌细胞肥大过程为了观察ET-1对tTG酶活性的影响,本研究分别采用[³H]-putrescine掺入二甲基酪蛋白的方法、活性炭吸附法及光亲和标记法来测定心肌细胞转谷氨酰胺酶（transglutaminase,TGase）、GTPase及GTP-结合（GTP-binding）活性。结果显示:不同浓度的ET-1（0.1-100 nM）处理心肌细胞24 h,或单一浓度的ET-1（10 nM）处理不同时间（6-48 h）对基础TGase活性无显著影响,但可显著抑制0.5 mM Ca²⁺依赖性TGase活性,并显著升高tTG蛋白的GTPase活性和GTP-binding能力,并呈浓度和时间依赖性。这些结果提示:ET-1诱导的心肌细胞肥大可能与tTG蛋白的GTPase活性相关。研究还发现,ET-1诱导tTG mRNA表达的上调可被选择性的ET_A受体阻断剂BQ-123所阻断,而ET_B受体阻断剂BQ-788无此作用。提示:ET-1可通过激活ET_A受体来调节tTG基因的表达水平在缺氧诱导的大鼠肺动脉高压及相关效应:右心室肥厚及肺小动脉重构实验模型上,观察tTG对心肌肥厚及血管重构的作用。成年雄性大鼠随机分为空气对照组、空气对照+盐酸胱胺组、缺氧模型组、缺氧+盐酸胱胺组,缺氧组动物置于低压缺氧舱内,模拟海拔5000米高度连续缺氧2或4 wk,正常组动物则置于不缺氧的环境下。给药组动物缺氧的同时腹腔给予30 mg/kg盐酸胱胺,2次/天,共2 wk。缺氧完毕后对各组动物进行肺动脉压、右心室重量、肺小血管（直径:50-100μm）管壁厚度及右心室tTG蛋白表达水平的测量。结果显示:1-4 wk的慢性缺氧可导致大鼠右心室发生明显的肥厚症状,且右心室tTG蛋白表达水平较对照组相比也显著升高,并呈时间依赖性;tTG竞争性抑制剂盐酸胱胺可阻断慢性缺氧所致的大鼠右心室肥厚和肺小血管重构,抑制百分率分别达49%和59%。相同浓度下的盐酸胱胺对对照组大鼠的上述参数无显著影响。在上述实验模型上还观察了选择性的ET_A受体阻断剂ETP-508对大鼠右心室tTG mRNA表达水平的影响。结果发现2周的缺氧可导致大鼠右心室tTGmRNA表达水平较对照组相比显著升高,此升高可被ET_A受体阻断剂ETP-508（0.4 mg/h,9.6 mg/day）静脉缓释给药所阻断。结论:本研究在离体细胞实验模型上首次发现:tTG可参与调节ET-1诱导的乳鼠心肌细胞肥大,ET-1可通过激活ET_A受体上调tTG的GTPase活性,使tTG以正性调控作用促进心肌细胞的肥大;在整体动物实验模型上首次发现:tTG可参与调节缺氧所致的大鼠肺动脉高压的相关效应,即右心室肥厚和肺小动脉重构,缺氧所致tTG的激活与ET_A受体相关,tTG可能是ET_A受体下游通路上一个重要的调控因子。这些研究结果为tTG参与心肌肥厚和血管重构的发生提供了有力的证据,为tTG抑制剂作为预防和治疗心肌肥厚和血管重构的新靶标及可能性提供了初步的实验依据。更多还原

【Abstract】 Aims and significances:Cardiac hypertrophy（CH）is the common pathological change and an independent risk factor for many cardiovascular diseases such as hypertension,ischemic heart disease,arrhythmia and valvular disease.Persistent hypertrophy often leads to the development of congestive heart failure and death.CH and structure remodeling of pulmonary resistant artery are important pathological base for the development and maintenance of pulmonary hypertension（PH）.Reversal of CH and vascular remodeling is one of target for clinical therapy of cardiovascular disease,while the exact mechanism remains unclear.Over-expression tissue transglutaminase（tTG/Gh）in transgenic mice in a cardiac specific manner resulted in a unique hypertrophy phenotype,and tTG was involved in small artery remodeling, thus tTG was proved to be important in both the development of CH and vascular remodeling.Therefore,the purpose of this study was to investigate the effects of tTG on cardiomyocytes hypertrophy induced by ET-1 as well as CH and small pulmonary artery remodeling induced by hypoxia,so as to provide experimental data for the pathogenesis of CH and small artery remodeling.Methods and results:The effects of ET-1 on the expression of tTG mRNA,protein and tTG multifunction enzymic activity were studied in the neonatal rat cardiomyocytes hypertrophy model induced by ET-1.In the model of right heart hypertrophy and vascular remodeling secondary to pulmonary hypertension induced by chronic hypoxia in rats,the expression of tTG mRNA and protein were determined. In addition,the effects of tTG competitive inhibitor on the pathological process were investigated in these two models.Primary culture of neonatal rat cardiomyocytes was prepared by the method originally described by Simpson and Savion.At 4 d after culturing,cardiomyocytes were treated with 0.1%DMSO or ET-1（10 nM）for 24 h or 48 h,and then the morphology of cardiomyocytes was observed by optical microscopy and the surface area was determined by computer-assisted software;Protein synthesis was determined by incorporation of[³H]-leucine into cells;Cardiomyocytes sarcomeric reorganization was analyzed by laser scanning confocal microscope after the cells were stained with phalloidin-Rhodamine;Hypertrophic gene re-expression was determined by RT-PCR. The results showed that the cell surface area and the rate of protein synthesis was increased after cardiomyocytes were treated with ET-1 10 nM for 24 h,at the same, and an apparent sarcomeric reorganization and re-expression of ANF and MLC-2 mRNA were also obseved.In the model of cardiomyocytes hypertrophy induced by ET-1,the effects of ET-1 on the expression of tTG mRNA and protein were analyzed by RT-PCR and Western blot,respectively.Cardiomyocytes treated with ET-1 0.1-100 nM for 24 h or ET-1 10 nM for 6-48 h induced an increase in the expression of tTG mRNA and protein compared with control cells in a concentration and time dependent manner. This result suggested that tTG was involved in the development of cardiomyocytes hypertrophy induced by ET-1.The effects of tTG inhibitor on cardiomyocytes hypertrophy induced by ET-1 were studied in the cardiomyocytes model.The increase in cell surface area induced by ET-1（10 nM）was significantly inhibited after the cells were treated with Monodansyl cadaverine MDC（100μM）for 24 h,the inhibitory rate of which was 61.9%;MDC（0.1-100μM）inhibited ET-1-induced increases in the rate of protein synthesis,the inhibitory rate of which was 33%,59%and 76%,respectively; Sarcomere reorgonization and hypertrophic gene re-expression induced by ET-1 were also significantly inhibited by MDC;However,MDC（100μM）exhibited no effects on any of the parameters assayed for control cells.The results suggested that tTG was involved in the development of cardiomyocytes hypertrophy induced by ET-1 in a positive regulation manner.In the model of cardiomyocytes hypertrophy induced by ET-1,The activity of TGase,GTPase and GTP binding were determined by evaluating the incorporation of [³H]-putrescine into N,N’-dimethylated casein,charcoal absorption and direct photoaffinity labeling of[α-³²p]GTP method,respectively.The results showed that cardiomyocytes treated with ET-1 0.1-100 nM for 24 h or ET-1 10 nM for 6-48 h didn’t influence the basal TGase activity of caridomyocytes,but significant inhibited the 0.5 mM Ca²⁺-stimulated TGase activity,and significant elevated the activity of GTPase and GTP binding in a concentration and time dependent manner.These results suggested that cardiomyocytes hypertrophy induced by ET-1 was related to GTPase activity of tTG.Furthermore,ET-1-induced increases in tTG mRNA expression was significantly inhibited by selective ET_A receptor antagonist BQ-123 but not by ET_B receptor antagonist BQ-788,which suggested that ET-1 up-regulated the expression of tTG mRNA through activation ET_A receptor.In the right ventricular hypertrophy and small pulmonary artery remodeling model secondary to hypobaric hypoxia-induced pulmonary hypertension in rats, effects of tTG on CH and vascular remodeling were observed.Adult male rats were randomly divided into air controls,air + cystamine dihydrochloride group,hypoxia group,hypoxia + cystamine dihydrochloride group.The rats in hypoxia group were placed in hypoxic chamber in conditions equivalent to an altitude of 5000 m（O₂10%） for 2 - 4 wk.Air control animals were not exposed to hypoxic atmosphere.The rats were injected intraperitoneally（i.p.）with cystamine（30 mg/kg,2 times/day）for 2 wk. Then the pulmonary pressure,expression of tTG protein in right heart,right ventricular weight and the wall thickness of small lung artery（diameter:50-100μm） were determined,respectively.The result showed that 1- 4 wk chronic hypoxia significantly induced right ventricular hypertrophy and led to the increases in the expression of heart tTG protein in rats compared with air control groups in a time dependent manner.Right ventricular hypertrophy and small pulmonary artery remodeling induced by chronic hypoxia were also significantly inhibited by cystamine dihydrochloride（30 mg/kg,2 time/day,i.p.）,the inhibitory rate of which were 49% and 59%,respectively.Same dose of MDC did not influence the air control properties.In the above experimental model,the influence of ET_A receptor antagonist ETP-508 on the expression of tTG mRNA was observed by RT-PCR method.The result showed that 2 wk hypoxia induced increases in the expression of tTG mRNA from the rat heart tissues compared with air control,which was inhibited by ET_A receptor antagonist ETP-508（0.4 mg/h,9.6 mg/day）via intravenous infusion.Conclusions:This study demonstrates for the first time in vitro cell model that tTG was involved in modulating cardiomyocytes hypertrophy induced by ET-1,activation of ET_A receptor by ET-1 up-regulated GTPase activity of tTG;and accelerated cardiomyocytes hypertrophy in positive regulation manner;The current research also demonstrates for the first time in vivo model that tTG was involved in regulating chronic hypoxia-induced right ventricular hypertrophy and small pulmonary remodeling secondary to pulmonary hypertension in rats,tTG activation induced by hypoxia was correlated with ET_A receptor,and tTG was a crucial regulator in the downstream signal pathway of ET_A receptor.These results provided powerful evidences that tTG was involved in the development of cardiac hypertrophy and vascular remodeling,further suggested that tTG inhibitors could be taken as a new target for the prevention and therapy of cardiac hypertrophy as well as vascular remodeling.更多还原