激活cAMP信号通路抑制CTGF表达对小鼠心肌梗死后心肌纤维化调控的研究

发布时间：2018-03-18 20:37

  本文选题：环磷酸腺苷　切入点：结缔组织生长因子　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：第Ⅰ部分环磷酸腺苷抑制CTGF表达调控小鼠心肌梗死后心肌纤维化的作用观察 摘要：目的观察环磷酸腺苷(cAMP)对小鼠心肌梗死后结缔组织生长因子(CTGF)的调控作用以及对心肌纤维化的影响 方法72只SPF级C57BL雄性小鼠随机分为假手术组(MS组)、心肌梗死组(MI组)及药物干预组(MT组),每组各24只。MI组及MT组小鼠呼吸机辅助开胸结扎左冠状动脉前降支(LAD)建立心肌梗死动物疾病模型：MS组小鼠开胸后不结扎,其余操作同MI组及MT组。术后1周开始MT组腹腔注射环磷酸腺苷葡胺(MAC),连续给药4周；同时MS组及MI组注射等量生理盐水对照。术后第5周,各组随机抽取10只小鼠行心脏超声检测,测量心脏形态指标及心功能指标；超声完毕后,立即开胸收获心脏标本,行组织形态学HE染色、胶原纤维Masson染色及CTGF免疫组化染色。各组剩余小鼠按照5只、5只、4只随机分成3组,收获心脏标本,分离左心室梗死区及非梗死区,分别行羟脯氨酸含量测定、CTGF蛋白表达western blot检测及CTGF mRNA、TGF-β1mRNA表达量RT-PCR检测. 结果超声心动图提示MT组射血分数(EF)为(19.83±3.95)%,左室短轴缩短率(FS)为(9.07±1.9)%,均高于MI组[EF(13.65±2.14)%,FS(6.22±1.03)%](P0.05)；左心室每搏输出量(SV)MT组[(26.26±3.57)μL]优于MI组[(18.82±2.71)μL](P0.05)；MT组左心室前、后壁厚度及内径与MI组之间无明显差异(P0.05)。心脏Masson染色提示MT组胶原纤维向非梗死区心肌组织浸润减少；免疫组化CTGF阳性表达较MI组减少。MT组梗死区羟脯氨酸含量[(1388.98±86.26)u g/mg]低于MI组[(1673.12±199.97) μ g/mg](P0.05)；非梗死区羟脯氨酸含量[(662.92±98.05)μ g/mg]低于MI组[(855.84±83.3)u g/mg)](P0.05)。Western blot检测提示MT组CTGF蛋白表达量低于MI组(P0.05)。RT-PCR检测提示MT组CTGF mRNA表达量低于MI组(P0.05);TGF-β1mRNA表达量MT组与MI组之间在梗死区与非梗死区均无明显统计学差异(P0.05)。 结论环磷酸腺苷可以抑制CTGF的表达,降低小鼠心肌梗死后胶原纤维的表达,减轻梗死区胶原纤维向非梗死区心肌的浸润程度,改善心脏功能。 第Ⅱ部分激活cAMP信号通路调控小鼠心肌成纤维细胞结缔组织生长因子的表达 摘要：目的研究小鼠心肌成纤维细胞(MCFs)胞内环磷酸腺苷(cAMP)浓度提高对结缔组织生长因子(CTGF)表达的影响及其信号通路。 方法采用1周龄内C57BL乳鼠,获取心脏后分离培养原代MCFs,第3代细胞用于实验。使用转化生长因子-β1(TGF-β1)培养细胞,快速上调MCFs对CTGF的表达,模拟小鼠心肌梗死后CTGF高表达情况。使用腺苷酸环化酶激活剂Forskolin干预培养细胞后检测MCFs表达CTGF的变化,同时检测可能的信号分子蛋白激酶A(PKA)、p44/42丝裂素活化蛋白激酶(MAPK)及磷酸化p44/42MAPK表达的变化。分别给予PD98509及Rp-cAMPS抑制p44/42MAPK的磷酸化水平及PKA的表达,检测细胞PKA、p44/42MAPK、磷酸化p44/42MAPK表达的变化,并同时检测细胞内CTGF的表达。 结果TGF-β1可以快速上调MCFs对CTGF的表达。Forskolin干预培养MCFs后,细胞内cAMP浓度明显提高、细胞活力下降；同时细胞中CTGF mRNA及蛋白表达均下降；其可能的信号分子PKA表达上升,磷酸化p44/42MAPK水平表达下降而p44/42MAPK总蛋白无明显改变。MCFs给予PD98509抑制p44/42MAPK磷酸化水平后,PKA表达上升,同时细胞中CTGF mRNA及蛋白的表达下调：给予Rp-cAMP抑制PKA活化后,p44/42MAPK磷酸化水平升高,细胞中CTGF mRNA及蛋白表达出现上调。 结论Forskolin可以通过提高MCFs胞内cAMP浓度抑制MCFs对CTGF的表达。其作用信号通路为高浓度cAMP上调PKA表达,降低下游p44/42MAPK磷酸化水平,抑制CTGF的表达。
[Abstract]:The effect of cyclic adenosine cyclic adenosine on CTGF expression in regulating myocardial fibrosis after myocardial infarction in mice
Abstract: Objective To observe the effect of cAMP on the regulation of connective tissue growth factor (CTGF) after myocardial infarction in mice and the effect on myocardial fibrosis
Methods 72 SPF male C57BL mice were randomly divided into sham operation group (group MS), myocardial infarction group (MI group) and drug intervention group (MT group), each group had 24 rats in group.MI and MT group were ventilator assisted ligation of left anterior descending coronary artery (LAD) to establish the model of animal myocardial disease infarction in MS group after thoracotomy without ligation, the same with the MI group and MT group. MT group received intraperitoneal injection of meglumine Cyclicadenylate started 1 weeks after operation (MAC), administered continuously for 4 weeks; at the same time, MS group and MI group were injected with normal saline. After fifth weeks, echocardiography 10 mice for each group were randomly selected, measuring heart shape index and heart function index; ultrasound after thoracotomy, immediately harvested heart specimens for histological HE staining, collagen Masson staining and CTGF immunohistochemical staining. All remaining mice according to 5, 5, 4 were randomly divided into 3 groups, the harvest of heart were left The content of hydroxyproline was measured, the expression of CTGF protein was detected by Western blot and CTGF mRNA, and the expression of TGF- beta 1mRNA was detected in the ventricular infarction area and non infarct area.
缁撴灉瓒呭０蹇冨姩鍥炬彁绀篗T缁勫皠琛，

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