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目的:观察心肌肌钙蛋白I在肥厚型心肌病小鼠心肌组织中的磷酸化与降解作用。方法:实验于2005-03/2006-01在南京医科大学第一附属医院心血管病研究所及南京医科大学动物中心SPF级动物房进行。取4周龄BALB/c雌鼠12只,随机分为模型组6只,正常对照组6只,适应性饲养1周后模型组小鼠转染带有EGFP报告基因的CTnIArg146Trp突变基因。饲养至20周龄时二维超声心动图检测证实模型组小鼠室间隔肥厚,断颈处死两组小鼠后,称取小鼠心脏质量(湿),心尖部心肌组织光镜病理学检查,小鼠心肌组织进行WesternBlotting检测重组蛋白、肌钙蛋白I的磷酸化及降解表达,GAPDH作为内参半定量。结果:12只小鼠进入结果分析。①模型组小鼠心脏明显增大,心脏质量(湿)/体质量比显著高于正常对照组(P<0.01);模型组小鼠心肌细胞肥大、排列紊乱、间质血管增生、少量炎性细胞浸润;两组血清肌钙蛋白I水平均正常(P>0.05)。②模型组小鼠心肌组织用抗EGFP单克隆抗体在Mr55000,24000检测到突变CTnI-EGFP及其降解片断表达。③模型组在Mr55000,30000,28000分别检测到磷酸化突变CTnI-EGFP、内源性肌钙蛋白I及其降解片断表达,正常对照组在Mr30000见磷酸化肌钙蛋白I表达,模型组内源性磷酸化肌钙蛋白I表达明显高于对照组(1.18±0.15,0.97±0.15,P<0.05)。④去磷酸化抗肌钙蛋白I单克隆抗体在Mr55000,28000,检测到去磷酸化突变肌钙蛋白I-EGFP、内源性肌钙蛋白I降解片断表达,正常对照组未见去磷酸化肌钙蛋白I表达;用针对不同位点的抗肌钙蛋白I单克隆抗体3E3,2I-14,8I-7,MF4分别在Mr55000,30000,28000检测到突变CTnI-EGFP、内源性肌钙蛋白I及其降解片断表达,正常对照组在Mr30000,见肌钙蛋白I表达,未见降解片断。结论:AdcCTnIArg146Trp-EGFP突变基因在小鼠体内成功转染心肌组织并有明显表达及降解片断出现,小鼠心脏呈现明显室间隔肥厚;小鼠内源性肌钙蛋白I磷酸化表达增强、降解与心肌肥厚的发生、发展及心肌功能的改变可能有关,但两者之间因果关系及确切机制还有待于进一步研究。
OBJECTIVE: To observe the phosphorylation and degradation of cardiac troponin I in the myocardium of hypertrophic cardiomyopathy mice. Methods: The experiment was performed at the Institute of Cardiovascular Diseases of the First Affiliated Hospital of Nanjing Medical University and SPF animal room of Nanjing Medical University from March 2005 to January 2006. Twelve BALB / c females aged 4 weeks were randomly divided into model group (n = 6) and normal control group (n = 6). One week after the adaptive feeding, the model group mice were transfected with the CTnIArg146Trp mutant gene with EGFP reporter gene. After feeding to 20 weeks of age by two-dimensional echocardiography test confirmed that the model group of mice with interventricular septal hypertrophy, neck severed after killing two mice, weighed mice heart mass (wet), apical myocardial tissue by light microscopy pathology, Western blotting was used to detect the phosphorylation and degradation of recombinant protein, troponin I and GAPDH as internal standard. Results: Twelve mice entered the result analysis. ① The heart of model group increased significantly, the heart mass (wet) / body mass ratio was significantly higher than that of the normal control group (P <0.01); the model group had a marked increase of cardiomyocyte hypertrophy, disordered arrangement, interstitial vascular hyperplasia, Cell infiltration; serum troponin I levels were normal (P> 0.05). (2) The expression of mutant CTnI-EGFP and its degradation fragment detected by Mr55000 and 24000 in anti-EGFP monoclonal antibody in myocardium of model mice. ③The expression of phosphorylation CTnI-EGFP, endogenous troponin I and its degradation fragment were detected in Mr55000, 30000 and 28000 respectively in model group, while in control group, Mr30000 showed phosphorylated troponin I expression. In model group, endogenous The phosphorylated Troponin I expression was significantly higher than that of the control group (1.18 ± 0.15,0.97 ± 0.15, P <0.05). ④ dephosphorylated anti-Troponin I monoclonal antibody in Mr55000, 28000, detected dephosphorylation of mutant troponin I-EGFP, endogenous troponin I degradation fragment expression, the normal control group no dephosphorylated muscle Troponin I was detected by using different anti-Troponin I monoclonal antibodies 3E3, 2I-14, 8I-7, and MF4 at different sites, Mr55000, I and its degradation fragment expression, the normal control group in Mr30000, see the expression of troponin I, no degradation fragments. CONCLUSION: The gene of AdcCTnIArg146Trp-EGFP was successfully transfected into myocardium of mice with obvious expression and degradation fragment. The mouse heart showed obvious interventricular septum hypertrophy. The expression of endogenous troponin I phosphorylation was enhanced, The occurrence and development of myocardial hypertrophy and the changes of myocardial function may be related, but the causal relationship between the two and the exact mechanism remains to be further studied.