【作者】 吴岚；

【导师】 孙景辉；

【作者基本信息】 吉林大学 ， 内科学， 2010， 博士

【摘要】 病毒性心肌炎（viral myocarditis,VMC）是小儿心血管系统常见疾病,其发病机制尚未完全阐明,病情轻重不一,虽然大多数急性VMC患者心功能可完全恢复,但部分患者可能出现反应性心肌纤维化,心室重塑,发展成为慢性VMC、扩张性心肌病（DCM）,心肌纤维化的病理过程伴随着VMC的始终,至今国内外对其尚无特效的治疗手段。Periostin是日本学者发现的一种具有调节成骨细胞黏附和分化的功能骨黏附分子,当时被命名为成骨细胞特异性因子-2（osteoblast-specific factor-2,OSF-2）,现已归类于细胞外基质（extracellular matrix,ECM）蛋白。近年来研究表明periostin在心梗时作为TGF-β1的效应因子具有趋化心肌成纤维细胞,促进胶原合成和成熟,形成稳定胶原网,参与心室重构的作用。有体外实验表明,periostin与心肌成纤维细胞相互作用,并可调节胶原-I的交联和成熟,与心梗后心肌纤维化密切相关。但目前periostin与病毒性心肌炎心肌纤维化的关系报道尚少。本研究通过构建柯萨奇B₃型病毒（CVB3）BALB/C小鼠VMC模型,运用半定量RT-PCR方法、免疫组织化学方法确定了periostin在VMC发病过程中的表达及可能作用,证实了periostin的表达与心肌纤维化程度、血清血管紧张素II水平,TGF-β1表达呈正相关,提示periostin可能作为血管紧张素II,TGF-β1的下游因子参与VMC心肌纤维化过程。冬虫夏草是我国传统中药,具有调节免疫、清除自由基、抗心律失常及直接抑制病毒复制等作用,本研究主要探讨冬虫夏草是否有抗病毒性心肌炎小鼠心肌纤维化的作用,同时以血管紧张素受体拮抗剂（AT1）厄倍沙坦作为疗效对比,结果表明二者均可通过对periostin的抑制,改善心肌纤维化,同时小剂量冬虫夏草效果与厄倍沙坦相当,大剂量冬虫夏草则优于厄倍沙坦,冬虫夏草的抗心肌纤维化作用,呈量效依赖性,本研究为病毒性心肌炎的临床治疗提供理论和实验依据。本研究创新点:1. periostin在病毒性心肌炎的表达少有报道,本研究探讨periostin在病毒性心肌炎各期的表达及可能作用。2.本研究探讨了冬虫夏草对periostin表达的影响,及其抗病毒性心肌炎心肌纤维化作用和可能机制。更多还原

【Abstract】 Viral myocarditis (VMC) is one of the most common infective diseases in cardiovascular diseases. Most of the patients are annoyed with acute VMC, but someone develops into the chronic VMC, dilated cardiomyopathy with survival rate about five years is around 50%. VMC has been threatening children health severely. Myocardial fibrosis refers to the excessive accumulation of collagen fibers in the normal tissues and structures, marked elevation of collagen concentration, or the changes of collagen constituents. This pathological change exists in every stage of VMC. Now, it is believed that this change is associated tightly with cardiac arrhythmia, cardiac dysfunction and dilated cardiomyopathy. The periostin gene was initially cloned from a mouse calvarial cell line (MC3T3-E1) and originally named osteoblast specific factor-2 (OSF-2). Periostin is a member of a growing family of matricellular proteins. Periostin has been shown to play a crucial role during tissue morphogenesis. Periostin is attended as a novel factor responsible for ventricular dilation. Perostin has been shown to be involved in the progress of Myocardial fibrosis after myocardial infarction It is believed that the abnormal expression of periostin may be related with the occurrence and development of VMC.(1) The expression and function of periostin in mice model with viral myocarditisMaterials and methods : In this study we first established VMC models through BALB/c mice infected with coxsackie virus B3. 100 pure bred male Balb/c mice aged 4 weeks and weighted 18±1g, were divided into control group(n=40) and VMC group (n=60) randomly. VMC model was founded by injecting intraperitoneally 0.1ml CVB3 Nancy solution one time a day for three days, while the control group was injected with 0.1ml Engeal’s fluid excluding virus. The model mice were sacrificed by extirpating eyeballs on day 7, day 14,day 28, and day 56 respectively, after injecting CVB3 ,0.8-1.0ml blood were extracted by burettes. The serums were separated and stored at -70℃, preparing for the test of Ang II and CK-MB. Then all mice were killed by cutting necks, obtaining hearts and putting them partly into 10% formaldehyde solution for biopsy, the other part was stored at -70℃for RT-PCR. The mice of control group were treated by the same methods. After virus inoculation until day 0,day 7,day14,day28,day56, we examined the contents of periostin in the myocardium by SP and RT-PCR . Meanwhile, we examined the score for myocardial necrosis, the level of CK-MB and AngII in serum, CVF and the contents of TGF-β1 mRNA in the myocardium. Based on the results, we further studied the relationship between periostin and other index. Our study would provide new theoretic base and experimental evidence for prevention and therapy of VMC.Results: We found that the model mice displayed tarnishable furs, reduced movements, been weary spirits and poor appetites ,vaulted backs, showed indifference to stimulation or tended to irritation and been lower temperature since the third day. Death occured on day 4, and reached the top from day 7 to day 14. Among 60 mice in model group, 21 died. The death rate was 35.0%. None of the control mice had any symptom and death. The serum CK-MB concentrations of the VMC group were much higher than that of the control mice at each time spot(P<0.01).The peak is in day 14 , after that day the serum CK-MB concentrations decreased. The other indexs include serum AngII concentrations ,CVF, TGF-β1 increased gradually in the course of disease. The expression of periostin was detected in myocardial tissue at the day0, day7, day14, day28, and day56 after CVB3 infection quantitatively by semiquantitative analysis RT-PCR. The results showed that the expression of periostin increased slightly at the 7 day after infection, then the expression was increased continuously, and the expression of periostin was highest at the 56th day after infection. Linear correlation analysis showed the contents of periostin in the myocardium with the level of AngII in serum, CVF and the contents of TGF-β1 in the myocardium is positive correlation remarkably, but score for myocardial necrosis and level of CK-MB in serum have no correlation with the contents of periostin in the myocardium.Conclusions: It suggested that periostin may be involved in the myocardial fibrosis in myocardium of VMC. Ang II and TGF-β1 maybe increase periostin expression, and eventually contributed to the myocardial fibrosis in VMC mice model. Periostin may be a new target in the therapy of VMC. (2): The therapeutical effect and possible mechanism of [Cordyceps sinensis (Berk.) Sacc.] (CS) and Irbesartan in the VMC mice models in chronic phase.Materials and methods :70 pure bred male Balb/c mice aged 4 weeks and weighted 18±1g, were divided into control group(n=10) and VMC group (n=60) randomly. VMC model was founded by injecting intraperitoneally 0.1ml CVB3 Nancy solution one time a month for three monthes , while the control group was injected with 0.1ml Engeal’s fluid excluding virus.Then we successfully established the models of viral myocarditis in chronic phase through BALB/C mice infected with CVB3 .42 models of viral myocarditis in chronic phase were divided into 4 groups. They were VMC-control group (n=12), Irbesartan group (n=10), CS-large group (n=10) and CS-small group(n=10). VMC-control group was treated intragastric with water 1ml/d . Irbesartan group was treated intragastric with Irbesartan at a dose of 10mg/kg.d . CS-large group was treated intragastric with CS at a dose of 7.5g/kg.d CS-small group was treated intragastric with CS at a dose of 2.5g/kg.d .After feeding drugs for 60 days, we weighed the mice, examined the heart function changes, observed Myocardial ultrastructural changes and myocardial collagen fiber distribution,measured CVF and the level of AngII in blood serum. At the same time, the contents of collagen I and collagen III were detected by immunohistochemical techniques. TGF-β1and periostin were detected by reverse transeription polymerase chain reaction Then the therapeutic effect of CS and Irbesartan were discussed.Results:Compared with VMC-control group, the mortality matched Irbesartan group, CS-large group showed significant decrease, while the parameters of heart function were improved (P<0.01), but the parameters is not normal yet (P<0.01). Compared with VMC-control group, the level of Ang II in serum was significantly inhibited by CS (P<0.01). But Irbesartan seemed not act(P>0.05). Compared with VMC-control group, Irbesartan group and CS-large group have lower parameters of CVF(p<0.05). The effect of CS large group is better than the effects of Irbesartan group and CS-smallgroup (p<0.05). Compared with VMC-control group, the expressions of TGF-β1and periostin were attenuated in Irbesartan group and CS-large group (p<0.05). There was no evident difference between CS-small group and VMC-control group(P>0.05). Compared with VMC-control group ,the expression of collagen I was decreased in Irbesartan group, CS-large group and CS-small group (p<0.05). AT the same time, the expression of collagen III was decreased in CS-large group and CS-small group (p<0.05).Conclusions: periostin play an important role in the process of viral myocarditis. CS can offer some protection to myocardial fibrosis in viral myocarditis mice . The inhibition of the expression of periostin may be one of the mechanisms .更多还原