【作者】 王东进；

【导师】 施广飞；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 以往认为,心脏为终末分化器官,其细胞本身不具有增殖分化功能,因此无法修复损伤心肌。但随着细胞生物学技术的进步以及人们研究的深入,心脏中存在心肌干细胞(CSC)的观念已经越来越多地为医学工作者们所接受。本研究将含不同浓度生脉注射液的培养基用于培养心肌组织来源细胞,观察该药能否在体外诱导心肌组织内干细胞分化,并通过临床试验进一步证实生脉注射液具有减轻心肌缺血-再灌注损伤的作用,同时探讨其体外循环心肌保护作用是否与诱导心肌组织内干细胞分化存在关联。第一部分生脉注射液促进人心肌组织内干细胞分化为心肌细胞的体外实验目的：成功培养人心肌组织来源细胞,尝试提取人CSC,观察生脉注射液是否具有诱导干细胞分化的作用,为后期的临床研究奠定基础；方法：选择行心脏手术的病人,将术中切除的心肌组织保留,用组织块消化法提取细胞,通过体外扩增后用免疫磁珠法分选人心肌组织来源干细胞,并尝试用不同浓度生脉注射液干预细胞,利用免疫荧光的方法检测心肌特异性转录因子和心肌特异性蛋白,进一步验证生脉注射液的诱导干细胞分化作用；结果：从人心肌组织中成功提取细胞,并在体外扩增,但未能成功分选人CSC,免疫荧光法证实5μl/ml和10μl/ml生脉注射液可以诱导心肌组织内干细胞分化,浓度增大后诱导效率稍有下降；结论：适当浓度的生脉注射液可以诱导心肌组织内干细胞分化为心肌细胞,但由于CSC本身含量极低,因此未能成功将其分选出来。第二部分生脉注射液在体外循环心肌保护中的作用目的：通过临床研究,证实生脉注射液可以减轻体外循环诱发的缺血再灌注损伤,观察体内试验中生脉注射液是否可以诱导心肌组织内干细胞分化为心肌组织；方法：选择行心脏手术的病人,分别选择术前、术中及术后的六个时间点抽取血液,检测血清中SOD、MDA、cTnl和CK-MB水平,术中切除小块心肌组织,用免疫荧光的方法检测其中心肌特异性转录因子(GATA4、Nkx2.5)和特异性蛋白(Cx 43、cTnI);结果：入院时两组患者血清SOD活力、MDA水平无明显差异,而手术开始时试验组血清SOD活力显著高于对照组,术后一天时试验组血清MDA水平显著低于对照组,而试验组和对照组血清cTnI和CK-MB水平有显著差异；试验组GATA-4和NKx2.5荧光强度显著强于对照组,试验组的cTnT和Cx43仍然趋向较强的荧光信号,但无显著统计学差异；结论：生脉注射液可以增强血清中SOD活力及减少脂质过氧化反应,抑制体外循环过程中的缺血再灌注损伤,而心肌组织内干细胞分化为心肌细胞可能也参与了体外循环心肌保护。第三部分我国大陆地区主动脉夹层发病特征——文献综合分析及与主动脉夹层国际注册研究资料进行对比目的：急性主动脉夹层(AAD)是一种致死率和致残率高的临床急症。为了加深对该病的认识,提高诊治水平,国际主动脉夹层注册研究(IRAD)于1996年成立。中国大陆(MC)是世界上人口最多的地区,每年新发AAD病人越来越多,由于IRAD病例均来源于发达的西方国家,医疗体制、经济发展水平和人种都与MC有所不同,因此该病在IRAD和MC两个群体之间的差异可能被忽略。方法：我们通过检索PubMed数据库和中国全文期刊数据库,通过一定的标准,分别选取了来自IRAD和MC的临床报告,以求助或者购买来获得全文。根据数据性质不同,分别采用t检验、卡方检验或Fisher精确概率计算等方法进行统计,所有统计过程应用SPSS 13.0软件进行。结果：来自MC的AAD病人较IRAD组病人显著年轻,且前者中男性病人比例也高于后者(80.7% vs 68.6%,P<0.001)。中国病人更可能出现非典型症状和体征。与IRAD相比,中国医师更愿意选择磁共振来进行AAD的诊断(45.5% vs 11.6%,P<0.001)。总体上来说,两组住院期间死亡率类似,但是由于研究方法的限制,相当一部分来自MC的病例未能参与统计,并且中国病人接受治疗的比例也较低。结论：通过初步观察和分析,我们可以发现IRAD和MC之间在AAD临床特征上存在一定的差异,但是这些结果需要更深入细致地研究加以证实。更多还原

【Abstract】 In the past, the adult heart had been regarded as terminally differentiated organ, cells isolated from which did not have the capability of generation and differentiation. With the development of technology in cell biology and studying much further, the opinion that there are a cluster of cells with the characteristics of stem cells in the heart have been accepted. In this study, four kinds of cell culture medium containing different concentration of Shengmai injection were used for cell expansion in order to assess whether Shengmai injection could induce stem cells in myocardium to differentiate into cardiomyocytes. Then a clinical trial would be carried out to confirm that Shengmai injection was able to extenuate myocardium ischemical reperfusion injury and to explore the ralationship between cardiac stem cells (CSC) differentiation and myocardial preservation in cardiopulmonary bypass.Part one Shengmai injection inducing the stem cells in myocardium to differentiate into cardiomyocytes in vitroObjective To isolate cells from myocardium and try to abstract cardiac stem cells, then assess whether Shengmai injection could induce stem cells in myocardium to differentiate into cardiomyocytes.Methods After patients’written consent, myocardium discarded in cardiac operation were collected and digested to dissociate and extract heart cells. A few weeks later, the cells were expanded and used to separate the stem cells by the way of immunomagnetic beads sorting. Then four kinds of cell culture medium containing different concentration of Shengmai injection were applied to induce stem cells in myocardium to differentiate into cardiomyocytes and the specificity transcription factors and protein of cardiomyocytes were detected by the method of immunocytochemical staining.Results Heart cells were isolated and expand in vitro successfully and the cell culture medium containing concentration of 5μl/ml and 10μl/ml Shengmai injection could induce stem cells in myocardium to differentiate into cardiomyocytes, but regrettably the cardiac stem cells were not able to sort from heart cells.Conclusion The cell culture medium containing appropriate concentration can induce stem cells in myocardium to differentiate into cardiomyocytes, but the cardiac stem cells were not able to sort from heart cells because of their much low number.Part Two Shengmai performing myocardia) protection during cardiopulmonary bypassObjective To comfirm that Shengmai injection can extenuate ischemia-reperfusion injury caused by cardiopulmonary bypass and determine whether Shengmai injection could induce stem cells in myocardium to differentiate into cardiomyocytes in vivo.Methods After patients’written consent, six blood samples were acquired at six time points in perioperative period which were used to test the contents of SOD、MDA、cTnl and CK-MB in serum. A small size of myocardium was taken in every patient and cardiac-specific transcription factors(GATA4、Nkx2.5)and cardiac-specific proteins(Cx 43、cTnI) were detected by the method of immunocytochemical staining.Results There was no significantly difference in serum SOD activity and MDA content between the two groups on admission. But before operation, serum SOD activity was significantly higher in the experimental group and in the day after surgery serum MDA level was significantly lower than that in the control group. And Serum cTnl and CK-MB levels were significantly different between the experimental and control group. Fluorescence intensity of NKx2.5 GATA-4 in the experimental group was significantly stronger than that of the control group, and fluorescence intensity of cTnT and Cx43 in experimental group was also stronger than that of the control group but no significant difference.Conclusion Shengmai injection could enhance the SOD activity and inhibit lipid peroxidation which extenuate ischemia-reperfusion injury caused by cardiopulmonary bypass during. Stem cells in myocardium differentiation into cardiomyocytes might also be involved in myocardial protection during cardiopulmonary bypass.Part three Preliminary characterization of acute aortic dissection in China’s mainland——Comprehensive analysis of the literature in comparison with the International Registry of Acute Aortic Dissection (IRAD)Objective Acute aortic dissection (AAD) is a clinical emergencies with high morbidity and mortality. In order to more fully understand the characteristics of AAD, the International Registry of Acute Aortic Dissection (IRAD) was established but the registry had only enrolled cases from developed countries, which could not be sufficient to know the full extent of this condition. And there may also be some differences in the characteristics of AAD between IRAD countries and China’s mainland(MC).Methods Reports from IRAD and MC were collected by searching in the PubMed Databases and the Chinese Journal Full-text Database, and patients’data were abstracted. The 2 groups were compared using the chi-square test, Fisher’s exact test and Student’s t test as appropriate.Results AAD patients from MC were significantly younger than IRAD and the percentage of male patients in the Chinese group was higher than IRAD (80.7% vs 68.6%, P<0.001). Patients in MC were less likely to present with typical symptoms and signs except for any focal neurological deficits. Different from the IRAD group, Chinese patients were prone to undergo magnetic resonance imaging (MRI) to make the diagnosis of AAD (45.5% vs 11.6%, P<0.001). The in-hospital mortality was similar between 2 groups but only smaller proportion of AAD patients in MC underwent surgical or medical treatment.Conclusions From this paper, the general characteristics of AAD patients in MC were shown and differences in some clinical variables between MC and IRAD groups still existed. However, the results need to be proved by much further studies.更多还原