【作者】 张健瑜；

【导师】 许顶立；

【作者基本信息】 南方医科大学 ， 心血管内科（专业学位）， 2014， 博士

【摘要】 背景及目的：老年退行性心脏瓣膜病(Senile degenerative heart valvular disease,SDHVD)又称为老年性心脏钙化性瓣膜病,发病率随着年龄的增长而增加,病变程度随着时间的推移而加重,引起心脏形态及功能变化,导致心脏血流动力学紊乱,诱发严重心血管系统并发症,是老年心血管疾病致死的重要原因之一。我国渐入老龄国家,心血管钙化性疾病的发病率及患病人数均呈上升趋势。心脏瓣膜钙化主要累及左心瓣膜,以主动脉瓣病变最多见,其次是二尖瓣。病理特点是瓣膜中脂质和胶原沉积,炎症细胞如T淋巴细胞和巨嗜细胞浸润,及包含类平滑肌细胞和类成骨细胞的钙化组织。然而目前退行性心脏瓣膜病的机理仍未完全阐明。既往认为该病的发展是与老化有关的退行性变的过程,但是最近的研究表明心脏瓣膜钙化是可能是瓣膜组织对某种启动因子的反应及多因素参与的主动过程。Klotho基因是一个与衰老密切相关的基因,该基因在生命体成熟与健康过程中发挥着重要作用。该基因缺陷的小鼠表现出动脉粥样硬化、异位钙化、低血糖及严重的高磷酸盐血症等与人类衰老相似的症状,而小鼠重新表达该基因后以上症状均得到缓解。目前关于Klotho与退行性心脏瓣膜病的研究报道尚不多。成纤维细胞生长因子23(Fibroblast growth factor23, FGF23)是近年来新发现的一种参与血磷代谢的细胞因子,属于多肽激素。Klotho蛋白是FGF23信号转导通路的关键因子,FGF23通过Klotho蛋白的介导发挥生物学作用。然而国内外关于FGF23/Klotho在退行性心脏瓣膜病患者中的表达及其与愈后关系的研究报道尚少。本课题就Klotho蛋白、FGF23因子及脂蛋白(a)(Lipoprotein (a),Lp(a))等在退行性心脏瓣膜病患者的表达水平进行分析研究,探索其在退行性心脏瓣膜钙化性疾病的作用及可能机制,并且对部分患者进行了随访,以分析影响患者生存的因素。第一部分：退行性瓣膜病变患者相关危险因素分析方法：随机选取佛山市第一人民医院2012年6月份至2013年7月份住院的45岁以上病人253例,其中男144例,女109例。有下列情况者不纳入本研究：(1)风湿性心脏病伴心脏瓣膜病变者；(2)有先天性心脏瓣膜畸形者；(3)感染性心内膜炎所致的瓣膜病变者；(4)合并恶性肿瘤者；(5)胸部经放射性治疗者；(6)合并免疫性疾病患者；(7)慢性血液透析患者；(8)梅毒患者。登记各研究对象的相关临床资料,包括性别、年龄,高血压、糖尿病、冠心病史及吸烟史,并抽取静脉血检测总胆固醇(Total cholesterol,TCH)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol, LDL-c)、高密度脂蛋白胆固醇(High-density lipoprotein cholesterol,HDL-c)、甘油三酯(Triglycerides,TG)、Lp(a)等,根据MDRD公式计算肾小球滤过率(Glomerular filtration rate,GFR) eGFR (ml/min/1.73m2)=170×[Scr]-0.999×(年龄)-0.176×[女性×0.762]×[BUN]-0.17×[Alb]×0.318。所有病例均进行了完整的超声心动图检查,包括二维、M型和多普勒超声心动图。根据声心动图检测结果分为瓣膜钙化及无瓣膜钙化组。判定心瓣膜退行性钙化病变的标准为：瓣膜或瓣环局限性增厚、回声增强,瓣叶活动受限,开放幅度减少。所有数据分析均用统计软件SPSS16.0完成,计量资料用均数±标准差描述,组间比较采用独立样本t检验,若方差不齐则采用t’检验；计数资料采用例(%)表示,用卡方检验比较组间差异,检验水准定为a=0.05；影响因素采用多元Logistic回归分析,并计算出比值比(Odds ratio,OR)。结果：1.瓣膜钙化组病例中单纯主动脉瓣钙化发生率最多,共检出91例(81.98%),其次是主动脉瓣合并二尖瓣钙化,检出17例(15.31%),单纯二尖瓣钙化3例(2.7%),未检出三尖瓣和肺动脉瓣钙化病变。2.钙化组111例患者中,无瓣膜功能障碍的有26例(23.42%)；单纯瓣膜关闭不全的有69例(62.16%),所占比例最大；狭窄合并关闭不全的有12例(10.81%)；单纯狭窄的有4例(3.60%),所有瓣膜狭窄的病例均为主动脉瓣钙化病例,二尖瓣钙化的病例中未发现有二尖瓣狭窄。3.瓣膜钙化组与无瓣膜钙化组两组间的性别、冠心病、高血压、糖尿病、吸烟、TCH、TG、LDL-C、HDL-C、尿酸无显著差异(p>0.05),两组间的Lp(a)差异有显著性(372.82±418.87mg/L比256.92±317.89mg/L,p=0.018),两组间的年龄差异有显著性(73.39±8.76岁比69.81±9.42岁,p=0.002)；两组间的eGFR有显著性差异(58.33±19.10ml/min比68.08±19.59ml/min, p=0.000).4.经Logistic多因素回归分析,年龄、Lp(a)、eGFR是瓣膜钙化的危险因素(OR值分别为1.038、1.001和0.983,β值分别为0.037、0.001和—0.017,p值分别为0.030、0.014和0.027)。结论：退行性瓣膜病主要累及主动脉瓣与二尖瓣,主动脉瓣钙化发生率最高,其次是主动脉瓣合并二尖瓣钙化。瓣膜退行性病变的患者Lp(a)水平较无瓣膜退行性变患者高,Lp(a)是发生瓣膜退行性病变的危险因素,Lp(a)的增加可能会增加瓣膜钙化的风险。年龄是退行性瓣膜病的危险因素,本研究显示年龄每增长1岁,退行性瓣膜病的发病危险增高了1.038倍。瓣膜退行性病变的患者eGFR水平较无瓣膜退行性变患者低,eGFR是发生瓣膜退行性病变的危险因素,eGFR下降会使心脏瓣膜钙化的风险增高。第二部分：退行性心脏瓣膜病患者血清FGF23、Klotho蛋白水平的改变在第一部分我们证实了年龄、脂蛋白(a)和eGFR是退行性心脏瓣膜病的危险因素,但尚未明确Klotho蛋白、FGF23、高敏C反应蛋白(High-sensitivity C-reactive protein,hs-CRP)等在退行性心脏瓣膜病患者中的水平,因此,本部分研究旨在通过检测Klotho蛋白、FGF23因子等在退行性心脏瓣膜病患者的表达水平进行分析研究,探索其在退行性心脏瓣膜钙化性疾病的作用及可能机制。方法：随机选取佛山市第一人民医院2012年8月份至2013年4月份住院的50岁以上病人71例(男41例,女30例),有下列情况者不纳入本研究：(1)风湿性心脏病伴心脏瓣膜病变者；(2)有先天性心脏瓣膜畸形者；(3)感染性心内膜炎所致的瓣膜病变者；(4)合并恶性肿瘤者；(5)胸部经放射性治疗者；(6)合并免疫性疾病患者；(7)慢性血液透析患者；(8)梅毒患者；(9)急性心肌梗死患者。登记各研究对象的相关临床资料,包括性别、年龄,高血压、糖尿病、冠心病史及吸烟史,并抽取静脉血检测钙(Calcium,Ca)、磷(Phosphorus,P)、全段甲状旁腺激素(Intact parathyroid hormone,iPTH)、hs-CRP等,根据MDRD公式计算eGFR。抽取血液标本并用酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)测定血清Klotho蛋白及FGF23的水平。所有入选患者行经胸超声心动图检查明确心脏瓣膜情况。根据声心动图检测结果分为瓣膜钙化及无瓣膜钙化组。判定心脏瓣膜退行性钙化病变的标准为：瓣膜或瓣环局限性增厚、回声增强,瓣叶活动受限,开放幅度减少。所有数据分析均用统计软件SPSS16.0完成。计量资料用均数±标准差描述,组间比较采用独立样本t检验,若方差不齐则采用t’检验,计数资料用卡方检验比较组间差异,检验水准定为α=0.05。结果：1.瓣膜钙化组有39例(男25例,女14例),无瓣膜钙化组32例(男16例,女16例)；瓣膜钙化组与无瓣膜钙化组两组间年龄有显著差异(73.67±7.88岁比69.41±8.40岁,p=0.031),两组间冠心病、糖尿病、高血压及吸烟无显著差异(p>0.05)。2.瓣膜钙化组与无瓣膜钙化组两组间的Kloth蛋白、钙、磷及iPTH无显著差异(p>0.05)。3.瓣膜钙化组与无瓣膜钙化组两组间的hs-CRP水平有显著差异(19.06±31.23mg/L比5.06±7.23mg/L p=0.010)4.瓣膜钙化组与无瓣膜钙化组两组间的FGF23水平有显著差异(85.43±128.92pg/ml比11.82±25.43pg/ml,p=0.001)；按年龄分层后,瓣膜钙化组的FGF23水平均仍显著高于无瓣膜钙化组(p<0.05)；按肾小球滤过率分层后,瓣膜钙化组的FGF23仍显著高于无瓣膜钙化组(p<0.05)。5.FGF23浓度与Klotho蛋白、钙、磷、iPTH浓度不相关。结论：退行性瓣膜病变患者的FGF23及hs-CRP水平显著高于无退行性瓣膜病变患者,钙、磷、iPTH、Klotho水平并未显示两组差异,且FGF23浓度与Klotho蛋白、钙、磷、iPTH浓度不相关,FGF23对瓣膜退行性变的影响可能独立于FGF23/Klotho轴。第三部分：退行性心脏瓣膜病患者的生存分析在第二部分研究的结果显示退行性瓣膜病变患者的FGF23及hs-CRP水平显著高于无退行性瓣膜病变患者,但我们尚不清楚退行性心脏瓣膜病患者的生存情况及影响因素。因此,在第三部分,我们通过对患者进行了随访,以分析影响患者生存的因素。方法：对第二部分入选患者进行为期32周的随访,主要为电话随访,并结合电子病历系统了解部分再住院病人的病情,以死亡作为终点事件,应用SPSS16.0软件进行数据录入及统计。生存资料对比采用Kaplan—Meier法,Log—rank检验,绘制生存曲线；生存危险因素采用COX回归模型进行分析；P<0.05认为有统计学差异。结果：1.32周时无瓣膜钙化组生存率为100%,瓣膜钙化组生存率为86.5%,两组间有显著差异(p=0.045)；瓣膜钙化组中死亡5例,其中4例死于心力衰竭,1例家属不愿意透露死因。2.将年龄、hs-CRP、Klotho蛋白、FGF23.eGFR.Lp(a)纳入COX回归模型,结果显示hs-CRP.FGF23为影响生存的主要危险因素(RR=1.033p=0.004；RR=1.007p=0.048)。结论：瓣膜钙化较无瓣膜钙化患者的生存率下降,心力衰竭是退行性心脏瓣膜病患者的死亡的主要原因。hs-CRP及FGF23水平是患者死亡的独立危险因素,血清hs-CPR及FGF23水平的升高增加了死亡的风险。全文总结：在本研究中,我们探讨Klotho蛋白、FGF23因子等在退行性心脏瓣膜病患者的表达水平,对退行性心脏瓣膜钙化相关危险因素进行多变量分析,并且对部分患者进行了随访,现总结如下：(1)退行性心脏瓣膜病变与年龄、肾小球滤过率、Lp(a)有关；年龄的增加及肾小球滤过率下降会使心脏瓣膜钙化的风险增高。(2) FGF23与退行性心脏瓣膜病有关,FGF23可能参与了瓣膜退行性变的过程,其机制可能独立于FGF23/Klotho轴。(3)瓣膜钙化患者生存率下降,心力衰竭是其死亡的主要原因；血清hs-CRP及FGF23水平的升高增加了死亡的风险。更多还原

【Abstract】 Background and Objective:Senile degenerative heart valvular disease (SDHVD) is also known as senile heart calcific valvular disease. The morbidity of SDHVD increases with age, and the valvular stenosis aggravate with disease duration. SDHVD is associated with an increased risk of cardiovascular events and mortality in the elderly which can leads to cardiac morphological changes and dysfunction, cardiac hemodynamics disorder and severe cardiovascular complication. With the arriving of aging society the incidence and prevalence of SDHVD is exponentially increasing in China. Valvular calcification mainly involves left heart valve, in which aortic valve is the most common one, followed by the mitral valve. The pathological feature of SDHVD encompass deposition of lipid and collagen in valve and infiltration by inflammatory cells such as T lymphocytes and macrophage, and the valve interstitial cells who adopt an osteoblast-like phenotype leading to calcium accumulation are also evident. The mechanisms of SDHVD are still unclear. Previously, SDHVD was thought to be a chronic degenerated process with aging, but some studies recently indicate that valvular calcification may be a multiple-factors involved and active process which valve tissues react to some initiative factors. Klotho is a gene associated closely with aging, which plays an important part in maturation of body and health. Klotho-deficient mice show some symptoms similar to aging such as atherosclerosis, ectopic calcification, hypoglycemia and severe hyperphosphatemia. And these symptoms would ease when this gene re-express in mice. At present,the research of Klotho protein and senile degenerated heart valvular disease was few.Fibroblast growth factor23(FGF23) belongs to the polypeptide hormone family.It is a cytokine newly discovered in recent years involved in the metabolism of phosphorus. Klotho protein is closely related with FGF23. It is a key factor in FGF23signaling, Klotho proteins mediate FGF23to exert biological function. The studies concerning expression of Klotho protein and FGF23in SDHVD patients are sill rare.This study aim to analyze the expression of Klotho protein、FGF23and Lp(a) etc in SDHVD patients, investigate their role in valvular calcification. We followed up some patients in order to analyze the related factors influencing survival of patients.Part Ⅰ:Related risk factors analysis on senile degenerative valvular heart diseaseMethods:We identified253SDHVD patients of age45or above who hospitalized in the fisrt people’s Hospital of Foshan between June2012and July2013at random. The following cases were excluded in this study:(1) diagnosed as rheumatic heart disease with valvular stenosis;(2) diagnosed as congenital valve malformation;(3) valvular stenosis caused by infective endocarditis;(4) complicated with malignant tumor;(5) had radiotherapy at chest;(6) complicated with immunologic diseases (7) chronic hemodialysis patients (8) diagnosed as syphilis. Record related clinical data of all patients enrolled including sex, age, history of hypertension, diabetes,coronary heart disease and smoking; obtain vein blood sample for TCH, LDL-C, HDL-C, Lp(a) test. Caculate glomerular filtration rate(GFR) according to the MDRD formula eGFR (ml/min/173m2.)=170×[Scr]-0.999×(Age)-0.176×[Female×0.762]×[BUN]-0.17×[Alb]×0318.All patients enrolled underwent transthoracic echocardiographic examination to make clear the heart valve condition. According to the test results divide the patients into echocardiographic valvular calcification and without valvular calcification group. Criteria for determining heart valve calcification:valvular membrane or annulus become thickened in local site, echo become stronger, valvular leaflets opening range decreased. Statistical analysis was performed using the statistical package SPSS16.0. Continuous variables are expressed as mean±SD. We used Student’s t-test for differences between independent continuous variables. Whereas categorical data are described as percentages, and we used the χ2test to test for categorical variables. P value less than0.05was accepted as statistically significant. Multiple logistic regression analysis was used to evaluate the association between SDHVD and influence factors. Odds ratios were calculated.Result:1. The incidence of simple aortic valvular calcification is the highest in SDHVD group,91cases(81.98%), followed by the incidence of aortic valvular calcification combining mitral valvular calcification,17cases(15.32%).3cases have simple mitral valvular calcification.Tricuspid valvular calcification and pulmonary valvular calcification were not found.2. There were111patients with valvular calcification,69patients with simple valvular regurgitation,12patients with valvular regurgitation and valvular stenosis,4 patients with simple valvular stenosis.The proportion of simple valve regurgitation in SDHVD group is the highest(62.16%), all valve stenosis were aortic valvular calcification, mitral stenosis were not found in patients with mitral valvular calcification.3. There were no significant differences between SDHVD group and non-SDHVD group in sex, coronary heart disease, hypertension, diabetes, smoking, TCH, TG, LDL-C, HDL-C and uric acid(p>0.05). There were significant differences in Lp(a)(372.82±418.87mg/Lvs256.92±317.89mg/L,p=0.018), age(73.39±8.76years vs69.81±9.42years, p=0.002) and eGFR(58.33±19.10ml/min vs68.08±19.59ml/min, p=0.000) between two groups.4. Multivariate logistic regression analysis demonstrated that age, Lp(a) and eGFR were the risk factors of SDHVD (OR=1.038β=0.037p=0.030, OR=1.001β=0.001p=0.014and OR=0.983β=-0.017p=0.027, respectively).Conclusion:Degenerative heart valvular disease is mainly involved with aortic and mitral. The incidence of simple aortic valvular calcification is the highest in SDHVD,followed by the incidence of aortic valvular calcification combining mitral valvular calcification. The Lp(a) was higher in SDHVD group than in non-SDHVD group. Lp(a) is a risk factor for SDHVD. Increase of Lp(a) can increase the risk of heart valvular calcification. Age also is a risk factor for SDHVD, This study shows that for each increase of1year of age, the incidence of SDHVD increased the risk of a1.038-fold. GFR was lower in SDHVD group than in non-SDHVD group.eGFR is a risk factor for SDHVD. Decreasing of GFR increase the incidence risk of SDHVD. Part Ⅱ:The levels of serum FGF23and Klotho protein on senile degenerative heart valvular diseaseIn the first part we confirmed age,lipoprotein(a) and eGFR were the risk factors for SDHVD. But the levels of Klotho protein, FGF23in patients with SDHVD were not clear yet. Therefore,we analyzed the expression of Klotho protein and FGF23in SDHVD patients, investigated their role in valvular calcification.Methods:We identified71SDHVD patients of age50or above who hospitalized between August2012and April2013at random. The following cases were excluded in this study:(1) diagnosed as rheumatic heart disease with valvular lesions;(2) diagnosed as congenital valve malformation;(3) valvular stenosis caused by infective endocarditis;(4) complicated with malignant tumor;(5) had radiotherapy at chest;(6) complicated with immunologic diseases (7) chronic hemodialysis patients;(8) diagnosed as syphilis;(9) Acute myocardial infarction.Record related clinical data of all patients enrolled including sex, age, history of hypertension, diabetes,coronary heart disease and smoking; obtain vein blood sample for calcium, phosphorus, iPTH, hs-CRP test. Calculate GFR with MDRD formula; obtain blood sample to detect the level of Klotho and FGF23by ELISA. All patients enrolled underwent transthoracic echocardiographic examination to make clear the heart valve condition. According to the test results divide the patients into echocardiographic valvular calcification and without valvular calcification group. Statistical analysis was performed using the statistical package SPSS16.0. Continuous variables are expressed as mean±SD. We used Student’s t-test for differences between independent continuous variables and the χ2test to test for categorical variables. P value less than0.05was accepted as statistically significant.Result: 1. There were39patients with SDHVD.There were no significant differences in coronary heart disease, hypertension, diabetes and smoking between SDHVD group and non-SDHVD(p>0.05).There were significant differences in age(73.67±7.88years vs69.41±8.40years, p=0.031) between two groups.2. There were no significant differences in and the level of Klotho protein, calcium, phosphorus and iPTH between SDHVD group and non-SDHVD group(P>0.05).3. There were significant differences in the level of hs-CRP(19.06±31.23mg/L vs5.06±7.23mg/L,P=0.010) between two groups.4. There were significant differences in the level of FGF23(85.43±128.92pg/ml vs11.82±25.43pg/ml, P=0.001) between two groups. When stratified by age, the SDHVD group had a higher lever of FGF23(P<0.05). And when stratified by glomerular filtration rate the lever of FGF23was still higher in SDHVD group(P<0.05).5. The level of FGF23was not associated with the level of Klotho protein, calcium, phosphorus and iPTH.Conclusion:The levels of FGF23and hs-CRP in patients with SDHVD were significant higher than in non-SDHVD. There were no significant differences in the level of Klotho protein, calcium, phosphorus and iPTH between two groups. The level of FGF23was not associated with the level of Klotho protein, calcium, phosphorus and iPTH. FGF23may be involved in the process of the SDHVD, whose mechanism may be independent of the FGF23/Klotho axis. Part Ⅲ:Survival analysis of the patients in senile degenerative heart valvular diseaseThe study in the second part showed the levels of FGF23and hs-CRP in patients with SDHVD were significantly higher than in non-SDHVD patients.But we do not know the survival of patients with SDHVD,and the factors affecting it. Therefore we followed up the patients to analyze the related factors influencing the survival of patients.Methods:The enrolled patients in Part Ⅱ were followed up for32weeks, mainly by telephone follow-up. In addition, get information about the disease process of re-hospitalized patients from hospital records and use death as end point. Statistical analysis was performed using the statistical package SPSS16.0. Survival curves were calculated by the Kaplan-Meier method, and comparison was performed by the log-rank test. A Cox proportional hazards regression model was used for the multivariable analysis of independent prognostic factors for survival. P value less than0.05was considered statistically significant.Result:1. The survival rate of non-SDHVD group in32weeks was100%, and the survival rate of SDHVD group was86.5%, there was significant difference between two groups(P=0.045).5patients died in the SDHVD group,4patients died of heart failure.2. A multivariate Cox regression model was used to analyze the association between survival and clinical parameter or molecular risk factor including age, hs-CRP, Klotho protein, FGF2, GFR, Lp(a). The hs-CRP, FGF23was identified as an independent prognostic factor for survival (RR=1.033P=0.004; RR=1.007P=0.048,respectively). Conclusion:The survival rate of SDHVD patients decreased.Heart failure was the leading cause of mortality in degenerative valvular heart disease.The hs-CRP, FGF23was identified as an independent prognostic factor for survival.Summary:In this study,we analyzed the expression of Klotho protein、FGF23and Lp(a) etc in SDHVD patients, investigated their role in valvular calcification. We followed up some patients in order to analyze the related factors influencing survival of patients.Now we summarize below:(1) Age and decrease of glomerular filtration rate can increase the risk of heart valvular calcification.(2) FGF23may be involved in the process of the SDHVD, whose mechanism may be independent of the FGF23/Klotho axis.(3) The survival rate of SDHVD patients decrease. Heart failure is the leading cause of mortality in degenerative valvular heart disease. The hs-CRP, FGF23was identified as an independent prognostic factor for survival.更多还原