【作者】 张琴；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 研究背景：心脑血管疾病是严重危害人类健康的主要慢性非传染性疾病。其中冠心病和脑卒中是世界上最常见的死亡原因。在我国随着人口的老龄化,以冠心病,脑卒中为代表的心脑血管疾病其发病率,致死率及致残率呈逐年上升的趋势。尽管现在已经发现了许多心脑血管疾病的危险因素,如高血压,糖尿病,血脂代谢的异常,男性,年龄,体重,体重指数等。然而这些危险因素只能解释部分病人,因此寻找新的心脑血管疾病危险因素将具有重要的临床意义。基础研究证明,激肽-缓激肽系统(Kallikrein-Kinin/bradykinin system, KKS)与肾素血管紧张素系统(Renin-Aangiotensin System, RAS)相互作用并在心脑血管疾病的发生发展中具有重要的作用。人组织激肽释放酶(TK)是一类丝氨酸蛋白酶,能促进低分子激肽原释放血管活性肽从而发挥一系列的生物活性作用,命名为KLK1。在人和动物的高血压、心血管疾病和肾脏疾病的模型中,均可见到人组织激肽释放酶的含量降低。我们在转基因的动物模型中发现人组织激肽释放酶具有降低血压,改善胰岛素抵抗和改善肾功能的作用。另外,大量研究还证明人组织激肽释放酶具有抑制细胞凋亡,炎症,纤维化和肥大,促进血管生成和神经发生的作用。因此,人组织激肽释放酶的水平在人的心脑血管疾病的发生发展的过程中可能具有重要的临床意义。由于人组织激肽释放酶在心血管疾病的保护作用已得到基础研究及动物实验的充分证明,而其在冠心病和脑卒中的临床研究中国际上还未涉及。因此,对人组织激肽释放酶的含量与冠心病和脑卒中的关系的病例-对照研究和队列研究具有较好的应用前景。鉴于人组织激肽释放酶的含量检测的应用前景,国外已有关于人组织激肽释放酶含量检测的ELISA试剂盒,但是其操作过程繁琐并且价格昂贵难于获得,而目前国内尚无此类的高效检测试剂盒。而建立具有高灵敏度、高特异性的人组织激肽释放酶含量测定ELISA试剂盒,需要制备特异性强,效价高并可以大量生产的抗人组织激肽释放酶的抗体及较纯的人组织激肽释放酶的蛋白或融合蛋白。本研究首先制备了高纯度并方便大量生产,具有高特异性及高灵敏度的抗人组织激肽释放酶单克隆抗体,同时制备了人组织激肽释放酶融合蛋白并将其纯化。然后建立了针对人组织激肽释放酶的高效ELISA检测试剂盒,同时建立检测血浆中人组织激肽释放酶的ELISA法。我们同时采用病例-对照研究和队列研究在评估人组织激肽释放酶与脑卒中的关系的同时判定其对卒中再发的预测价值。另外,我们采用病例-对照研究的方法评估了血浆中人组织激肽释放酶与冠心病的发病率及严重程度的关系。我们还对血浆中人组织激肽释放酶的含量和血脂的关系进行了评估。最后,我们对冠心病患者中ACEI类药物的使用情况及与血浆人组织激肽释放酶的含量进行了评估。方法：(1)利用表位设计软件设计一段位于KLK1尾端的特异性12肽序列并采用单抗制备技术筛选出杂交瘤细胞株HTK并扩大培养杂交瘤细胞将其注入小鼠腹腔内诱生腹水并纯化出高效价、高特异性的抗人组织激肽释放酶的单克隆抗体。(2)利用原核表达的方法表达并纯化人组织激肽释放酶融合蛋白(TK-4T1)。采用双抗体夹心技术和生物素-亲和素放大系统建立高效的针对人组织激肽释放酶的ELISA检测试剂盒。(3)采用病例-对照研究的方法收入1,268例脑卒中患者和1,210例正常对照,检测其血浆中人组织激肽释放酶的含量,并做5年随访研究。评估血浆中人组织激肽释放酶的含量与脑卒中及再发的关系。(4)采用横截面的病例-对照研究收入890例冠心病患者和905例正常对照。检测其血浆内人组织激肽释放酶含量并评估其与冠心病及其严重程度的关系。同时,采用判别分析和ROC曲线判定其对冠心病的临床应用价值。(5)评估了社区人群中血浆人组织激肽释放酶含量与血脂的关系。(6)评估了ACEI类药物与血浆人组织激肽释放酶的含量的关系。结果：(1)我们获得了8株可分泌抗HK的杂交瘤细胞株,命名为1A,1B8,1E4,2E4,2F8,3B7,4D5,4E2。纯化后的2F8具有较高的效价(1：25600)。经12% SDS-PAGE和Western blotting证实,该单克隆抗体制备成功并具有较高的纯度。(2)表达的融合蛋白经SDS-PAGE蛋白电泳及考马斯亮蓝染色显示,大约在55KD处有一条浓染的新增蛋白条带。经Western bloting鉴定,能与人组织激肽释放酶单克隆抗体发生强阳性反应,HK-4T1融合蛋白具有较强的免疫原性。生物素-亲和素双抗体夹心ELISA法的结果提示：以0.5ug/ml,0.25 ug/ml,0.125 ug/ml,0.062 ug/ml,0.031 ug/ml, 0.016 ug/ml,0.008 ug/ml为横坐标,其对应的OD值为纵坐标绘制标准曲线,其线性关系良好(r=0.9987)。利用此试剂盒检测了124例正常人的血浆中HK1的含量,每组做三个复孔,间隔6个月后再重复一次。其组内差异为1.7%,组间差异为8%。正常人的HK1的血浆含量为252±93 ng/ml。(3)脑卒中患者血浆中人组织激肽释放酶的含量明显低于对照组(0.163±0.064 mg/l versus 0.252±0.093 mg/l, P<0.001),并且其与脑卒中的发病率及5年无事件生存率呈剂量依赖性的负相关关系。服用阿司匹林的脑卒中患者其血浆中人组织激肽释放酶的含量高于对照组(0.172±0.064 mg／lvs.0.162±0.065 mg／l,P=0.003)。(4)冠心病患者血浆中人组织激肽释放酶的含量明显高于对照组(0.347±.082 versus 0.256±0.087 mg/L, P<0.001)。升高的血浆人组织激肽释放酶的含量与冠心病呈正相关关系,而与其严重程度呈负相关关系。(5)血浆人组织激肽释放酶的含量与高密度脂蛋白(HDL)成正相关关系(r=0.069,P=0.003),与低密度脂蛋白(LDL)成负相关关系(r=-0.051,P=0.030)。(6)ACEI能明显提高冠心病患者血浆人组织激肽释放酶水平(0.388±0.076 mg/l vs. 0.320±0.079 mg/l, P<0.001; OR=3.20,95%CI 2.59-3.95, P<0.001)。结论：我们首先成功制备了人组织激肽释放酶的单克隆抗体和ELISA试剂盒。然后,本研究证明了降低的血浆人组织激肽释放酶含量具有较高的脑卒中发生率和再发率。升高的血浆人组织激肽释放酶含量与冠心病发生呈正相关关系而与冠心病的严重程度呈负相关关系。因此,人组织激肽释放酶可能具有重要的心脑血管保护作用,其在血浆中含量的可能预测脑卒中及冠心病的发生及再发。同时,人组织激肽释放酶与心脑血管疾病的传统危险因素之间存在一定的联系,尤其是其与血脂代谢的关系较为密切。更多还原

【Abstract】 Background:cardiovascular and cerebrovascular diseases were serious and diminished quality of life in the world. Stroke and coronary heart disease(CHD) were leading causes of death in the world and absolute numbers of the events will increase dramatically in China due to a growing and aging population alone. In the last half-century, a number of important risk factors of stroke have been recognized, including hypertension, age, sex, weight, Body Mass Index (BMI), diabetes and smoking, as well as dyslipidemia. However, these risk factors can explain only part of patients, suggesting that one needs to find more risk factors to explain the remaining patients.Basic study proved that the interactions among kallikrein-Kinin/bradykinin system (KKS) and renin-Aangiotensin System (RAS) paly important role in the development of cardiovascular and cerebrovascular diseases. Tissue kallikrein (TK), a serine protease, processes low molecular weight kininogen substrates to release vasoactive bradykinin and kinin peptides. Kinins bind to bradykinin receptors present in the endothelium, the smooth muscle cells of vascular walls and elsewhere, and activate signaling pathways such as NO-cAMP and prostacyclin-cAMP, which trigger a broad spectrum of biological effects including vasodilatation, smooth muscle contraction and relaxation, inhibition of apoptosis, inflammation, proliferation, hypertrophy and fibrosis, and promotion of angiogenesis and neurogenesis. These indicate a potential predictive value of TK in cardiovascular and cerebrovascular diseases. As the protective effect of TK on cardiovascular disease has been fully proved in basic research, the role TK on CHD and stroke in clinical research has not yet refer to in the world. Therefore, it is of interest to perform case-control study and cohort study to investigate the associations among TK, CHD and stroke. In view of the clinical utility of TK, the ELISA kit special to TK with cumbersome and expensive had developed abroad. Because of the expensive kits and complex method of the kit it is necessary to prepare monoclonal antibody (mAb) against tissue kallikrein, prepare pure protein or fusion protein with TK and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in plasma, serum and urine.Monoclonal antibody against TK with high specific and titer first and the fusion protein of TK were prepared. We then employs double antibody sandwich ELISA technique and Biotin-Avidin System to develop the ELISA kit. A case-control study was used to evaluated the association among TK levels and the presence and severity of CHD. Another case-control study and prospective study were used to assess the value of TK on stroke and recurrecce in 5-years fellow-up.Methods:(1) BALB/c mice were immunized with the conjugate of hemocyanin (KLH) and polypeptide special for HK, and then mAb was prepared by hybridoma technique. (2) Preparing and purifying the fusion protein (TK-4T1) by prokaryotic expression and then developing ELISA kit. This kit employs double antibody sandwich ELISA technique and Biotin-Avidin System. (3) This is a case-control study with 1,268 stroke patients (941 cerebral infarction,327 intracerebral hemorrhage) and 1,210 controls. Plasma TK levels were measured using enzyme-linked immunosorbent assay and a five-year follow-up was performed. To assess whether TK levels contribute to the risk of stroke and reoccurrence, logistic regression and Cox proportional-hazards models were used. (4) We enrolled 890 CHD patients and 905 controls in this cross-sectional study. Plasma TK levels were measured using enzyme-linked immunosorbent assay and traditional risk factors of CHD were registered in all participants. Associations among plasma TK levels, CHD and the severity of CHD were examed considering traditional risk factors. Discriminat analysis and receiver-operator characteristic (ROC) curves evaluated the clinical utility of plasma TK levels on CHD. (5) double antibody sandwich with biotin-avidin ELISA were used to detected plasma TK levels in 1881 normal person. We described the distribution of plasma TK and perfumed analysis with bivariate correlation and partial correlation. (6) plasma TK levels in 464 CHD patients were detected by double antibody sandwich with biotin-avidin ELISA.162 of them were with history of ACEI taking,302 of them were controls. We described the distribution of plasma TK and analyzed the data by Independent sample T test and unconditional logistic regression analysis.Results:(1) 8 hybridoma cell lines secreting mAbs special to HK, labeled 1A,1B8, 1E4,2E4,2F8,3B7,4D5,4E2.2F8 was purified and of high titer (1:25600).12% SDS-PAGE and Western blotting demonstrated successful preparing and purification of mAbs. (2) The fusion proteins identified by SDS-PAGE and Western bloting, results showed that a stain of additional protein bands was at the department of 55KD. The Western bloting proved TK-4T1 was the interst protein. This assay recognizes both recombinant and native HK. The linearity of this ELISA kit is demonstrated (r=0.9987).Among 124 people with three TK measurements during a six-month period, the intra-individual coefficient of variation was 1.7 percent, reflecting the long-term stability of TK levels. The range of detection of the assay is 0.007 to 0.5 mg per liter. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing of samples. (3) Plasma TK levels were significantly lower in stroke patients (0.163±0.064 mg/1 versus 0.252±0.093 mg/l, P<0.001), especially in ischemic stroke. Higher plasma TK levels were directly associated, in a dose-response manner, with a lower risk of the first-ever stroke after multivariate adjustment of traditional risk factors. As compared with the first quarter, the odd ratios for stroke were as follows: second quarter,0.78 (95% CI,0.56 to 1.07); third quarter,0.23 (95% CI,0.17 to 0.32); fourth quarter,0.04 (95% CI,0.03 to 0.07). Furthermore, stroke patients with higher plasma TK levels, below the cutoff point (0.211 mg/1), had significantly lower risk of stroke reoccurrence and longer event-free survival time during 5-years of follow-up visits. (4) Compared with controls, plasma TK levels were significantly higher in CHD patients (0.347±.082 versus 0.256±0.087 mg/L, P<0.001) and elevated plasma TK levels were positive associated, in a dose-response manner, with the presece of CHD and negtive associated with the severity of CHD with respect to traditional risk factors of cardiovascular diseases. comparing with other biomarkers of CHD, discriminat analysis and ROC curves also indicated that elevated plasma TK level and decreased HDL were with the best diagnosis value on CHD. (5) Plasma TK levels had direct correlation with high density lipoprotein(r=0.069, P=0.003) and inverse correlation with low density lipoprotein(r=-0.053, P=0.025). Considering the influence factor of lipid, plasma TK levels still had direct correlation with high density lipoprotein(r=0.055, P=0.001) and inverse correlation with total cholesterol (r=-0.069,P=0.018) and low density lipoprotein(r=-0.053, P=0.025). (6) plasma TK in CHD patients subjected to positively skewed distribution (Skewness=0.617, SE=0.090, Kurtosis=0.774, SE=0.181). ACEI elevated the plasma TK levels in CHD significantly (0.388±0.076 mg/l vs. 0.320±0.079 mg/l, P<0.001; OR=3.20,95%CI 2.59-3.95, P<0.001)Conclusion:We prepared monoclonal antibody and ELISA kit specified to tissue kallikrein successed. Reduced TK is directly and dose-dependent associated with first-ever stroke, especially ischemic stroke, and may be an independent protector against stroke in Chinese. Elevated TK is positve associated with the presence of CHD and negtive associated with the severity of CHD and may be an independent risk factor or a strong biamarker of CHD in Chinese as well. Therefore TK could be a a useful prognostic tool for the evaluation of risk for stroke and its recurrence and a useful diagnostic tool for the evaluation of the presence and severity of CHD.更多还原