| 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.
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