CN106596925A - Use of MMPs and TIMPs in screening of medicines for diagnosing or treating myocardial matrix reconstruction related diseases - Google Patents

Abstract

The invention discloses the use of matrix metalloproteinases MMPs and matrix metalloproteinase tissue inhibitors TIMPs as markers in screening drugs for diagnosing or treating diseases related to myocardial matrix remodeling, and belongs to the medical field. Result of the present invention shows that congenital heart disease group (CHD group), rheumatic heart disease group (PHD group) MMP-3, MMP-9, TIMP-1 content in blood before operation are all higher than coronary heart disease group (COR group) The increase of MMP‑9 was more significant; moreover, MMP‑3, MMP‑9 and TIMP‑1 in CHD group and PHD group were more widely distributed than those in COR group, MMP‑1, MMP‑9, The mRNA expression of TIMP‑1 was higher than that of COR group. Therefore, the present invention reveals that the dynamic balance of MMP-1, MMP-9 and TIMP-1 can be used as an important indicator of myocardial matrix remodeling, and regulating the activity of MMPs and TIMPs is expected to become a new direction for heart disease treatment.

Description

MMPs and TIMPs are used in screening for the diagnosis or treatment of diseases related to myocardial matrix remodeling use in disease medicine

technical field

The invention relates to the use of matrix metalloproteinases MMPs and matrix metalloproteinase tissue inhibitors TIMPs as markers in screening drugs for diagnosing or treating diseases related to myocardial matrix remodeling, and belongs to the medical field.

Background technique

In the past, it was believed that cardiac remodeling was only due to endogenous changes in cardiomyocytes, but now it is recognized that changes in the quantity, composition and structure of collagen in the extracellular matrix of cardiomyocytes are also involved in cardiac remodeling, that is, the process of matrix tissue repair and regeneration, and is the ventricular An important cause of remodeling, also known as myocardial matrix remodeling. Ventricular remodeling is one of the main factors that determine the cardiac function and prognosis of patients with heart disease. It is the result of an imbalance in the synthesis or degradation of cardiac matrix components. The cardiac matrix plays an important role in maintaining the structural and functional integrity of the heart. Regeneration of stromal tissue repair causes myocardial fibrosis and progressive ventricular dilation, eventually leading to heart failure. Matrix metalloproteinases (MMPs), which can degrade all cardiac matrix components in the myocardium, are the main factor of cardiac matrix degradation during the remodeling process. In the failing heart, the increased activity of MMPs leads to the degradation of fibrous collagen and the remodeling of extracellular matrix, leading to the progressive dilation of the left ventricle and the gradual decline of systolic function. The interaction between substances is regulated by endogenous physiological inhibitors, namely tissue inhibitors of metalloproteinases (TIMps). Therefore, the interaction between MMPs, TIMPs and their regulators determines the progression of myocardial fibrosis. Regulating the expression and activity of myocardial MMPs and TIMPs has become an important therapeutic approach to control the tissue repair and regeneration of the cardiac matrix in the progression of heart failure. Therefore, an in-depth understanding of the degradation and remodeling of myocardial matrix is very important to clarify the mechanism of matrix metalloproteinases and their inhibitors in remodeling.

At present, the research on vascular reconstruction after myocardial injury is relatively in-depth at home and abroad. Hojo et al. conducted a large number of studies on the expression of matrix metalloproteinases in acute myocardial infarction, and concluded that many matrix metalloproteinases are elevated in acute myocardial infarction. When Creemers et al. conducted research on heart failure, they found that some metalloproteinase inhibitors were of great help in preventing the progression of heart failure and inhibiting heart failure. This greatly inherits and develops SPinale et al.'s basic research on cardiomyocyte protein molecules and gene expression in the early stage. This gives a huge hint for our current research. Myocardial injury and heart surgery caused by many heart diseases inevitably cause some damage to the myocardium. Although the research on myocardial protection has achieved remarkable results in recent years, there are still relatively many complications caused by myocardial injury. The detection method is still relatively simple, outdated, and complicated. It must synthesize various inspections and test results, and the final specificity is not satisfactory. At present, there is still a lack of prospective research on the role of the dynamic balance of MMPs and TIMPs in maintaining the repair and regeneration of myocardial cells. The research on matrix remodeling caused by myocardial injury is still in its infancy at home and abroad. Research on protein molecules and biopharmaceuticals is imperative.

Contents of the invention

The object of the present invention is to reveal MMP-1, The dynamic balance of MMP-9 and TIMP-1 can be used as an important marker of myocardial matrix remodeling, thus revealing that matrix metalloproteinases MMPs and matrix metalloproteinase tissue inhibitors TIMps are used as markers of heart failure in screening for diagnosis or treatment of myocardial matrix remodeling. It provides new research and treatment directions for heart disease treatment.

In order to achieve the above object, the technical scheme adopted by the present invention is: use of matrix metalloproteinases MMPs and tissue inhibitors of matrix metalloproteinases TIMPs as markers in screening drugs for diagnosing or treating diseases related to myocardial matrix remodeling.

As a further improvement to the above technical solution, the MMPs are MMP-3 and MMP-9.

As a further improvement to the above technical solution, the TIMPs are TIMP-1.

As a further improvement to the above technical solution, the dynamic balance of MMP-3, MMP-9 and TIMP-1 is related to myocardial matrix remodeling.

As a further improvement to the above technical solution, the disease related to myocardial matrix remodeling is heart failure.

As a further improvement to the above technical solution, the heart failure is caused by myocardial hypertrophy or thickening caused by myocardial matrix remodeling.

The beneficial effect of the present invention is that: the present invention studies the tissue repair and regeneration of myocardial matrix from the dynamic balance of MMPs and TIMps by understanding myocardial injury and myocardial matrix remodeling in patients after surgery, and explores the effects of myocardial matrix tissue repair and Regenerative biological agents and drugs provide effective biological detection methods, new detection proteins, biological agents, and drug treatment and prevention for heart failure caused by myocardial hypertrophy and thickening caused by matrix remodeling after myocardial injury.

Description of drawings

Figure 1 is a comparison chart of the contents of MMP-3, MMP-9, and TIMP-1 in the blood of the three groups of patients. Compared with the CHD group and the COR group, ^# P<0.05, compared with the HD group and the COR group, *P<0.05;

Figure 2 is the immunohistochemical morphology distribution diagram of MMP-3, MMP-9, TIMP-1, where A is CHD group, B is PHD group, C is COR group, 1 is MMP-3, 2 is MMP-9, 3 is TIMP-1.

detailed description

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

1. Materials and methods

1.1 Clinical data

1.1.1 Research objects and grouping Patients who underwent cardiopulmonary bypass cardiac surgery in Guangzhou Red Cross Hospital from January 2012 to April 2014 were included as research objects. Preoperative heart function class (NYHA) Ⅱ-Ⅲ, American Society of Anesthesiologists (ASA) Ⅱ-Ⅲ, no history of rheumatic activity and coronary heart disease before operation, no hormones and non-steroids used 1 month before operation Anti-inflammatory analgesics etc. Preoperative hemoglobin, blood coagulation function, electrolyte, liver, kidney and lung function were all in the normal range, according to the method of random block grouping and grouping, according to the different cardiac operations, the research subjects were divided into 3 groups, the first group was congenital heart disease disease group (CHD group); the second group was rheumatic heart disease group (RHD group); the third group was coronary heart disease group (COR group), and preoperative echocardiographic examination showed no atrioventricular size change, Let it be the control group.

1.1.2 Specimen collection and determination

1.1.2.1 Specimen collection (ELISA) Collect blood before operation, (1) use vacuum tube containing EDTA to collect 4-5ml of venous blood from selected patients; (2) stand at room temperature for 1h; (3) centrifuge at 3000r/min for 15min (4) In the vertical ultra-clean bench, use a pipette to move the supernatant to a 1.5ml centrifuge tube, cover the tube cap in time, and move 1 ml of plasma into each centrifuge tube. Do not suck the liquid too hard to avoid If contamination is caused, a small amount of supernatant can be properly left at the end to ensure the purity of the plasma. The centrifuge tube and pipette tip are all sterilized at high temperature; (5) Label and store at -80°C.

1.1.2.2 Specimen collection (Rt-PCR) Excision of abnormal muscle bundles in the right ventricle, about 3 mm × 3 mm myocardial tissue was immediately placed in a cryopreservation tube, labeled, placed in liquid nitrogen for quick freezing, and stored in a -80°C refrigerator. Ethics committee review and approval.

1.1.2.3 Specimen collection (immunohistochemical method) Another myocardial tissue was taken, about 2mm×2mm ² was wrapped with gauze together with the label, and fixed with a pin (fix the pin to avoid damage to the sample). Put it in a jar filled with fixative and store it at room temperature. (The obtained myocardium is the abnormal muscle bundle of the right ventricle, which has been reviewed and approved by the ethics committee).

1.2 Experimental steps

1.2.1ELISA

(1) Determine the number of wells of the antibody-coated ELISA plate required for this detection; (2) Set up blank wells, standard wells, and sample wells to be tested respectively. Add 0.1 ml of BSA (2%) to the blank well, add 0.1 ml of standard solution or sample to be tested to the remaining wells, mix gently, cover the microplate with tinfoil, 37°C, 120min; (3) Reaction Finally, use an automatic plate washer to absorb the liquid in the microplate, or shake off the liquid in the microplate, and then pat it against the absorbent paper for a few times, and wash it twice; (4) press the prepared biotin antibody working solution Add 0.1 ml to each well one by one, and react at 37°C for 60 minutes; (5) Wash with PBS three times, soak for about 1 minute each time, and spin dry; (6) Add the prepared ABC (substrate) working solution at 0. Add 1 ml in turn, react at 37°C for 30 minutes; (7) Wash 5 times with PBS, soak for about 1-2 minutes each time, and spin dry; (8) Add 0.09ml of TMB chromogenic solution equilibrated at 37°C for 30 minutes in turn in each well , 37 ℃ dark reaction, during the reaction, should be observed frequently, when the first 3 to 4 wells of the standard can be seen with a clear gradient blue, and when the difference is not obvious in the last 3 to 4 wells, you can add TMB stop solution 0 .1 ml/well, (the longest color reaction should not exceed 30min); (9) Measure the OD value at 450nm with a microplate reader; (10) Find the corresponding concentration on the coordinates according to the absorbance value of the sample. Since the sample is diluted by N times, its actual concentration should be ×N.

1.2.2 Immunohistochemical method to determine the morphology and distribution of collagen in myocardial matrix

(1) Preparation of conventional paraffin sections for the excised myocardial tissue; (2) HE staining, soaking in xylene and alcohol in turn, staining with Harris hematoxylin solution and eosin in turn, soaking in alcohol and xylene, and using medium (3) Perform immunohistochemical staining with SP method, rinse with PBS 3 times for 3 minutes each time after routine dehydration; restore tissue antigens according to antibody requirements; add a drop of 3% H2O2 50 μl to the section, Incubate at room temperature for 10 minutes, wash with PBS for 3 times, each time for 10 minutes; add antibody and polymerization enhancer in turn, rinse with PBS and shake off, add DAB or AEC color developing solution, when the color is red or brown under the microscope, wash with distilled water , counterstained with hematoxylin 0.1% hydrochloric acid alcohol differentiation, after washing, washed with PBS to turn blue, developed color with DAB, dehydrated and dried with gradient alcohol, and sealed with neutral resin.

1.2.3RT-PCR technology is used to detect the mRNA of mmps and timps in myocardial tissue, and check the mRNA expression

(1) Extraction of RNA by guanidine isothiohydrogen-chloroform classical method; (2) Synthesis of cDNA; (3) Conventional PCR reaction; (4) Fluorescent quantitative PCR reaction, the preparation of the reaction system is strictly in accordance with the operation of the kit order.

1.3 Calculation

Take the concentration of the standard substance as the abscissa (logarithmic coordinate), and the OD value as the ordinate (ordinary coordinate), draw the standard curve on the semi-logarithmic coordinate paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; Then multiply by the dilution factor; or use the concentration and OD value of the standard to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample. Then SPSS17.0 was used for data analysis, and Dunnett's method was used to test the comparison between multiple experimental groups and control groups, taking α=0.05.

2. Conclusion

2.1 Comparison of clinical data among CHD, RHD, and COR groups

After statistical analysis, there was no significant difference in age, height, and weight among the three groups of patients (P<0.05). The effects of age, height, and body weight on the results were excluded. The echocardiographic results of the three groups of patients showed that after the variance analysis of the completely randomized design data, using the Dunnett method of comparing multiple experimental groups with a control group, the RAD, LVEF and COR groups in the CHD group can be considered as α=0.05. The difference in the mean is statistically significant, indicating that the RAD and LVEF of the CHD group have changed to varying degrees compared with the COR group; while the mean difference of the LAD, LVED, LVEF, IVS of the RHD group and the COR group is statistically significant, It is clear that LAD, LVED, LVEF, and IVS in the RHD group have also changed to varying degrees compared with the COR group (Table 1).

Table 1 Comparison of clinical data of the three groups of patients

Note: LAD (left atrial diameter), LEVD (left ventricular end diameter), RAD (left atrial end diameter), IVS (ventricular septal thickness), LVEF (ejection fraction), LVPW (left ventricular posterior wall), *( CHD vs. COR), ^# (RHD vs. COR)

2.2 ELISA test results for MMP-3, MMP-9, TIMP-1

Through the ELISA test of the matrix metalloproteinase in the blood of the three groups of patients, using the variance analysis of the completely random design data, and the Dunnett test comparing multiple experimental groups with a control group, according to the level of α = 0.05, the blood of the CHD group and the PHD group The MMP-3, MMP-9, and TIMP-1 levels in blood were significantly different from those in the COR group (P<0.05, Figure 1). The -1 content was higher than that in the COR group, but there was no significant difference between the CHD group and the PHD group in the experimental group (P>0.05), indicating that the heart matrix of the CHD group and the PHD group had undergone changes and remodeling to varying degrees.

2.3 Immunohistochemical test results

Under the microscope, MMP-3, MMP-9, and TIMP-1 showed diffuse brown-yellow or yellow-brown granules, while normal cardiomyocytes showed uniform, sparse light-brown or pink granules, which were quantitatively analyzed by Image-proplus image analysis software: The positive expression results of A1 and B1 were higher than those of C1, and the difference was significant and statistically significant (P<0.05). Under the microscope, it can be seen that the brownish yellow or yellowish brown particles of A1 and B1 were significantly more than those of C1 group, and they were diffusely distributed; The positive expression results of B2 and B2 were higher than those of C2, and the difference was statistically significant (P<0.05). The diffuse brown-yellow or yellow-brown particles under the microscope were more abundant than those of C2; although the expressions of A3 and B3 were higher than those of A1, B1, A2, and B2 However, its positive expression was still higher than that of C3, and the difference was significant (P<0.05). Under the microscope, brownish-yellow or yellowish-brown granules were still abundant, while C3 showed diffuse light brown and pink granules (Figure 2).

2.4PCR reaction results

After quantitative detection by polymerase chain reaction, the mRNAs of MMP-3, MMP-9, and TIMP-1 in the CHD group and PHD patients were highly expressed. After multiple experimental groups and a control group were compared by Dunnett's method and Levene's homogeneity test, according to α =0.10 level, the variances of the three groups of data are equal. According to the level of α=0.05, it can be considered that the mRNA expression of MMP-3 and MMP-9 in CHD group and PHD group is higher than that in COR group (Table 2).

Table 2

^# (comparison between CHD group and COR group), *(comparison between PHD group and COR group)

3. Conclusion analysis

In the present invention, we have adopted a variety of methods to detect the matrix remodeling caused by myocardial injury, and explored the MMP-3 and MMP-9 proteins of myocardial matrix degradation and fragmentation in blood and tissues from both qualitative and quantitative aspects. The expression of the expression, and the expression of its mRNA was quantitatively detected by PCR technology, and the matrix metalloproteinase inhibitor TIMP-1, which prevents and inhibits the degradation and remodeling of the cardiac matrix, was quantitatively, qualitatively and mRNA expressed. The quantitative and qualitative changes caused by MMP-1, MMP-9 and TIMP-1 in the whole process of myocardial matrix changes were analyzed comprehensively. It was found that the expressions of MMP-3, MMP-9 and TIMP-1 in the congenital heart disease group (CHD group) and rheumatic heart disease group (PHD group) with cardiac matrix remodeling were significantly higher than those without cardiac matrix remodeling. coronary heart disease group (COR group). This shows that the dynamic balance of matrix metalloproteinases and their inhibitors may affect the degradation of cardiac matrix and the fragmentation of collagen. It plays a huge role in maintaining the shape of the myocardium and the structure of collagen. How to grasp this key point, maintain this dynamic balance, prevent cardiac remodeling after myocardial injury, and find out the target of gene expression. Targets or protein molecules to develop drugs or protein-like inhibitors that can prevent and inhibit myocardial remodeling will become the driving force and goal of our further research.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention can be modified or equivalently replaced without departing from the essence of the technical solution of the present invention.

Claims (6)

1. matrix metalloproteinase MMPs and matrix metalloprotease tissue depressant TIMPs is used to examine as mark in screening Purposes in medicine that is disconnected or treating the disease related to myocardium matrix reconstruction.

2. purposes as claimed in claim 1, it is characterised in that the MMPs is MMP-3 and MMP-9.

3. purposes as claimed in claim 1, it is characterised in that the TIMPs is TIMP-1.

4. purposes as claimed in claim 2 or claim 3, it is characterised in that the dynamic equilibrium of MMP-3, MMP-9 and TIMP-1 and cardiac muscle Matrix reconstruction is relevant.

5. purposes as claimed in claim 1, it is characterised in that the disease related to myocardium matrix reconstruction is heart failure Exhaust.

6. purposes as claimed in claim 5, it is characterised in that the heart failure is the cardiac muscle caused by myocardium matrix reconstruction Hypertrophy thickens initiation.