CN101200721B - Human myocardium protecting gene and uses thereof - Google Patents

Abstract

The invention belongs to the field of biological technology, and relates to a heat shock transcription factor (HSF1) of a human heart muscle protection gene and a screening method and application thereof. The present invention adopts the method of "death trap" to screen the gene with resistance to cardiomyocyte death from the human heart gene bank, and clones the heat shock transcription factor 1, a human heart muscle protection gene, from the obtained gene. The results of transfected cells and animal experiments show that the obtained human cardioprotective gene has the effect of anti-cell death, and also has the effect of resisting ischemia, protecting cardiomyocytes and preventing the occurrence and development of heart failure in the living heart. The invention can prepare medicines for treating ischemic heart disease and preventing cardiac insufficiency, provide new target genes for further functional research and development of genetic engineering medicines, and are important for early prevention and treatment of heart failure, and research and development of related medicines and means significance.

Description

Human cardioprotective gene and its use

technical field

The invention belongs to the field of biological technology and relates to a human cardioprotective gene, in particular to a human cardioprotective gene heat shock transcription factor (HSF1) and its screening method and application.

Background technique

Heart failure is a group of complex clinical syndromes, which is the severe stage of most patients with organic heart disease, and the 5-year survival rate is similar to that of malignant tumors. Once heart failure occurs in heart disease patients, the 1-year mortality rate is 43%, and the 5-year mortality rate reaches 75%, seriously endangering human health. Epidemiological data show that the number of heart failure patients in the world is as high as 22.5 million, and there are 2 million new cases every year. The rapid aging of the population and the increase of various risk factors make ischemic heart disease the most common cause of heart failure. In developed countries, the increase in the survival rate of myocardial infarction is the main reason for the increase in heart failure. In developing countries, epidemiology has changed from The transition from infection, tuberculosis to heart disease is also an important issue. Consistent with the global epidemic trend, ischemic heart disease is the most common cause of heart failure in my country. In particular, there are about 130 million people with high blood pressure in my country, and it is still in the status quo of low awareness rate, low treatment rate, and low compliance rate. Ischemic heart disease in my country will be the main cause of heart failure now and for a considerable period of time in the future, and it is on the rise. It is a major problem facing public health in our country and has caused a heavy burden on the national economy.

With the gradual elucidation of the pathogenesis of heart failure and the research of large-scale evidence-based medicine, the treatment model of heart failure is also constantly updated and evolving. From the 1950s to the 1980s to correct hemodynamic abnormalities and improve symptoms to long-term restorative strategies and improve prognosis after the 1990s. However, the current treatment methods for heart failure are all palliative treatments for damaged or necrotic myocardium, and the degree of recovery is limited. Cardiomyocytes are generally considered to be end-stage cells, and the loss of functional cardiomyocytes is the direct cause of low myocardial contractility and heart failure. Hypoxia is a common link in cell damage and death caused by a variety of pathogenic factors. The outcome of cell survival and death under the stimulation of damage depends on the dynamic balance between its endogenous protection mechanism and death signals. Early regulation of the balance between its protective factors and death signals and activation of protective mechanisms are the key issues to protect functional myocardial tissue and prevent heart failure.

With the development of genetics and bioinformatics, changing gene function through gene modification has once become the hope of treating heart failure. The results of coordination and co-regulation, and heart failure is a clinical syndrome caused by the joint participation of multiple factors, and the treatment method targeting a single gene cannot achieve the desired effect. In the prior art, gene chips are used to find differentially expressed genes, and then the functional identification of these genes (often hundreds or thousands) is often felt like looking for a needle in a haystack. Although a large amount of sequence information about genes can be obtained, not many of them play a key role in elucidating their functions. Vito P reported in 1996 a method for screening protective genes from induced death cultured cells, called the method of death trap (Death Trap). There is no research on the application of death trap gene screening method to heart disease.

Contents of the invention

The purpose of the present invention is to provide a human cardiomyocyte protection gene, in particular to a gene capable of protecting the myocardium against hypoxic injury at the very early stage of cell injury. More specifically it relates to the human cardioprotective gene heat shock transcription factor (HSF1).

A further object of the present invention is to provide a screening method for the above-mentioned gene and its use.

The present invention adopts the "Death Trap" method to screen functional genes, and screens out genes that resist cardiomyocyte death from the human heart gene bank, and clones heat shock transcription factor 1 (HSF1) with the structure of sequence 1 ).

The HSF1 of the present invention has the effect of resisting cell death as confirmed by cell experiments; the results of animal experiments prove that it has the effects of resisting ischemia, protecting cardiomyocytes and preventing the occurrence and development of heart failure.

The object of the present invention is achieved through the following technical solutions,

1. Use the death trap functional gene screening method to screen the gene that protects the myocardium against hypoxic injury and clone its cDNA;

2. Further verify the genes with cardioprotective effect in the cloned cDNA through cell and animal experiments;

3. Based on the basic elucidation of the interaction and function of the screened genes, seek the optimal interaction mode, and verify whether the best cardioprotective effect can be achieved through cell and animal experiments;

4. Predict the product form according to the screened gene sequence with cardioprotective function, transform Escherichia coli with the gene that can produce secreted protein, and conduct cytokine activity screening and identification;

5. Compare the expression changes of serum and heart-related protective genes in patients after myocardial ischemia, screen for early predictive indicators with prognostic value, and conduct clinical verification.

The present invention has carried out a cell experiment, transfected cells with the HSF1 gene, and the result shows that more than 60% of the cells transfected with the gene can resist death caused by hypoxia, confirming that the HSF1 has the effect of resisting cell death.

The present invention has carried out animal experiments. First, a cardiac ischemia/reperfusion injury model was made with a transgenic mouse overexpressing the heart HSF1 gene. The results showed that after cardiac ischemia/reperfusion injury, the area of myocardial necrosis in the HSF1 transgenic mouse Compared with non-transgenic mice, it was reduced by more than 30%, and the number of cardiomyocyte apoptosis was reduced by more than 40%. At the same time, the present invention confirms that HSF1 protects the heart through heat shock proteins HSP27, HSP70 and HSP90 instead of HSP110. These protective effects are also reflected in the electrocardiogram of transgenic mice at the same time - faster recovery of ST-T changes after ischemia/reperfusion injury; in addition, the present invention uses cardiac HSF1 gene overexpressed transgenic mice and cardiac HSF1 gene A model of pressure overload heart failure was made in the removed mice. The results showed that, compared with the control group, the former delayed the occurrence and development of heart failure, and at the same time showed that it could promote the formation of new cardiac microvessels, and the latter did the opposite. The results of animal experiments have proved that HSF1 gene also has the function of resisting ischemia, protecting cardiomyocytes and preventing the occurrence and development of heart failure in the living heart.

The present invention applies the direct functional gene screening method of the death trap to the heart, which is more purposeful and accurate. The screened genes can be recombined in a targeted manner, which is more conducive to finding the best gene therapy method.

In the experiment of the present invention, the COS7 cell line (purchased from ATCC, USA) was used for gene screening; the human heart gene bank was purchased from Life Technologies, USA; HSF1 transgenic mice (Department of Applied Medical Engineering, Yamaguchi University, Japan) were used for animal experiments.

In the present invention, the human heart gene library is transfected into cultured cells, and hydrogen peroxide is used to induce death, and the heat shock transcription factor (HSF1) is cloned from the gene extracted from the surviving cell line. Further studies have shown that HSF1 not only resists cell death, but also has a protective effect in the living heart. It is also found that HSF1 activates the protein kinase Akt1, which has a cytoprotective effect, and at the same time inhibits the kinase JNK and the protein caspase3 that can cause apoptosis. activity, thereby reducing the apoptosis of cardiomyocytes and playing a role in cardioprotection.

The present invention screens out related genes from cells in different stages of death or heart tissue of different degrees of heart failure, artificially produces cDNAs of these genes, introduces these cDNAs into cultured cells or animal hearts, and observes their interrelationships and effects . It can provide new target genes for further functional research and genetic engineering drug development. Through the "death trap" functional gene screening method, a new gene that can reverse myocardial death and protect the myocardium in the very early stage of myocardial injury is screened out, and its interaction mode is clarified, which is helpful for the early prevention and treatment of heart failure and the further development of related drugs and drugs. means are important.

The invention has important significance for finding medicines and means for treating ischemic heart disease and preventing cardiac insufficiency.

Description of drawings

Fig. 1 is a technical roadmap of the present invention.

Figure 2 is a graph of the measured values of left ventricular ejection fraction in the pressure overload model.

Figures 3 and 4 are graphs of the measured values of the number of new blood vessels in the pressure overload model.

Figure 5 shows the relationship between ASK1 and HSF1.

Figure 6 shows the protective effect of HSF1 on cells.

Detailed ways

Example 1

1. Use the death trap method to screen the genes that protect the myocardium against hypoxic injury

Rat cardiac fibroblasts were used as the research object, and the human heart gene library (pCMVSPORT heart cell expression cDNA library) was transfected by electroporation. After 48 hours, the transfected cells were stimulated with lethal hypoxia to induce cell death, and the surviving cell clones may contain genes that protect the myocardium against hypoxic injury. The subgene pool was extracted from the surviving cell clones and then transfected into cardiac fibroblasts, and the experimental procedure was repeated 4 times. The most obviously altered genes were screened from the last surviving cell clones, and their cDNAs were cloned.

2. Verify whether the screened cDNA has cardioprotective effect and optimize its interaction mode

1. Cytological level

The cloned cDNA was transfected into cardiomyocytes respectively, and after giving lethal hypoxia stimulation, the survival and death of the cells were observed, and it was clarified which genes screened by the death trap method had the effect of protecting cardiomyocytes against hypoxia stimulation.

In the present invention, several genes that can protect cells against hypoxic damage are co-transfected into cardiomyocytes in different combinations, and after giving lethal hypoxic stimulation, the effects of different transfection methods on cell survival and death are compared, and different cell levels are optimized. The intercombination pattern of genes.

Ser 967 of ASK1 is autophosphorylated under physiological conditions, and the test results show that when the temperature rises, p967 is dephosphorylated, and ASK1 is activated at this time (prone to apoptosis). The results showed that the cultured neonatal rat cardiomyocytes were placed at 42°C for 2 hours. At this time, the activity of HSF1 was the highest. When it was higher than 37°C, the expression of ASK1 decreased more obviously at 42°C than at 37°C, while the expression of total ASK1 had no obvious effect. Variety. The above results indicated that the activation of HSF1 can inhibit the expression of ASK1, which may reduce the apoptosis and protect the myocardium.

Neonatal rat cardiomyocytes were cultured, 0.1M hydrogen peroxide induced apoptosis, ROS was selected as an indicator, ROS was marked with DCFH-DA, ROS was detected by flow cytometry, and the cells were transfected with HSF1. The results showed that cardiomyocyte apoptosis increased significantly after adding H2O2. Compared with non-transfected cells, cells transfected with HSF1 can reduce ROS levels by more than 20% for H2O2-induced apoptosis. For cells without hydrogen peroxide-induced apoptosis, transfection of HSF1 does not necessarily reduce intracellular ROS level.

The above cell culture results showed that: HSF1 can inhibit the expression of ASK1, inhibit the level of ROS, and protect the myocardium.

2. Animal level

To replicate the acute myocardial infarction model in rats, immediately after coronary artery ligation, 30min, 3h, 6h, and 12h after acute ischemia, the cloned cDNA was transfected separately or co-transfected in the ischemic myocardium. At 1 month and 6 months, echocardiography was performed to evaluate cardiac function, myocardial tissue was taken for pathological examination to observe morphological changes, and to evaluate which gene or combination of several genes could protect the myocardium against acute hypoxic stimulation at the animal level.

Replicate the chronic myocardial ischemia model of coronary artery coarctation, transfect the cloned cDNA separately or co-transfected locally, perform echocardiographic examination to evaluate cardiac function at 1 month, 3 months, and 6 months after operation, and collect myocardial tissue for disease progression. Morphological changes were observed by physical examination, and the effect of the gene or the combination of several genes on protecting the myocardium against chronic hypoxia stimulation was evaluated at the animal level.

Replicate the mouse model of heart failure induced by mechanical pressure load, transfect the cloned cDNA locally or co-transfected, perform echocardiography to evaluate cardiac function at 1 week, 2 weeks and 4 weeks after operation, and take myocardial tissue for pathological examination Observe the morphological changes, and evaluate the effect of the gene or the combination of several genes on the prevention and treatment of heart failure at the animal level.

The results show that:

1. After the successful establishment of the pressure overload model, the left ventricular ejection fraction (LVEF) in the wild mouse (NO) group began to decline at the 3rd week, and decreased significantly at the 4th week, and heart failure occurred; compared with the NO group, the gene knockout The LVEF of the mice (KO) decreased from the first week until the fourth week, and heart failure occurred, while the LVEF of the gene transfected mice (TG) group did not decrease until the fourth week, and no heart failure occurred. Its specific measurement values are shown in Figure 2.

2. After the pressure overload model was successfully established, the number of new blood vessels was detected by immunohistochemistry at the end of the fourth week. It can be observed that the number of new blood vessels in the wild mouse (NO) group and the gene knockout mouse (KO) group decreased, but Gene transfected mice (TG) group increased. See charts 3 and 4 for the specific measured values and charts.

The above animal experiments preliminarily show that HSF1 can promote angiogenesis and improve heart function.

3. Predict the product form according to the screened gene sequence with cardioprotective function, transform the gene that produces secreted protein into Escherichia coli, and conduct cytokine activity screening and identification to provide source innovative targets for further development of growth factors and protein drugs Gene.

4. Compare the expression changes of serum and heart-related protective genes in patients after myocardial ischemia, and screen for early predictive indicators with prognostic value

1. Collect serum samples from healthy volunteers and patients with acute myocardial infarction 6h, 12h, and 24h after infarction, and extract genomic cDNA. Probes were designed and synthesized based on the protective gene sequences screened in the above experiments, and were hybridized with cDNA of serum samples to observe the expression changes of the screened genes in the serum of healthy volunteers and at different time periods after acute hypoxia.

2. Collect myocardial samples from patients who died unexpectedly and underwent heart transplantation due to end-stage heart failure due to ischemic heart disease, and extracted genomic cDNA. The synthesized probes were hybridized with cDNA of myocardial tissue, and the expression changes of screened genes in chronic hypoxic myocardial tissue were observed. By comparing and analyzing the expression changes of related protective genes in serum and myocardial tissue samples, the screening may be used as a very early indicator for judging the prognosis of ischemic heart disease, and further clinical follow-up verification.

SEQUENCE LISTING

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Claims (2)

1. the human myocardium protecting gene heat shock transcription factor of sequence 1 is preparing the purposes for the treatment of in the heart failure drugs.

2. the purposes of the human myocardium protecting gene heat shock transcription factor of sequence 1 in preparation prophylaxis of heart failure medicine.

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