Application of Fam198B gene or protein coded by Fam198B gene in myocardial infarction treatment

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a Fam198B gene or a protein coded by the Fam198B gene in preparation of a medicine for preventing, relieving or / and treating myocardial infarction. It is found for the first time that the expression level of Fam198B in myocardial tissue of a myocardial infarction mouse induced by coronary artery ligation is remarkably increased; in addition, after myocardial infarction of the mouse with the Fam198B gene specifically knocked out of the myocardial cells is carried out for one day, the myocardial cell apoptosis is obviously increased, and the systolic function of the heart becomes poor; after 10 days of myocardial infarction of the mouse with the Fam198B gene specifically knocked out of the myocardial fibroblasts, the systolic function of the heart becomes poor, collagen secretion of the fibroblasts is increased, and the death rate of the mouse is remarkably increased. The results show that Fam198B participates in dual regulation and control of myocardial cell survival and fibroblast activation, and plays a role in heart protection in the pathological process of myocardial infarction. Therefore, the Fam198B gene, the Fam198B protein and the activator expressed by the Fam198B gene can be used for preparing the medicine for preventing, relieving or / and treating the myocardial infarction, and the Fam198B gene or the Fam198B protein can also be used as a medicine target for screening the medicine for preventing, relieving or / and treating the myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of Fam198B gene or protein encoded by it in the treatment of myocardial infarction. Background Art

[0002] Cardiovascular disease (CVD) is the leading cause of death in the world, and the number of deaths from CVD is increasing worldwide due to population growth, population aging, and changes in disease epidemiology. Damage to coronary arteries caused by atherosclerotic plaques accounts for approximately 70% of all myocardial infarction events, and other causative factors include coronary artery spasm, embolism, and dissection. Harmful complications of acute myocardial infarction include cardiac rupture, cardiogenic shock, arrhythmias, pericardial disease, and heart failure, which are also the reasons for the low survival rate of patients. Although the development of precision medicine and the optimization of clinical treatment strategies have significantly increased the survival rate of patients after acute myocardial infarction, they still face a 40% mortality rate after surgery. In clinical studies, Bolli et al. have proposed various therapeutic strategies to alleviate the risk of myocardial infarction, but only a few have shown positive clinical research results. Therefore, it is particularly important to identify new therapeutic strategies, such as identifying and identifying new cardioprotective candidate drugs that can be used for acute myocardial infarction intervention.

[0003] After acute myocardial infarction occurs, prolonged ischemia and hypoxia will cause a series of pathological reactions, which can be roughly divided into three periods: the early stage (about 1-3 days after myocardial infarction) is characterized by the loss of myocardial cells mainly due to damage and apoptosis, causing progressive decline in cardiac function; the middle stage (about 3-7 days after myocardial infarction) is characterized by rapid proliferation and repair of fibroblasts that rapidly proliferate and activate into myofibroblasts, massive secretion of collagen fibers, and matrix deposition; the late stage (about 10 days to 4 weeks after myocardial infarction) is characterized by chronic and continuous activation of senescent fibroblasts in the infarct edge zone, leading to excessive deposition of fibrous matrix, promoting continuous negative remodeling of the myocardium, and further damaging cardiac function. It can be seen that the apoptosis of myocardial cells in the acute phase after infarction and the continuous activation of fibroblasts after scar maturation play a decisive role in the pathological process of myocardial remodeling.

[0004] Protein kinases are signals that play a key role in many disease networks and are key regulators of intracellular signal transduction. Protein kinases involved in regulating cell metabolism, proliferation and differentiation have become an important class of drug targets. Fam198B protein belongs to protein kinase, also known as Golgi associated kinase 1B (Gask1B). It can be expressed in the heart and intestine as early as 9.5 days of embryonic development. As the embryo matures, its expression can also be detected in organs such as the peripheral nervous system, kidneys, skin and testes. It is worth noting that at a specific embryonic period, the expression level of Fam198B in ventricular muscle is the highest among 214 mouse organs. Fam198B protein is highly conserved in humans and is homologous to Drosophila Fj kinase, including Fjx1 (Four-jointed box protein 1), Fam198A, Fam20A, Fam20B and Fam20C. It is a member of the Four-jointed associated protein kinase family. The protein structures of the family members are similar. They all contain a signal peptide sequence (SP) at the amino terminus and a kinase domain at the carboxyl terminus. Among them, Fam198B is the most unique. It is the only member with a transmembrane sequence (type II single transmembrane region), so it is speculated that it may be a membrane-bound glycoprotein located in the Golgi apparatus. These domains may be involved in protein-protein interactions, signal transduction or other biological functions. The Fam198B gene is located on chromosome 4 in humans, specifically in the 4q32.1 region. The Fam198B protein encoded by the Fam198B gene consists of 519 amino acids and is expressed at a high level in the heart. In the protein kinase Fam198B, the signal peptide is highly selective and specific, and can bind to specific receptors to trigger specific cell signal transduction pathways. The kinase active sequence has the ability to catalyze the transfer of phosphate groups and can convert substrates into their phosphorylated forms, thereby changing the activity, stability or interaction of the substrate with other molecules. Current studies have shown that the type II single transmembrane region of secretory protein kinases plays an important role in cell signal transduction, catalytic regulation, and intercellular communication. The type II single transmembrane region can anchor it to the cell membrane, ensuring that the protein kinase is located in a specific cell membrane region, thereby effectively interacting with other molecules on the cell membrane or signal molecules outside the cell. At the same time, triggering the signal transduction process within the cell helps the cell to respond promptly and accurately to changes in the external environment. Through interactions with other membrane proteins or regulatory factors within the cell, this transmembrane region can affect the activity, selectivity, and substrate specificity of the kinase, and can interact with corresponding receptors or molecules on neighboring cells to promote intercellular communication and signal transmission.Protein kinase Fam198B can participate in the body's intercellular signal transduction through the above mechanism, thereby triggering intracellular signal cascade reactions and ultimately affecting the physiological functions and behaviors of cells.

[0005] However, current research on the exact function and biological significance of the Fam198B gene is still limited. Mainly limited to the field of tumors, previous studies have shown that protein kinase Fam198B is associated with the survival of lung adenocarcinoma and can effectively inhibit tumor cell invasion, migration, migration, proliferation and anchorage-independent growth. Later studies found that protein kinase Fam198B can regulate the polarization of macrophages, tumor immune microenvironment, and other immune responses. Recently, bioinformatics studies have pointed out that protein kinase Fam198B has been identified as a key regulator of acute aortic dissection. These studies have shown that protein kinase Fam198B can reduce cell apoptosis on the one hand and regulate extracellular matrix remodeling on the other. However, there are still no relevant reports on the effect of protein kinase Fam198B on myocardial infarction. Therefore, the Fam198B gene as a new drug target has important clinical and practical significance for the future treatment of cardiovascular diseases. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide an application of Fam198B gene or a protein encoded by it in the treatment of myocardial infarction.

[0007] Based on the above purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of Fam198B gene or Fam198B protein in the preparation of a drug for preventing, alleviating or / and treating myocardial infarction.

[0009] The second aspect of the present invention provides the use of Fam198B gene or Fam198B protein as a drug target for screening drugs for preventing, alleviating or / and treating myocardial infarction. Wherein, Fam198B gene is located on human chromosome 4, and its site information is as follows: Homo sapiens chromosome 4, GRCh38.p14 Primary Assembly, NC_000004 (158124474..158173025), and its transcript (NM_001128424.2) nucleotide sequence is shown in sequence 1; Fam198B protein consists of 519 amino acid residues, and its amino acid sequence is shown in sequence 2.

[0010] The nucleotide sequence shown in SEQ ID NO: 1 is as follows:

[0011] acactataaatgtctttccttatctgtgtgtactcttatctcactgttctattttttctcctcatttatattaactctttcttacctttttttctgaacttctagg

[0012] ccttctctttccagaactggtggaagacaaatgaaacggccaagatggtaagaaacaagccgcatttctccttggggagactgataatttaaaaggt

[0013] ttgttgtgtcagaaacattcccagcttcatcaccaaccctttccttccacctctgcccactggagaccacttatatcccgaagcggacgcggcagctg

[0014] aagtcaggaaaccatgcatcacattagcaggagccaactgcagactttaaactccgttcaacatgtggatgcggcagagaaatgacctgtccaga

[0015] caagccggggcagctcataaactggttcatctgctccctgtgcgtcccgcgggtgcgtaagctctggagcagccggcgtccaaggacccggag

[0016] aaaccttctgctgggcactgcgtgtgccatctacttgggcttcctggtgagccaggtggggagggcctctctccagcatggacaggcggctgaga

[0017] aggggccacatcgcagccgcgacaccgccgagccatccttccctgagatacccctggatggtaccctggcccctccagagtcccagggcaatg

[0018] ggtccactctgcagcccaatgtggtgtacattaccctacgctccaagcgcagcaagccggccaatatccgtggcaccgtgaagcccaagcgcag

[0019] gaaaaagcatgcagtggcatcggctgccccagggcaggaggctttggtcggaccatcccttcagccgcaggaagcggcaagggaagctgatg

[0020] ctgtagcacctgggtacgctcagggagcaaacctggttaagattggagagcgaccctggaggttggtgcggggtccgggagtgcgagccggg

[0021] ggcccagacttcctgcagcccagctccagggagagcaacattaggatctacagcgagagcgccccctcctggctgagcaaagatgacatccga

[0022] agaatgcgactcttggcggacagcgcagtggcagggctccggcctgtgtcctctaggagcggagcccgtttgctggtgctggaggggggcgc

[0023] acctggcgctgtgctccgctgtggccctagcccctgtgggcttctcaagcagcccttggacatgagtgaggtgtttgccttccacctagacaggat

[0024] cctggggctcaacaggaccctgccgtctgtgagcaggaaagcagagttcatccaagatggccgcccatgccccatcattctttgggatgcatcttt

[0025] atcttcagcaagtaatgacacccattcttctgttaagctcacctggggaacttatcagcagttgctgaaacagaaatgctggcagaatggccgagta

[0026] cccaagcctgaatcgggttgtactgaaatacatcatcatgagtggtccaagatggcactctttgattttttgttacagatttataatcgcttagatacaaa

[0027] ttgctgtggattcagacctcgcaaggaagatgcctgtgtacagaatggattgaggccaaaatgtgatgaccaaggttctgcggctctagcacacatt

[0028] atccagcgaaagcatgacccaaggcatttggtttttatagacaacaagggtttctttgacaggagtgaagataacttaaacttcaaattgttagaagg

[0029] catcaaagagtttccagcttctgcagtttctgttttgaagagccagcacttacggcagaaacttcttcagtctctgtttcttgataaagtgtattgggaaa

[0030] gtcaaggaggtagacaaggaattgaaaagcttatcgatgtaatagaacacagagccaaaattcttatcacctatatcaatgcacacggggtcaaag

[0031] tattacctatgaatgaatgacaaaagaatcttctggctagggtgttagatatatttatgcatttttggttttgtttttaaatcaagcacatcaacctcaagcc

[0032] cgtttagcaatgaggcagtgtagatgaatacgtaaaataaatgactttaaccaagtagctataatgggacttagcactgtatgcatacttaaaaaggtt

[0033] ttgaaaaacaaactacttgagaaatatttgtttatatttttctctaacatcatgctatgtgtcagtctgaacatctgacaacagaaatttcagttattattcta

[0034] gctaagttttgaaaacatttgtcatgctgtttaatagaaaactgcaaaccagagacactgactccattaataaaccatattttgtgccgttttgactgttct

[0035] gaccaaatactaatgggaacaattcttgacgtttttctgttgctgattgttaacatagagcagtctctacactaccctgaggcaactctacattggaaca

[0036] ctgaggcttacagcctgcaagagcatcagagctgaccatacatttaaacagaaatgctggtttatttgcaaaatcaccagtatattttctattgtgtctat

[0037] aaaaaatcagtcatttaagtacaagaatcatattttccattcctttttagaaatttattttgttgtccctatggaaatcattcacatctgacaatttatatgttaa

[0038] agagttttactctctctattttggtccaatttgtatctagtggctgagaaattaaataattctaaagtatgaagttacctatctgaaaatgtacttacagagt

[0039] atcattttaaaatggatgtctctttaaaaattttgttacttttaccaacaatgtaatataatttatgtatattttattaataatagtgaattccttaaaatttgttcta

[0040] tgtacttatatttaatttgatttaatggttactgcccagatattgagaattggttcaaatattgagtgtgtttcaatatattatctggcttatttcaacatgagta

[0041] atatgagcaaaataagttaaaacctgcgtctgatcaattttcctcatgactagaactaaaacagtaaatttggacaatattaagcctcaaataatcatct

[0042] ccaaactccttctaacactttttaaatcagattggaagacatggacaaatcaggttcatgtgttgcatctttatgtcctttgccaatatccaagatcatcac

[0043] atatggtagatattcacatggagtttcaaattcagaatagattaccattaccttcctgcccttacacatcctactccttatttaaaagttctatttgtgactttt

[0044] catttcctgaaagtttaaaaatacaatttgagaatgtttataatacattctctcctgtcttttcacggttacgtctgttattgctgaaatacaccacattttcttt

[0045] gttctggtcaaggttaactcaatatctgtgtgaaagagaactactaacaacgttacaaatagaggctagatttgaaaaaaaaaatctatagatctaattg

[0046] atacaattgtagaacaaaatgtcaaaataatgttttaagtaagagaagatggaccaaggagagagagatcatttgaaaatctaattgtagcttttct

[0047] aggctcacattcatgtactacttttagcacccttatgggctgtgctcgccccctggacagttgagctttggattatcttcctcttcaattttccctctattga

[0048] cccgagtgtctccctctgcttctacagatttatagtactccttggctcttttgagtctccacttttactcactgtctctgggatttttaagatccttttcttctctt

[0049] ataaatcatcctcttaatgaaaattagcctaacaaaagtttggagactggaatcctactttgagccactgacttgaaataactcttttggcaagttgcct

[0050] gacatcctgtcttaccaaggtggcatatttgcattttttactgcttaaaacatttttttttttttaccatctttatccaaatttatcatattgatggtaggactaaca

[0051] ggctttttagaagctggctttaactttgagtctcaagctacaatgctgttgggcagcctggtcttcccacgtgagggtttaactttgtttatttgcctccag

[0052] ttattccaaaatgcttattaaatgaaagtcccaggaacatgtttattttagtcacctttgctttttaacaattttgttttgtaatcaatgagtaattcatgatgaa

[0053] ttatttttgactaatggatagccgaaggccaggcttttaattctaataggtaatgttcttcttttgtcttattgaaacaatgagaatactctgtgcatttcaaa

[0054] tgcactccgattatgctgtggttttattcacataagcacaatatgtgttttatttataacttcataacaaacttataatataataatttaccttagcagacatg

[0055] caaaagcttattcttgtgtgacttactttctttaagctaataatataaaaataaatatgtatcttaaaaatctataataaaacattagaattaaagatatgtg

[0056] ctttttattttgcagatgagttcatttgcttttgtagatgtgttttcagagctaggtacagaggaatgtttgctacctttagcggtgaaaaaagaaagagag

[0057] tcaagaattttgttggattgtgtttgtgtgtgcatatatttgatatcatcattatatttgtaatctttggacttgtaatcatagcctgtttattctactgtgccatta

[0058] aatatactttaccttatacataacgaataaaatacctagaagtagatttatttacacactgtcttatgaaattcttatgtttatgtcagcacagaacaagca

[0059] cacatttagagcattcatctgaaaattatatggaaattgaacatgggtcaaataaagatg。

[0060] The amino acid sequence shown in Sequence 2 is as follows:

[0061] MTCPDKPGQLINWFICSLCVPRVRKLWSSRRPRTRRNLLLGTACAIYLGFLVSQVGRASL

[0062] QHGQAAEKGPHRSRDTAEPSFPEIPLDGTLAPPESQGNGSTLQPNVVYITLRSKRSKPANIRG

[0063] TVKPKRRKKHAVASAAPGQEALVGPSLQPQEAAREADAVAPGYAQGANLVKIGERPWRLVR

[0064] GPGVRAGGPDFLQPSSRESNIRIYSESAPSWLSKDDIRRMRLLADSAVAGLRPVSSRSGARLL

[0065] VLEGGAPGAVLRCGPSPCGLLKQPLDMSEVFAFHLDRILGLNRTLPSVSRKAEFIQDGRPCPII

[0066] LWDASLSSASNDTHSSVKLTWGTYQQLLKQKCWQNGRVPKPESGCTEIHHHEWSKMALFD

[0067] FLLQIYNRLDTNCCGFRPRKEDACVQNGLRPKCDDQGSAALAHIIQRKHDPRHLVFIDNKGF

[0068] FDRSEDNLNFKLLEGIKEFPASAVSVLKSQHLRQKLLQSLFLDKVYWESQGGRQGIEKLIDVI

[0069] EHRAKILITYINAHGVKVLPMNE.

[0070] The third aspect of the present invention provides the use of a promoter of Fam198B gene expression in the preparation of a drug for preventing, alleviating or / and treating myocardial infarction.

[0071] According to the above application, preferably, the promoter is a substance that increases the expression level of the Fam198B gene.

[0072] According to the above application, preferably, the promoter comprises an expression vector carrying a nucleotide sequence encoding Fam198B protein or a recombinant virus containing a nucleotide sequence encoding Fam198B protein.

[0073] The fourth aspect of the present invention provides a medicine for preventing, alleviating or / and treating myocardial infarction, wherein the medicine contains Fam198B gene or Fam198B protein or Fam198B gene expression promoter.

[0074] According to the above-mentioned medicine, preferably, the medicine also contains a pharmaceutically acceptable carrier / excipient.

[0075] According to the above-mentioned medicine, preferably, the promoter is a substance that increases the expression level of the Fam198B gene.

[0076] According to the above-mentioned drug, preferably, the promoter comprises an expression vector carrying a nucleotide sequence encoding Fam198B protein or a recombinant virus containing a nucleotide sequence encoding Fam198B protein.

[0077] Compared with the prior art, the present invention has the following beneficial effects:

[0078] The expression level of Fam198B was significantly upregulated, and the expression of Fam198B continued to increase in the myocardial infarction tissue of myocardial infarction mice, indicating that Fam198B plays a specific function in the process of myocardial infarction. In order to further confirm the role of Fam198B in myocardial infarction, the inventors constructed a cardiomyocyte-specific knockout mouse of Fam198B gene and studied the effect of Fam198B gene-specific knockout on myocardial infarction in mice. The results showed that compared with the cardiomyocyte-non-knockout mice of Fam198B gene, the cardiomyocyte apoptosis of mice in the cardiomyocyte-specific knockout mice was significantly increased 1 day after myocardial infarction. The infarction area increased significantly, and the heart contraction function deteriorated; 4 weeks after myocardial infarction, the myocardial tissue remodeling of mice was significantly aggravated, and the mortality rate of mice increased significantly; the inventors also constructed a mouse with Fam198B gene knockout in myocardial fibroblasts to study the effect of Fam198B gene knockout on myocardial infarction in mice. The results showed that compared with mice with Fam198B gene knockout in myocardial fibroblasts, the myocardial infarction area of ​​Fam198B gene knockout in myocardial fibroblasts increased significantly, the heart contraction function deteriorated, the collagen secretion of fibroblasts increased, and the mortality rate of mice increased significantly 10 days after myocardial infarction. The above results show that Fam198B is involved in the dual regulation of myocardial cell survival and fibroblast activation, and plays a cardioprotective role in the process of myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is the result of Western blotting test on the expression level of Fam198B protein in the IZ region of myocardial tissue in a mouse myocardial infarction model;

[0080] Figure 2 The results of cardiac ultrasound, TUNNEL staining, TTC staining of myocardial tissue and mouse survival curve of mice with in vivo cardiomyocyte-specific knockout of Fam198B gene are shown in Figure 1. A is a representative picture of mouse cardiac ultrasound detection; B is the statistical results of left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) of each group of mice evaluated by mouse cardiac ultrasound; C is a representative picture of TUNNEL staining; D is the quantitative statistical result of TUNNEL staining; E is a representative picture of TTC staining, and F is the quantitative statistical result of myocardial infarction area by TCC staining; G is the statistical result of mouse survival curve;

[0081] Figure 3The immunofluorescence staining, collagen quantitative statistics, cardiac ultrasound and mouse survival curve of mouse myocardial tissue after the Fam198B gene was specifically knocked out in vivo; A is a representative picture of immunofluorescence staining of myocardial tissue of mice in each group, and the fluorescence indicates the collagen fibers secreted by fibroblasts; B and C are quantitative statistical results of immunofluorescence staining of type I collagen (Collagen I) and type III collagen (Collagen III), respectively; D is a representative picture of mouse cardiac ultrasound detection; E is the statistical results of left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) of each group of mice evaluated by mouse cardiac ultrasound; F is the statistical results of mouse survival curve. 。 DETAILED DESCRIPTION

[0082] The following examples are only for further explanation of the present invention. It should be noted that all technical and scientific terms used in the present invention have the same meanings as in the technical field to which the present invention belongs unless otherwise specified. The experimental methods in the following examples without specifying specific conditions are all based on conventional techniques in the technical field, or according to the conditions recommended by the manufacturer; the reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0083] In order to make the purpose, technical solution and advantages of the present invention clearer and more specific, the present invention is further described in detail by examples below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0084] Molecular biochemistry reagents:

[0085] β-Mercaptoethanol, sodium dodecyl sulfate, and Tris base were purchased from Sigma, USA;

[0086] BCA quantitative kit was purchased from ThermoScientific, USA;

[0087] 30% methylene bisacrylamide was purchased from Bio-Rad, USA;

[0088] Triethanolamine buffer (TBS), Tween-20, phosphate buffered saline (PBS), and Fam198B primers were purchased from Sangon Biotechnology (China);

[0089] Horseradish peroxidase chemiluminescent solution was purchased from Millipore, USA;

[0090] Isoflurane was purchased from Lunanbet, China;

[0091] RNA reverse transcription kit and DNA amplification kit were purchased from Takara Japan;

[0092] Fam198B antibody was purchased from abcam, USA;

[0093] GAPDH antibody was purchased from Cell Signaling Technology, USA;

[0094] Horseradish peroxidase secondary antibody was purchased from Jackson, USA;

[0095] Tunel kit was purchased from Roche;

[0096] TTC (2,3,5-triphenyltetrazolium chloride) staining solution was purchased from MesGen.

[0097] Example 1: Study on Fam198B expression in a mouse myocardial infarction model

[0098] 1. Experimental animals:

[0099] Forty male mice aged 8-12 weeks were divided into two groups, the experimental group (MI) and the sham operation group (Sham), with 20 mice in each group. The mice were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0100] 2. Experimental methods:

[0101] Establishment of mouse myocardial infarction model: The mice were anesthetized with 3% isoflurane, and the anesthetic effect was evaluated by observing the disappearance of reaction and the degree of muscle relaxation. After anesthesia, the mice were fixed on the operating table in supine position with their limbs fixed, the chest area was depilated, the surgical area was disinfected, the trachea was intubated at the tracheotomy site, and the ventilator was used for assisted breathing (using volume control mode, tidal volume 3ml / 100g, respiratory rate 60-70 times / min, and respiratory ratio of 1:1).

[0102] A purse was made at the left edge of the sternum with No. 3.0 surgical sutures. After longitudinal incision of the skin, the subcutaneous tissue and muscles were bluntly separated. The intercostal space was opened at the strongest point of the apical beat, the pericardium was quickly located and the pericardium was punctured to fully expose the heart. The left anterior descending branch of the mice in the experimental group (MI) was ligated with No. 5.0 sutures, while the mice in the control group (Sham) were threaded but not ligated. The heart was then immediately replaced, the chest cavity was quickly closed after squeezing out the residual gas in the chest cavity, and the purse was sutured. The endotracheal tube was removed after surgery. In order to reduce airway edema and asphyxiation caused by excessive secretions, the trachea and neck incision were not sutured.

[0103] At different time points after surgery (1 day, 3 days, 1 week, 2 weeks, and 4 weeks), myocardial tissues of the infarct zone (IZ) of the left ventricular free wall were obtained, and Western blotting was used to detect the expression level of Fam198B protein in the myocardial tissues of the IZ zone.

[0104] 3. Experimental results:

[0105] The expression level of Fam198B protein in the IZ region of myocardial tissue in the mouse myocardial infarction model was detected by Western blotting. Figure 1 shown.

[0106] Depend on Figure 1 It can be seen that one week after coronary artery ligation and infarction, the expression of Fam198B protein in the IZ region of mouse myocardial tissue continued to increase with the increase of infarction time, and the difference was statistically significant.

[0107] Example 2: Knockout of Fam198B gene in myocardial cells in vivo to study the effect of Fam198B on myocardial infarction

[0108] 1. Breeding of mice with cardiomyocyte-specific knockout of Fam198B gene:

[0109] Breeding Fam198B flox / flox The offspring were hybridized with the parent mice and the target mice Fam198B were obtained according to the genotype identification results. fl / fl Myh6 Cre+ (Myocardial cell-specific knockout of Fam198B gene mice). The above-mentioned genetically engineered mice have been constructed and continuously bred at Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0110] 2. Experimental grouping and treatment:

[0111] Experimental setup for Fam198B fl / fl Myh6 Cre+ group and Fam198B fl / fl Myh6 Cre- groups, each with 20 mice; Fam198B fl / f l Myh6 Cre+ Group mice and Fam198B fl / fl Myh6 Cre- The mice in the two groups underwent coronary artery ligation to establish the mouse myocardial infarction model.

[0112] 3. Experimental methods and results:

[0113] (1) Cardiac ultrasound evaluation

[0114] One day after coronary artery ligation, Fam198B fl / fl Myh6 Cre+ Group and Fam198B fl / fl Myh6 Cre- The mice in the two groups were examined by cardiac ultrasound.

[0115] The specific operation of cardiac ultrasound detection is as follows: use 2% isoflurane for continuous inhalation anesthesia and fix the mice in a supine position on an ultrasound examination table with a self-heating function. First, remove the mouse hair in the precordial area with a depilatory cream. Use the VisualSonics VeVo770 ultrasound diagnostic instrument and a 30MHz high-frequency probe to collect parasternal long-axis sections on the chest wall, record electrocardiograms, and measure heart rate. A two-dimensional ultrasound section image is obtained at the level of the papillary muscle under the mitral valve in the parasternal left ventricular short-axis section, and the left ventricular ejection fraction (LVEF) and shortening fraction (LVFS) are measured. The instrument parameter settings for the examination of each group of mice remain the same, and the same person performs image acquisition and calculation. The average value of 4 to 5 different cardiac cycles is selected for each measurement index.

[0116] The results of cardiac ultrasound examination 1 day after coronary artery ligation are as follows Figure 2 As shown in A and B. Figure 2 As shown in A and B, Fam198B fl / fl Myh6 Cre- Compared with the control group, the cardiac contractile function of mice with cardiomyocyte-specific knockout of Fam198B gene deteriorated and the cardiac function was significantly impaired one day after myocardial infarction modeling.

[0117] (2) TUNNEL staining

[0118] One day after coronary artery ligation, Fam198B fl / fl Myh6 Cre+ Group and Fam198B fl / fl Myh6 Cre- The myocardial tissues of mice in the two groups were subjected to TUNNEL staining.

[0119] The specific operation of TUNNEL staining is as follows: take the paraffin sections of mouse myocardial tissue and place them in a staining jar, wash them twice with xylene, 5 minutes each time, wash them twice with anhydrous ethanol, 3 minutes each time, wash them once with 95v% and 75v% ethanol, 3 minutes each time, wash them with PBS for 5 minutes, add proteinase K solution (20ug / ml), hydrolyze them at room temperature for 15 minutes, and remove tissue protein. Then wash them 4 times with distilled water, 2 minutes each time, add PBS containing 2% hydrogen peroxide, and react at room temperature for 5 minutes. Then wash them twice with PBS, 5 minutes each time, carefully absorb the excess liquid around the tissue on the slide with filter paper, immediately add 2 drops of TdT enzyme buffer on the section, and place it at room temperature for 1 to 5 minutes. Then carefully absorb the excess liquid around the section with filter paper, immediately add 54μl TdT enzyme reaction solution on the section, and place it in a humidified box at 37C for 1 hour. Then place the slices in a staining jar, add the washing and termination reaction buffer preheated to 37°C, keep warm at 37°C for 30 minutes, and gently lift and put down the slide every 10 minutes to stir the liquid slightly. Then wash the tissue slices with PBS 3 times, 5 minutes each time, and directly add two drops of peroxidase-labeled anti-digoxigenin antibody on the slices, and react in a wet box at room temperature for 30 minutes. Then wash with PBS 4 times, 5 minutes each time, and directly add freshly prepared 0.05% DAB solution on the tissue slices, and color at room temperature for 3 to 6 minutes. Then wash with distilled water 4 times, 1 minute each time for the first 3 times, and 5 minutes for the last time, and counterstain with methyl green at room temperature for 10 minutes. Then wash with distilled water 3 times, lift and put down the slide 10 times for the first two times, and let it stand for 30 seconds for the last time, and wash it three times with 100% n-butanol in the same way. Then, the samples were dehydrated with xylene for 3 times, 2 min each time. After sealing and drying, the experimental results were observed and recorded under an optical microscope, and the proportion of apoptotic cells was calculated.

[0120] The results of TUNNEL staining of mouse myocardial tissue 1 day after coronary artery ligation are as follows Figure 2 As shown in C and D. Figure 2 As can be seen from C and D, Fam198B fl / fl Myh6 Cre- Compared with the control group, the apoptosis and necrosis of myocardial cells in the Fam198B gene knockout mice were significantly increased one day after myocardial infarction modeling, and the difference was statistically significant.

[0121] (3) TTC staining

[0122] One day after coronary artery ligation, Fam198B fl / fl Myh6 Cre+ Group and Fam198B fl / fl Myh6 Cre-The myocardial tissues of mice in each group were stained with TTC, and the myocardial infarction lesions and infarction areas of mice in each group were observed.

[0123] The specific operation of TTC staining is as follows: prepare TTC (2,3,5-triphenyltetrazolium chloride) staining solution, usually using 1% TTC solution, that is, dissolve 1 gram of TTC powder in 100 ml of phosphate buffer (PBS), adjust the pH to 7.4, check the color of the solution, ensure it is bright red, and store it away from light. Take myocardial tissue and pre-treat it. Select healthy or diseased myocardial tissue as a sample, quickly take the sample, avoid long-term exposure of the tissue to the air to cause oxidation, use physiological saline to wash the surface of the tissue, and remove blood and impurities. Place the tissue block in a culture dish containing an appropriate amount of physiological saline, trim it to a size suitable for slicing with a blade, and use a tissue slicer to cut the trimmed myocardial tissue into thin slices, usually 1-2 mm thick. Keep the blade sharp during slicing to ensure that the slices are flat, without curling or tearing. Place the cut myocardial tissue slices in the pre-prepared TTC staining solution, ensure that the tissue slices are completely immersed in the staining solution, and avoid bubbles. Staining is performed under light-proof and constant temperature (usually 37°C) conditions, and the time is generally 15 to 30 minutes. When the staining time is reached, the tissue slices are taken out, the staining solution on the surface is gently rinsed off with physiological saline, and the tissue slices are placed in a fixative containing an appropriate amount of 4% paraformaldehyde for fixation.

[0124] The results of TTC staining are as follows Figure 2 As shown in E and F. Figure 2 E and F show that Fam198B fl / fl Myh6 Cre- Compared with the control group, the myocardial infarction area of ​​the mice with cardiomyocyte-specific knockout of Fam198B gene increased significantly one day after myocardial infarction modeling, and the difference was statistically significant.

[0125] (4) Mouse survival curve statistics

[0126] Fam198B after coronary artery ligation fl / fl Myh6 Cre+ Group and Fam198B fl / fl Myh6 Cre- The survival curve of the mice in the Figure 2 As shown in G.

[0127] Depend on Figure 2 As can be seen from the middle G, it is similar to Fam198B fl / fl Myh6 Cre- Compared with the control group, the survival time of mice with myocardial infarction model was significantly shortened after myocardial infarction was modeled by myocardial cell-specific knockout of Fam198B gene. This indicates that myocardial cell-specific knockout of Fam198B gene will accelerate the death of mice with myocardial infarction and significantly increase the mortality rate of mice with myocardial infarction.

[0128] Example 3: Specific knockout of Fam198B gene in cardiac fibroblasts in vivo to study the effect of Fam198B on myocardial infarction

[0129] 1. Breeding of mice with Fam198B gene knockout specifically for cardiac fibroblasts:

[0130] Breeding Fam198B flox / flox The mice were crossed with Col1a2-CreERT mice, and the offspring were obtained by hybridization. The offspring were hybridized with the parent mice, and the target mice Fam198B were obtained according to the genotype identification results. fl / fl Col1a2 Cre+ Tamoxifen induces Cre enzyme expression, causing the Fam198B gene in fibroblasts to be specifically knocked out. The above-mentioned genetically engineered mice have been constructed and continuously bred by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. fl / fl Col1a2 Cre+ Mice with Fam198B fl / fl Col1a2 Cre- Mice underwent coronary artery ligation.

[0131] 2. Experimental grouping and treatment:

[0132] Experimental setup for Fam198B fl / fl Col1a2 Cre+ Group and Fam198B fl / fl Col1a2 Cre- groups, each with 20 mice; Fam198B fl / fl Col1a2 Cre+ Group mice, Fam198B fl / fl Col1a2 Cre- The two groups underwent coronary artery ligation to establish mouse myocardial infarction models.

[0133] 3. Experimental methods and results:

[0134] (1) Immunofluorescence staining

[0135] Ten days after coronary artery ligation, Fam198B fl / fl Col1a2 Cre+ Group and Fam198B fl / fl Col1a2 Cre- Immunofluorescence staining was performed on the myocardial tissues of the mice in the two groups.

[0136] The specific operation of immunofluorescence staining is as follows: take paraffin sections of mouse myocardial tissue and bake them at 60°C for 30 minutes. Then dewax with xylene and hydrate with alcohol gradient, immerse in antigen repair solution (0.01mol / L sodium citrate, pH=6.0) to repair antigens under high pressure. Wash three times with PBS for 5 minutes each time, and then block with 5% BSA at room temperature for 30 minutes, and carefully absorb the blocking solution with absorbent paper. Use 5% BSA to dilute the primary antibody in an appropriate proportion, incubate at 4°C overnight, and wash three times with PBS for 5 minutes each time. Use 5% BSA to dilute the secondary antibody in an appropriate proportion, incubate at room temperature for 1 hour, and wash three times with PBS for 5 minutes each time. After sealing with a coverslip sealing agent, observe under a fluorescence microscope and take pictures.

[0137] Representative images of immunofluorescence staining are shown in Figure 3 As shown in A. Figure 3 As shown in A, Fam198B fl / fl Col1a2 Cre- Compared with the control group, the secretion of collagen in cardiac fibroblasts of Fam198B gene-specific knockout mice increased 10 days after myocardial infarction modeling.

[0138] The quantitative statistical results of immunofluorescence staining of type I collagen (Collagen I) and type III collagen (Collagen III) are as follows Figure 3 As shown in B and C, Figure 3 As shown in Figures B and C, the secretion of type I collagen and type III collagen increased significantly after the Fam198B gene was knocked out in cardiac fibroblasts. This indicates that after the Fam198B gene was specifically knocked out in fibroblasts, the secretion of collagen in fibroblasts increased, inhibiting remodeling, and Fam198B played a protective role in the long term of myocardial infarction.

[0139] (2) Cardiac ultrasound evaluation

[0140] Ten days after coronary artery ligation, Fam198B fl / fl Col1a2 Cre+ Group and Fam198B fl / fl Col1a2 Cre- The specific operation of cardiac ultrasound detection is the same as that in Example 2. The results of cardiac ultrasound detection are shown in Figure 3 As shown in D and E.

[0141] Among them, the results of cardiac ultrasound examination 10 days after coronary artery ligation were as follows Figure 3 As shown in D and E. Figure 3 As shown in D and E, Fam198B fl / fl Col1a2 Cre-Compared with the control group, the cardiac function of mice with Fam198B gene knockout in cardiac fibroblasts was significantly impaired 10 days after myocardial infarction modeling.

[0142] (3) Mouse survival curve statistics

[0143] Fam198B after coronary artery ligation fl / fl Col1a2 Cre+ Group and Fam198B fl / fl Col1a2 Cre- The survival rate of mice in the Figure 3 As shown in F.

[0144] Depend on Figure 3 It can be seen from the middle F that it is related to Fam198B fl / fl Col1a2 Cre- Compared with the control group, the survival time of mice with Fam198B gene knockout in cardiac fibroblasts was significantly shortened after myocardial infarction modeling. This indicates that the Fam198B gene knockout in cardiac fibroblasts can significantly increase the mortality rate of mice with myocardial infarction.

[0145] In summary, the present invention effectively overcomes the deficiencies in the prior art and has a high industrial utilization value. The above embodiments serve to illustrate the substantial content of the present invention, but do not limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of Fam198B gene or the protein encoded by it in the preparation of drugs for preventing, alleviating or / and treating myocardial infarction.

2. Application of Fam198B gene or Fam198B protein as a drug target for screening drugs for preventing, alleviating or / and treating myocardial infarction.

3. Use of a promoter of Fam198B gene expression in the preparation of a drug for preventing, alleviating or / and treating myocardial infarction.

4. The use according to claim 3, characterized in that: The promoter is a substance that increases the expression level of the Fam198B gene.

5. The use according to claim 3, characterized in that: The promoter comprises an expression vector carrying a nucleotide sequence encoding Fam198B protein or a recombinant virus containing a nucleotide sequence encoding Fam198B protein.

6. A drug, characterized in that The medicine is used for preventing, alleviating or / and treating myocardial infarction, and contains Fam198B gene or Fam198B protein or Fam198B gene expression promoter.

7. The drug according to claim 6, characterized in that The medicine also contains pharmaceutically acceptable carriers / auxiliaries.

8. The drug according to claim 6 or 7, characterized in that The promoter is a substance that increases the expression level of the Fam198B gene.

9. The drug according to claim 8, characterized in that The promoter comprises an expression vector carrying a nucleotide sequence encoding Fam198B protein or a recombinant virus containing a nucleotide sequence encoding Fam198B protein.

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