Application of HMGB2 in promotion of myocardial regeneration and repair after heart injury

Abstract

The invention discloses an application of a high mobility group protein B2 (HMGB2) in regeneration and repair of cardiac muscle after myocardial injury, and belongs to the technical field of biomedicine. Test results show that by overexpressing HMGB2 protein in myocardial cells, the in-vitro myocardial cell proliferation capacity is remarkably enhanced, and meanwhile, after acute myocardial ischemia injury in vivo, the myocardial cells can quickly start endogenous myocardial regeneration to play roles in promoting myocardial regeneration and repair, reducing scar formation, improving cardiac functions and the like. Besides, the invention designs a drug delivery mode based on a virus vector carrying HMGB2 gene, the problems of poor targeting, low biological safety, low treatment efficiency and the like of a traditional treatment method are solved, a new drug action target is provided for prevention and treatment of acute myocardial infarction and myocardial ischemia-reperfusion injury, and the drug delivery system has important clinical transformation value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to an application of high mobility group box protein 2 (HMGB2) protein in myocardial regeneration and repair after cardiac injury. Background Art

[0002] Cardiovascular disease, especially myocardial infarction, is one of the main causes of high mortality and significant health burden worldwide. Myocardial infarction occurs mainly due to the large-scale loss of cardiomyocytes, while the proliferation capacity of adult cardiomyocytes is extremely limited and they are unable to regenerate damaged myocardial tissue through their own repair mechanisms. This lack of regenerative capacity makes the heart prone to fibrosis and pathological remodeling after infarction, leading to a gradual decline in cardiac function and eventually developing into heart failure.

[0003] Studies in recent years have found that promoting cardiomyocyte proliferation may be a key path to repair heart damage and restore heart function. Although studies have shown that the heart of mammals in the newborn period has a certain regenerative ability, such as complete repair of damaged sites through the proliferation of cardiomyocytes, this ability is rapidly lost in adulthood. At present, strategies for intervening in cardiomyocyte proliferation are mainly concentrated in the fields of gene editing and stem cell transplantation. However, these methods have problems such as high cost, complex technology, donor shortage and immune rejection in practical applications, which greatly limits their clinical promotion.

[0004] In contrast, endogenous molecular regulatory factors have gradually become a hot topic in the study of promoting cardiomyocyte proliferation due to their potential targeting and operability. HMGB2 (High Mobility Group Box Protein 2) is a member of the high mobility group protein superfamily (HMG) and contains an HMG box motif. The four members of its family are HMGB1 to 4. HMGB1 and HMGB2 are both highly conserved nuclear proteins. As chromatin binding factors, they can bind to DNA and promote transcription protein assembly. Studies have confirmed that HMGB1 is an oncogene for colorectal cancer, and its high expression is a molecular marker for predicting the prognosis of colorectal cancer [Zhang W, An F, Xia M, et al. Increased HMGB1 expression correlates with higher expression of c-IAP2 and pERK in colorectal cancer [J]. Medicine (Baltimore), 2019, 98 (3): e14069.]. HMGB2 is highly expressed during embryonic development and is mainly expressed in adult testis and lymphoid organs [Kimura A, Matsuda T, Sakai A, et al. HMGB2 expression is associated with transition from aquiescent to an activated state of adult neural stem cells [J]. Dev Dyn, 2018, 247(1): 229-238.]. HMGB2 has been found to regulate cell proliferation, migration and apoptosis in prostate and ovarian cancer [Barreiro-Alonso A, Lamas-Maceiras M, García-Díaz R, et al. Delineating the HMGB1and HMGB2 interactome in prostate and ovary epithelial cells and its relationship with cancer [J]. Oncotarget, 2018, 9(27): 19050-19064.].In gastric cancer, high expression of HMGB2 is associated with poor prognosis of gastric cancer [Cui G, Cai F, Ding Z, et al. HMGB2 promotes themalignancy of human gastric cancer and indicates poor survival outcome [J]. Hum Pathol, 2019, 84: 133-141.]. HMGB2 widely affects gene expression in many ways, such as regulating chromatin structure and participating in the interaction between transcription factors and DNA.

[0005] By searching domestic and foreign literature, there is no literature report that HMGB2 protein improves cardiac function after myocardial infarction by promoting cardiomyocyte proliferation. Summary of the invention

[0006] In view of the high cost and technical complexity of gene editing and stem cell transplantation technologies, the main purpose of the present invention is to provide an application of HMGB2 in promoting myocardial regeneration and repair after cardiac injury.

[0007] The inventors unexpectedly discovered in the clinic that the expression of HMGB2 in the myocardial cells of patients with heart damage showed an upward trend. Based on this discovery, the inventors conceived that HMGB2 could be used as a new target for the treatment of myocardial damage, by overexpressing HMGB2 in myocardial cells to promote myocardial cell proliferation. In subsequent studies, the inventors verified this concept through a large number of experiments, and finally developed a new strategy based on HMGB2, that is, regulating myocardial cell proliferation and improving cardiac function through HMGB2, and promoting myocardial regeneration and repair after heart damage, which not only has important scientific value, but also provides a new direction for solving the clinical problem of functional recovery after myocardial infarction, and has important clinical application value.

[0008] Specifically, the purpose of the present invention is achieved through the following technical solutions: any of the following applications of the HMGB2 gene: (1) application in the preparation of a drug or reagent for promoting myocardial cell proliferation; (2) application in the preparation of a drug for promoting myocardial regeneration and repair after cardiac injury; (3) application in the preparation of a drug for inhibiting myocardial fibrosis and cardiac remodeling after cardiac injury. In a specific embodiment of the present invention, the above application is to overexpress the HMGB2 gene in the myocardial tissue of the body. Further preferably, the cardiac injury is acute myocardial infarction or myocardial ischemia-reperfusion injury.

[0009] In order to solve the problems of poor targeting, low biosafety, and low therapeutic efficiency in traditional drug administration, the inventors designed a drug administration method in which adenovirus and adeno-associated virus vectors carry human HMGB2 genes to prepare HMGB2 preparations. Therefore, the purpose of the present invention can also be achieved through the following technical solutions: any of the following applications of recombinant adenovirus or recombinant adeno-associated virus expressing HMGB2 protein: (1) application in the preparation of drugs or reagents for promoting myocardial cell proliferation; (2) application in the preparation of drugs for promoting myocardial regeneration and repair after cardiac injury; (3) application in the preparation of drugs for inhibiting myocardial fibrosis and cardiac remodeling after cardiac injury. Further preferably, the cardiac injury is acute myocardial infarction or myocardial ischemia-reperfusion injury.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the HMGB2 protein is shown in SEQ ID NO: 1 or SEQ ID NO: 3, which is as follows:

[0011] SEQ ID NO: 1 (mouse origin):

[0012] MGKGDPNKPRGKMSSYAFFVQTCREEHKKKHPDSSVNFAEFSKKCSERWKTMSAKE

[0013] KSKFEDLAKSDKARYDREMKNYVPPKGDKKGKKKDPNAPKRPPSAFFLFCSENRPKI

[0014] KIEHPGLSIGDTAKKLGEMWSEQSAKDKQPYEQKAAKLKEKYEKDIAAYRAKGKSE

[0015] AGKKGPGRPTGSKKKKNEPEDEEEEEEEEEEEDDEEEEEDEE

[0016] SEQ ID NO: 3 (human origin):

[0017] MGKGDPNKPRGKMSSYAFFVQTCREEHKKKHPDSSVNFAEFSKKCSERWKTMSAKE

[0018] KSKFEDMAKSDKARYDREMKNYVPPKGDKKGKKKDPNAPKRPPSAFFLFCSEHRPK

[0019] IKSEHPGLSIGDTAKKLGEMWSEQSAKDKQPYEQKAAKLKEKYEKDIAAYRAKGKSE

[0020] AGKKGPGRPTGSKKKKNEPEDEEEEEEEEDEDEEEEDEDEE

[0021] In a preferred embodiment of the present invention, the recombinant adenovirus or recombinant adeno-associated virus contains a gene encoding the HMGB2 protein, and the nucleotide sequence of the coding strand of the gene encoding the HMGB2 protein is shown in SEQ ID NO: 2 or SEQ ID NO: 4, specifically as follows:

[0022] SEQ ID NO: 2 (mouse origin):

[0023] atgggcaagggtgaccccaacaagccgcggggcaaaatgtcctcgtacgccttcttcgtgcagacctgccgcgaggagcacaagaa

[0024] gaagcatcccgactcgtcggtgaacttcgccgagttctccaagaaatgctccgagagatggaagaccatgtctgcaaaggaaaagtcc

[0025] aagtttgaagatttggccaagagcgacaaagctcgttatgacagggatgaagaactatgttcctcccaaaggggataagaaaggaa

[0026] agaaaaaagaccccaatgctccgaagagaccaccgtctgccttcttcctgttttgctctgaaaatcgcccaaagatcaaaattgaacacc

[0027] caggcctgtctattggagatactgcgaagaaactgggtgagatgtggtctgagcaatctgccaaagataaacaaccgtatgagcagaa

[0028] agcagctaaactaaaggagaagtatgaaaaggatattgctgcataccgtgccaagggcaaaagtgaagcaggaaagaagggtcctg

[0029] gtaggccaacaggctcaaagaagaagaacgaaccagaagatgaggaggaggaagaagaggaggaagaggaggaagatgacga

[0030] ggaagaagaggaggatgaagaataa

[0031] SEQ ID NO: 3 (human origin):

[0032] atgggtaaaggagaccccaacaagccgcggggcaaaatgtcctcgtacgccttcttcgtgcagacctgccgggaagagcacaagaa

[0033] gaaacacccggactcttccgtcaatttcgcggaattctccaagaagtgttcggagagatggaagaccatgtctgcaaaggagaagtcg

[0034] aagtttgaagatatggcaaaaagtgacaaagctcgctatgacagggagatgaaaaattacgttcctcccaaaggtgataagaagggga

[0035] agaaaaaggaccccaatgctcctaaaaggccaccatctgccttcttcctgttttgctctgaacatcgcccaaagatcaaaagtgaacacc

[0036] ctggcctatccattggggatactgcaaagaaattgggtgaaatgtggtctgagcagtcagccaaagataaacaaccatatgaacagaaa

[0037] gcagctaagctaaaggagaaatatgaaaaggatattgctgcatatcgtgccaagggcaaaagtgaagcaggaaagaagggccctgg

[0038] caggccaacaggctcaaagaagaagaacgaaccagaagatgaggaggaggaggaggaagaagaagatgaagatgaggaggaa

[0039] gaggatgaagatgaagaataa

[0040] Further preferably, the present invention uses two recombinant viruses, namely recombinant adenovirus Ad5:cTNT-HMGB2 and recombinant adeno-associated virus AAV9:cTNT-HMGB2, so the adenovirus is Ad5 and the adeno-associated virus is AAV9; both viruses contain the myocardial-specific promoter cTNT, so that the purpose of specifically expressing HMGB2 protein in myocardial cells can be achieved by driving the myocardial-specific cTNT promoter.

[0041] Furthermore, the present invention also provides a drug for promoting myocardial regeneration and repair, wherein the active ingredient of the drug comprises a recombinant adenovirus or a recombinant adeno-associated virus expressing HMGB2 protein. Further preferably, the drug also comprises a pharmaceutically acceptable excipient or additive, so as to be prepared into an injection for in situ myocardial injection or intravenous administration. In certain specific embodiments, the drug achieves the effect of promoting myocardial regeneration and repair by upregulating the expression level of HMGB2 or enhancing its activity. Preferably, the drug comprises an effective ingredient capable of regulating the expression or function of HMGB2, such as a small molecule agonist, a protein drug or a gene editing tool. Preferably, the drug can also be used in combination with other drugs for treating myocardial injury, and the combined use of multiple drugs can significantly improve the therapeutic effect.

[0042] Compared with the prior art, the present invention has the following advantages and progress:

[0043] (1) The myocardial regeneration and repair function of HMGB2 protein is clarified for the first time: This invention reveals for the first time the role of HMGB2 protein in promoting myocardial regeneration and repair. By supplementing HMGB2 protein in myocardial cells, the proliferation capacity of myocardial cells in vitro and in vivo is significantly improved. After the adult heart is injured by acute myocardial ischemia, myocardial cells quickly activate the endogenous regeneration mechanism, thereby promoting the regeneration and repair of damaged myocardial tissue, reducing scar formation and improving heart function.

[0044] (2) Significant cardiac protection and repair effects: Studies have shown that local injection of HMGB2 into the infarct margin area can effectively promote endogenous proliferation of cardiomyocytes in adult mice, reduce chronic scars, inhibit ventricular pathological remodeling, and significantly improve the level of cardiac function recovery.

[0045] (3) cTNT-specific promoter enhances the precision and efficiency of treatment: The present invention uses a viral vector carrying the cardiomyocyte-specific promoter cTNT to achieve specific expression of the exogenous HMGB2 gene only in myocardial tissue. This design ensures that the treatment process is limited to myocardial cells, avoiding side effects caused by off-target tissues or cells. In addition, the cTNT-driven gene therapy strategy based on viral vectors provides an ideal precision treatment solution for myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 :The expression changes of HMGB2 protein level after myocardial injury;

[0047] Figure 2 : pH3 immunofluorescence staining and statistical graph of myocardial cells after HMGB2 preparation of the present invention was transfected into primary mouse myocardial cells;

[0048] Figure 3 : The cardiac EF and FS values ​​of mice after in situ injection of the HMGB2 preparation of the present invention at 1 and 28 days after MI injury;

[0049] Figure 4 : The expression of HMGB2 protein in the heart tissue of mice 2 weeks after in situ injection of the HMGB2 preparation of the present invention after MI and the statistical graph;

[0050] Figure 5 : The cardiac fibrosis area and statistical graph of mice injected orally with the HMGB2 preparation of the present invention 28 days after MI surgery;

[0051] Figure 6 : Ki67 immunofluorescence staining and statistical graph of heart tissue of mice injured by MI 2 weeks after in situ injection of the HMGB2 preparation of the present invention. DETAILED DESCRIPTION

[0052] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments are only partial examples of the present invention and cannot be regarded as limiting the scope of application of the present invention. Based on the embodiments of the present invention, other implementation methods that can be thought of by those skilled in the art without creative work all belong to the protection scope of the present invention. For specific technical operation steps and operators not clearly listed in the embodiments, they should be processed according to the conventional technical conditions described in the existing literature or relevant product manuals in the field.

[0053] Example 1: The expression level of HMGB2 showed an upward trend after cardiac injury in mice.

[0054] Construction of mouse apical resection model: Six newborn C57BL / 6J male mice were used in this experiment. These mice were purchased from the Medical Experimental Animal Center of Nanjing Medical University and divided into two groups, namely, the apical resection (AR) model group and the sham operation group (sham). The specific operation of the experiment is as follows: First, the mice were placed on ice for hypothermia anesthesia for about 3 minutes until the mice lost their pain response. After anesthesia, the mice were fixed under a stereomicroscope, and the surgical area and surrounding skin were disinfected with iodine. Then, the skin of the anterior heart area was cut open with microsurgical scissors, the intercostal muscles were bluntly separated, and the chest cavity was exposed. The mouse abdomen was gently pressed to make the apex protrude and expose it outside the chest cavity. Then, a microinjector equipped with a 36G needle was used to perform intramyocardial injections in the anterior, middle, and posterior walls of the left ventricle in the lower third of the heart, respectively, and a virus dilution containing trypan blue markers was injected.

[0055] The next step is to carefully cut away the myocardial tissue, which accounts for about 15% of the total weight of the ventricle, using surgical scissors under a stereo microscope. After surgery, the heart is gently returned to the chest cavity using a cotton swab containing normal saline, and the subcutaneous tissue and skin are sutured layer by layer with 6-0 sutures. Finally, the mouse is placed on a constant temperature table to restore its body temperature, and is returned to the cage after the mouse resumes normal activity. After all mice have completed the surgery, they are uniformly returned to their mothers.

[0056] Heart tissue sampling: For suckling mice within 7 days of birth, the heart was sampled after low-temperature anesthesia on ice; for mice over 7 days old and adult mice, 1.2% tribromoethanol (200μl / 10g body weight) was used to fully anesthetize the samples. After complete anesthesia, the mice were killed, and the chest was opened and the adhesions of the chest wall were removed with fine scissors. The heart was removed and immediately placed in PBS to squeeze out the residual blood to prevent coagulation. After the blood was completely squeezed out, the adhesion tissue on the surface of the heart was gently removed with microsurgical scissors and forceps, and the intact heart was retained. The heart tissue used for immunofluorescence and Masson staining experiments was fixed in 4% paraformaldehyde solution overnight, and then changed to 70% ethanol for storage; the tissue used for Western blot (WB) detection was placed in RIPA lysis buffer containing three protease inhibitors.

[0057] Protein extraction: Place the tissue on ice and use sterilized scissors to cut it as finely as possible. After adding the protein extract, use an ultrasonic device (4°C, energy 25%) to sonicate for 3 seconds and immediately cool in an ice bath. Repeat the sonication 2-3 times, the specific number of times depends on the state of the tissue. Let the sample stand on ice for 1 hour, and vortex mix for 5-10 seconds every 10 minutes. After standing, centrifuge in a high-speed centrifuge (4°C, 12,000 rpm, 1 hour). Collect the supernatant and place it in a new 1.5ml EP tube, and determine the protein concentration using the BCA method.

[0058] Western blot detection of HMGB2 protein expression:

[0059] 1. Sample processing: After the protein sample is thoroughly mixed with 5X loading buffer, boil it in a 99℃ metal bath for 10 minutes. After cooling, it can be used directly in the experiment or stored at -20℃.

[0060] 2. Gel preparation and electrophoresis: Prepare appropriate concentrations of concentrated gel and separation gel according to the SDS-PAGE gel preparation instructions. Sample loading: marker 5-10μl, protein sample to be tested 25-35μg. Electrophoresis conditions: concentrated gel 80V constant voltage for 25-30 minutes, separation gel 120V for about 100 minutes.

[0061] 3. Transfer: PVDF membrane was fully irradiated with methanol (size 8×5 cm), and the membrane was transferred at 300 mA constant current or 100 V constant voltage for 2 hours.

[0062] 4. Blocking: Prepare blocking solution with 5% BSA and block on a shaker (60 rpm) at room temperature for 2-3 hours.

[0063] 5. Primary antibody incubation: Add the membrane to the proportionally diluted primary antibody solution and incubate overnight at 4°C on a horizontal shaker (60 rpm).

[0064] 6. Secondary antibody incubation: Recover the primary antibody, wash the membrane with TBST for 30 minutes (change 3-5 times in the middle), then add the corresponding secondary antibody solution diluted in proportion, and incubate on a horizontal shaker at room temperature for 2 hours.

[0065] 7. Luminescence imaging: After incubation with the secondary antibody, wash with TBST for 120 minutes (replace TBST 3-5 times), and then rinse the foam with TBS. Prepare the developer according to the ECL developer instructions, expose it, and use a chemiluminescence imager to collect the band image. Western blot results showed that compared with the control group, the expression of HMGB2 protein in the heart of AR mice was significantly increased (see Figure 1 )

[0066] Example 2: Experimental study on HMGB2 promoting the proliferation of mouse cardiomyocytes in vitro.

[0067] In this case, neonatal rat primary cardiomyocytes (NMCMs) were used as experimental cells.

[0068] The myocardium-targeted HMGB2-overexpressing adenovirus (Ad5:cTNT-HMGB2) used in this example was synthesized by Shanghai Genetech Company.

[0069] Method for isolating primary cardiomyocytes from neonatal rats: Before isolating neonatal rat cardiomyocytes, prepare 10×ADS stock solution in advance and sterilize it using a 0.22μm filter membrane. On the day of extraction, dilute 10×ADS into 1×ADS working solution for later use. Select 0-1 day old neonatal rats, 100-150 for each extraction. Quickly immerse the neonatal rat in 75% ethanol for disinfection, remove the heart, and place it in a pre-cooled 1×ADS solution. The assistant uses sterile tweezers to remove the residual blood in the heart cavity and transfer it to a new 1×ADS solution for storage.

[0070] After collecting all the hearts, discard the 1× ADS solution and cut the myocardial tissue into pieces with ophthalmic scissors (about 1-2 minutes). Add 20 ml of preheated myocardial tissue digestion solution and place the sample in a 37°C constant temperature shaker for 6 minutes at 100 rpm. Aspirate the supernatant and transfer it to a sterile 50 ml centrifuge tube, add 4 ml HS to terminate the digestion, and place it on ice. Repeat the digestion process until all myocardial tissue is completely digested into a single cell suspension. Centrifuge all collected supernatants at 1000 rpm for 5 minutes, discard the supernatant and add an appropriate amount of myocardial cell culture medium. Filter out incompletely digested tissue and collagen fibers through a 40 μm mesh.

[0071] The filtered cardiomyocyte suspension was evenly inoculated in a culture dish pre-coated with coating solution, and placed in a 37°C constant temperature incubator for differential adhesion treatment for 45 minutes to remove adherent fibroblasts. After centrifugation, the non-adherent cells were resuspended with 1×ADS, and the cardiomyocytes (middle layer), fibroblasts (upper layer) and red blood cells (lower layer) were separated by Percoll density gradient centrifugation (3000rpm, 30 minutes, 22°C). After recovering the cardiomyocyte layer, it was resuspended with cardiomyocyte culture medium and centrifuged (1000rpm, 5 minutes), and then resuspended in culture medium for cell counting and inoculation and culture. The culture medium was replaced after 24 hours of culture, and viral transfection was performed after 36-48 hours of culture. The cardiomyocyte plating density involved in immunofluorescence staining in this study: 24-well plate, 1.2×10 5 / hole.

[0072] Reagent formulation

[0073] 1.10×ADS: Each 100 ml solution contains 6.8 g sodium chloride, 4.76 g HEPES, 0.138 g sodium dihydrogen phosphate, 0.6 g glucose, 0.4 g potassium chloride, 0.1 g anhydrous magnesium sulfate, and the pH is adjusted to 7.35-7.45. Store at 4°C and use within 2 weeks.

[0074] 2. Myocardial tissue digestion solution: 200 ml working solution is composed of 180 ml ddH2O and 20 ml 10×ADS, collagenase II concentration is 40 mg / 100 ml, and pancreatin concentration is 60 mg / 100 ml. Prepare on the day of extraction, incubate in a 36°C water bath for 30 minutes, and then sterilize with a 0.22 μm filter membrane.

[0075] 3. Percoll solution: Percoll stock solution and 10×ADS were mixed in a ratio of 9:1 to prepare Percoll solution; the upper layer solution was a mixture of Percoll solution and 1×ADS in a ratio of 9:11, and the lower layer solution was a mixture of Percoll solution and 1×ADS in a ratio of 13:7.

[0076] 4. Coating solution: Prepare by mixing 0.5 ml of gelatin with 9.5 ml of PBS.

[0077] 5. Fibroblast culture medium: Every 100 ml of DMEM contains 10 ml of FBS and 1 ml of double antibody (P / S).

[0078] 6. Cardiomyocyte culture medium: Every 100 ml of DMEM contains 5 ml of FBS, 10 ml of HS and 1 ml of double antibody (P / S).

[0079] Construction of primary cardiomyocyte OGD / R model: In order to simulate myocardial infarction injury in vitro, an oxygen glucose deprivation / reoxygenation (OGD / R) model of NMCMs was constructed. The specific operation is as follows: After completing the pretreatment of neonatal rat cardiomyocytes and culturing them for different periods of time, they were replaced with sugar-free and serum-free DMEM medium. Subsequently, the culture dish was placed in an anaerobic environment, specifically by placing it in a sealed wide-mouth bottle containing an anaerobic bag and incubating it at 37°C for 8 hours. Next, the sugar-free and serum-free culture medium was replaced with a complete culture medium containing sugar, and the cells were moved into an incubator at 37°C, 5% CO2 and air balance to continue culturing for 12 hours to complete the reoxygenation process.

[0080] Cell treatment and virus transfection: First, we used different titers of Ad5:cTNT-HMGB2 virus (MOI = 10, 25, 50 and 100) to transfect neonatal rat cardiomyocytes. After transfection for 48 hours, the brightness of GFP fluorescence was observed under a microscope to evaluate the transfection efficiency, and finally determined that MOI = 50 was the appropriate transfection titer. In subsequent experiments, MOI = 50 was used as a uniform transfection concentration. The experiment set up three treatment groups, including an empty virus group, a myocardial-specific HMGB2 gene expression vector group (Ad5:cTNT-HMGB2) and a blank control group. Transfection in all groups was performed at the same MOI = 50 concentration.

[0081] Transfection effect and immunofluorescence staining: 48 hours after transfection, use a microscope to check the transfection effect and perform immunofluorescence staining experiments. The specific steps are as follows:

[0082] Cell fixation and permeabilization: Take out the cell culture dish from the 37°C incubator and use a microscope to observe the growth status and density of the cells. After removing the culture medium, add PBS to the culture dish along the wall of the tube to wash the cells, and repeat 3 times. After removing the PBS, add 4% paraformaldehyde (for fixing cells) to the culture dish, and place it on a shaker at room temperature and shake gently (50 rpm) for 20 minutes. After removing the fixative, add PBS again and shake gently for 5 minutes, repeat 3 times. Next, add goat serum containing 0.2% Triton X-100 (goat serum: PBS = 1:10) for permeabilization, and shake at room temperature (50 rpm) for 30 minutes. After permeabilization, wash again with PBS, repeat 3 times. Then, add goat serum without Triton for blocking, and shake at room temperature for 2 hours.

[0083] Antibody incubation: For primary antibody incubation, use a dilution of the specific antibody (cTNT: goat serum = 1:200, pH3: goat serum = 1:200) and shake overnight at 4°C.

[0084] Secondary antibody incubation and imaging: The next day, after removing and washing the primary antibody dilution, add the corresponding secondary antibody dilution and incubate at room temperature for 2 hours with shaking. After removing the secondary antibody dilution, wash the cells with PBST (50 rpm, 5 minutes, repeated 3 times). Then, perform Hoechst nuclear staining, and finally use a laser confocal microscope to photograph and analyze. By analyzing the pH3 positive signals of each group of cells, the cell proliferation levels of different treatment groups were statistically analyzed (such as Figure 2 The results showed that the primary cardiomyocytes of newborn mice in the Ad5:cTNT-HMGB2 group had obvious cell cycle activity and increased mitosis, indicating that the preparation can effectively promote the proliferation of cardiomyocytes.

[0085] Example 3: Experimental study on the effect of HMGB2 on promoting myocardial regeneration and repair after myocardial infarction in mice

[0086] The myocardium-targeted HMGB2-overexpressing adenovirus (AAV9:cTNT-HMGB2) used in this example was synthesized by Shanghai Genetech Company.

[0087] Animal model establishment and virus injection: This experiment used 40 8-week-old C57BL / 6J male mice, which were purchased from the Medical Experimental Animal Center of Nanjing Medical University and divided into two groups to establish a myocardial infarction (MI) combined with intramyocardial virus injection model at the edge of infarction. The specific operation of the experiment is as follows:

[0088] First, mice were anesthetized to a fully sedated state by intraperitoneal injection of 1.2% tribromoethanol (200 μl / 10 g). Subsequently, the limbs of the mice were fixed to a flat plate using narrow tape, and the mice were intubated and assisted ventilation was performed using a small animal ventilator. The chest of the mice was disinfected and the hair in the precordial area was removed. The intercostal skin was cut open between the 3rd and 4th intercostal spaces on the left side, and the subcutaneous tissue was separated to expose the chest cavity and heart. The heart was gently pushed to the left with the left hand, and the left anterior descending artery (LAD) could be clearly seen under a cold light source. With a fine needle holder in the right hand, a suture needle with a 7-0 suture was used to pass through the myocardium on the left side of the middle segment of the LAD for ligation. At this time, the assistant used a microinjector with a 36G needle to intramyocardially inject AAV9:cTNT-HMGB2 preparation or control virus at the pale edge of the infarct area around the ligation site (including the left, right, and above). 3 μl of virus solution was injected at each injection point, and the total amount of virus was 1.1×10^9 v.g. Next, the subcutaneous tissue and skin were sutured layer by layer using a 3-0 suture needle. Finally, the endotracheal tube was removed and the mouse was placed on a constant temperature platform and returned to the cage after it was fully awake and resumed normal activities.

[0089] Experimental observation and cardiac function evaluation: On the 1st and 28th day after surgery, 5 mice in each group were randomly selected for cardiac function monitoring. The recovery of cardiac function in mice after myocardial infarction was evaluated by cardiac ultrasound. The results showed that on the 28th day after surgery, the left ventricular ejection fraction (EF value) and left ventricular shortening fraction (FS) of the AAV9:cTNT-HMGB2 group of mice were significantly improved, indicating that the preparation can effectively and continuously promote the recovery of cardiac function in mice after MI (see Figure 3 ).

[0090] All mice were euthanized 28 days after surgery, and fresh heart tissue was obtained for paraffin sectioning and stained using the Masson staining kit. The specific method is as follows:

[0091] Dewaxing and rehydration: After taking out the paraffin sections to be stained, place them in a 37°C oven and sequentially place them in xylene solution ① and xylene solution ②, with each solution treated for 20 minutes. Subsequently, place the sections in room temperature anhydrous ethanol ① and ② for 2 minutes each, and then sequentially treat them in 90%, 80%, and 70% ethanol for 2 minutes each. Finally, soak the sections in distilled water for 15 minutes, and change the water twice during this process to complete the rehydration process.

[0092] Masson staining: First, soak the rehydrated slices in iron hematoxylin staining solution for 10 minutes, and use acidic ethanol differentiation solution for differentiation for 15 seconds. Then, rinse with distilled water for 1 minute, incubate the slices with blueing solution for 3 minutes, and continue to wash with distilled water for 1 minute. After that, immerse the slices in Ponceau fuchsin staining solution for 5 minutes and wash with weak acid working solution (distilled water and weak acid solution are configured in a 2:1 ratio) for 1 minute. Next, differentiate with phosphomolybdic acid solution for 2 minutes, and then wash with weak acid working solution for 1 minute. Finally, place the slices in aniline blue staining solution for 2 minutes and wash with weak acid working solution again for 1 minute. After staining, dehydrate, first use 95% ethanol for rapid dehydration 3 times, then use anhydrous ethanol for rapid dehydration 3 times, and use xylene to make it transparent 2 times, 2 minutes each time, and finally use resin to seal the slices.

[0093] Covering and photographing: First, wipe off the excess water around the slice, then drop a little glycerol on the tissue surface on the slide, and gently cover the cover slip from right to left to avoid bubbles. If bubbles are encountered, they can be squeezed out carefully. Then, place the slide in a 37℃ oven to dry for 1 to 2 hours. After drying, take out the slice, observe the slice with a stereo microscope or an upright fluorescence microscope, and take images.

[0094] The results showed that the degree of myocardial fibrosis in mice in the AAV9:cTNT-HMGB2 group was significantly lower than that in the AAV9:cTNT-CON group (see Figure 5 ), indicating that the HMGB2 preparation has significant effects in reducing myocardial fibrosis, inhibiting cardiac remodeling and improving cardiac function.

[0095] Transfection efficiency detection: On the 14th day after surgery, 3 mice were randomly selected from each group, and the heart tissue was collected in the same way as above. The transfection efficiency was detected by Western Blot experiment. The results showed that the expression of HMGB2 protein in the heart tissue of mice in the in situ injection of AAV9:cTNT-HMGB2 group was significantly increased (see Figure 4 ).

[0096] Cell proliferation detection: Fresh myocardial tissues were obtained from the edge zone of injury, paraffin sections were prepared, and Ki67 staining was performed.

[0097] The specific steps are as follows:

[0098] Slice hydration: The slice hydration process is the same as the previous steps.

[0099] Antigen repair: Prepare the acid repair solution in advance, remove the slices from the distilled water, and place them in a repair box containing the acid repair solution. Heat the repair box in a microwave oven: heat on high for 3 minutes, then turn to low for 7 minutes. After heating, let the repair box cool naturally to room temperature. This process takes about 2 hours. After the repair solution cools to room temperature, pour it out and replace it with an equal volume of PBS solution. Wash the repair box in a 60 rpm shaker for 15 minutes, changing the PBS solution twice in between.

[0100] Blocking: During the PBS washing process, prepare 5% BSA + 1‰ Triton blocking solution and shake well. After washing, take out the slices and put them in a wet box, wipe off the excess water around the tissue, and use a histochemical pen to draw circles around the tissue. The diameter of the circle is 1 to 2 cm, and draw 3 circles. Then, use a pipette to absorb the blocking solution and drop it on the tissue to observe whether the blocking solution leaks out. If leakage occurs, use a histochemical pen to draw additional circles to ensure complete sealing.

[0101] Primary antibody incubation: Prepare the primary antibody diluent (Ki67 concentration = 1:300, cTNT concentration = 1:300) with 5% BSA + 1‰ Triton solution in advance. After blocking for 2 hours, shake off the blocking solution on the tissue, use a pipette to absorb the primary antibody diluent, and drop it on the tissue to ensure even distribution. After that, place the humidified box in a 4°C refrigerator for primary antibody incubation overnight.

[0102] Secondary antibody incubation: On the second day, remove the slides, shake off the primary antibody diluent, and place the slides in a repair box containing PBST solution. Wash on a shaker at 60 rpm for 30 minutes, changing the PBST solution 3 times. When washing, prepare the secondary antibody diluent with 5% BSA + 1‰ Triton solution and ensure that it is protected from light. After the primary antibody is washed, add the secondary antibody using the same method and incubate for 2 hours in the dark.

[0103] Sealing and observation: After the secondary antibody incubation is completed, the secondary antibody diluent is also shaken off and washed with PBST for 30 minutes. After washing, wipe off the excess water around the tissue and drop a small amount of glycerol on the surface of the tissue on the slide. Gently cover the tissue with a cover slip from right to left to avoid bubbles. After sealing, the slices are observed and photographed using a laser confocal microscope.

[0104] The results showed that the cardiomyocytes in the injured marginal zone of mice in the AAV9:cTNT-HMGB2 group had significant cell cycle activity and increased mitosis, indicating that this preparation can effectively promote the proliferation of cardiomyocytes in adult mice after MI injury (see Figure 6 ).

[0105] The above experimental results show that HMGB2 preparations can significantly promote myocardial regeneration and repair, reduce myocardial fibrosis, and improve cardiac function. This study provides a new strategy for regeneration and repair treatment after acute myocardial infarction.

Claims

1. Any of the following uses of the HMGB2 gene: (1) use in the preparation of drugs or reagents for promoting myocardial cell proliferation; (2) use in the preparation of drugs for promoting myocardial regeneration and repair after cardiac injury; (3) use in the preparation of drugs for inhibiting myocardial fibrosis and cardiac remodeling after cardiac injury.

2. The use according to claim 1, characterized in that: The HMGB2 gene is overexpressed in the myocardial tissue of the body.

3. Any of the following uses of a recombinant adenovirus or a recombinant adeno-associated virus expressing HMGB2 protein: (1) use in the preparation of a drug or reagent for promoting myocardial cell proliferation; (2) use in the preparation of a drug for promoting myocardial regeneration and repair after cardiac injury; (3) use in the preparation of a drug for inhibiting myocardial fibrosis and cardiac remodeling after cardiac injury.

4. The use according to claim 1 or 3, characterized in that: The heart injury is acute myocardial infarction or myocardial ischemia-reperfusion injury.

5. The use according to claim 3, characterized in that: The amino acid sequence of the HMGB2 protein is shown in SEQ ID NO: 1 or SEQ ID NO:

3.

6. The use according to claim 3, characterized in that: The recombinant adenovirus or recombinant adeno-associated virus contains the gene encoding the HMGB2 protein, and the nucleotide sequence of the coding chain of the gene encoding the HMGB2 protein is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

7. The use according to claim 3, characterized in that: The adenovirus is Ad5, and the adeno-associated virus is AAV9.

8. The use according to claim 7, characterized in that: The adenovirus Ad5 or adeno-associated virus AAV9 contains a myocardium-specific promoter cTNT.

9. A drug for promoting myocardial regeneration and repair, characterized in that: The active ingredient of the drug comprises a recombinant adenovirus or a recombinant adeno-associated virus expressing HMGB2 protein.

10. The drug for promoting myocardial regeneration and repair according to claim 9, characterized in that: The medicine also includes pharmaceutically acceptable excipients or additives.

Images