Medical application of MFAP2 in myocardial cell proliferation

Abstract

The invention relates to the technical field of medicines, in particular to a medical application of MFAP2 (Microfibril Associated Protein 2). It is found through a large number of experiments that the expression quantity of MFAP2 in heart tissue is reduced along with the increase of time points after newborn rats are born. A myocardial infarction model is established by adopting an anterior descending branch ligation method, an MFAP2 mouse in-vivo overexpression model is established by adopting a caudal vein AAV virus injection method, and it is found that reduction of the cardiac contraction function after myocardial infarction is relieved through mouse in-vivo overexpression of MFAP2. Meanwhile, the fibroblast overexpressing the MFAP2 promotes the proliferation of myocardial cells. The result shows that the MFAP2 protein reduces the cardiac infarction area and improves the cardiac systolic function of patients suffering from myocardial infarction and heart failure by stimulating myocardial cell proliferation and promoting heart regeneration and repair, and can be used as an effective factor for promoting heart repair of patients suffering from cardiovascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the medical use of MFAP2 (Microfibril Associated Protein 2), and further to the medical use of MFAP2 protein for promoting myocardial cell regeneration, and the application of MFAP2 protein in the preparation of a drug for repairing myocardial injury. Background Art

[0002] Among many diseases, cardiovascular disease (CVD) is the main cause of death. Among them, myocardial infarction and heart failure seriously endanger health and life, and are often related to the loss or dysfunction of cardiomyocytes. Drug therapy and medical intervention are recognized treatments for acute myocardial infarction. However, neither of these two methods can fundamentally solve the problem of cardiomyocyte loss. Therefore, exploring the mechanism of cardiomyocyte regeneration and how to control the process of cardiomyocyte proliferation is the key to successfully repairing heart damage.

[0003] Microfibril Associated Protein 2 (MFAP2), also known as Microfibril-Associated Glycoprotein-1 (MAGP1), is a protein component of extracellular matrix microfibrils and is involved in physiological and pathological mechanisms such as elastic fiber formation, vascular and bone integrity, lung disease and metabolic diseases. Its extracellular form binds to fibrin, collagen VI, elastin, core proteoglycan and diglycan, while its intracellular form upregulates the expression of downstream genes related to cell adhesion, motility and matrix remodeling. In adipose tissue, MFAP2 has a high affinity for members of the transforming growth factor (TGF)-β superfamily, and in the absence of MFAP2, the basal activity of TGF-β is increased, which is involved in the occurrence and development of metabolic diseases. It can also activate FAK, paxillin, ERK1 / 2 and other pathways through the integrin α5β1 / FAK / ERK1 / 2 pathway, thereby playing a role in the differentiation, proliferation and EMT of various tumor cells. However, the role and mechanism of MFAP2 in cardiovascular disease are still unclear. Therefore, the present invention aims to provide the use of MFAP2 protein in the preparation of drugs for repairing myocardial injury and in drugs for treating myocardial infarction and heart failure. Summary of the invention

[0004] The purpose of the present invention is to provide the use of MFAP2 protein in the preparation of a drug for repairing myocardial injury, so as to achieve myocardial cell proliferation and promote the regeneration and repair of the heart.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The invention discloses a medicine for treating acute myocardial infarction, characterized in that the medicine comprises an agent for improving the expression of MFAP2 protein.

[0007] Furthermore, the reagent for increasing the expression of MFAP2 protein includes the MFAP2 gene, or a plasmid containing the MFAP2 gene coding region, or a recombinant vector containing the MFAP2 gene coding region, or a host bacteria containing the recombinant vector, or a cell containing the recombinant vector.

[0008] Furthermore, the drug also includes a pharmaceutically acceptable carrier; the dosage form of the drug is a pharmaceutically acceptable dosage form.

[0009] The present invention also discloses a reagent for promoting the proliferation of cardiomyocytes, characterized in that the reagent comprises a reagent for increasing the expression of MFAP2 protein.

[0010] Furthermore, the reagent for increasing the expression of MFAP2 protein includes the MFAP2 gene, or a plasmid containing the MFAP2 gene coding region, or a recombinant vector containing the MFAP2 gene coding region, or a host bacteria containing the recombinant vector, or a cell containing the recombinant vector.

[0011] The present invention also discloses the use of a detection reagent for the expression level of the MFAP2 gene or its encoded protein in the preparation of an auxiliary diagnosis product for acute myocardial infarction.

[0012] The present invention also discloses the use of a detection reagent for the expression level of the MFAP2 gene or its encoded protein in the preparation of a product for diagnosing the proliferation of myocardial cells.

[0013] Furthermore, the product detects the expression level of the MFAP2 gene or its encoded protein in a sample by reverse transcription PCR, real-time quantitative PCR, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

[0014] Furthermore, the product contains specific primers for amplifying the MFAP2 gene, a probe that hybridizes with the MFAP2 gene nucleotide sequence, or an antibody that specifically binds to the MFAP2 protein; the specific primer sequence of the MFAP2 gene is as shown in SEQ ID NO.1-2.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] The present invention has found through a large number of experiments that the expression of MFAP2 in cardiac tissue decreases as the time point increases after the birth of newborn mice. The myocardial infarction model was established by the method of ligating the anterior descending branch, and the MFAP2 overexpression model in mice was constructed by the method of injecting AAV virus through the tail vein. It was found that overexpression of MFAP2 in mice alleviated the decrease in cardiac contractile function after myocardial infarction. At the same time, fibroblasts overexpressing MFAP2 promoted myocardial cell proliferation. The above results show that MFAP2 protein can promote cardiac regeneration and repair by stimulating myocardial cell proliferation, thereby reducing the area of ​​myocardial infarction and improving the cardiac contractile function of patients with myocardial infarction and heart failure, and can be used as an effective factor to promote cardiac repair in patients with cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 After the birth of newborn mice, the expression of MFAP2 was downregulated as the time points increased. Figures A and B are the results of Western blot and their statistical graphs; Figure C is the result of Real-time PCR detection; Figures D and E are immunohistochemical staining and their statistical graphs (n=3, ns: p>0.05, **p<0.01, ***p<0.001 vs. P1 group).

[0018] Figure 2 Overexpression of MFAP2 in mice does not affect body fat rate, fasting blood glucose, cardiac function, and blood pressure under physiological conditions. Figure A is a map of the vector constructed to overexpress MFAP2 in the whole body; Figure B is the result of Real-time PCR detection; Figures C and D are Western blot results and statistical graphs; Figure E is a statistical graph of body fat rate; Figure F is a statistical graph of fasting blood glucose; Figures G and H are cardiac function results and statistical graphs; Figures I and J are statistical graphs of blood pressure (n=5, ns: p>0.05, **p<0.01, ***p<0.001, vs. oe-NC group).

[0019] Figure 3 Overexpression of MFAP2 improves cardiac function damage in mice after long-term myocardial infarction. Figures AC show the cardiac function results and statistical graphs 35 days after myocardial infarction modeling (ns: p>0.05, *p<0.05 vs. oe-NC group).

[0020] Figure 4 Overexpression of MFAP2 in fibroblast culture supernatant leads to Ki67 in cardiomyocytes + Figures A and B are Western blot results and their statistical graphs; Figure C is Real-time PCR test results; Figures D and E are immunofluorescence staining results and their statistical graphs (n=3, *p<0.05, ***p<0.001 vs. ov-NC group). DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0022] Unless otherwise specified, the reagents and materials used in the present invention are commercially available. The experimental data of the present invention are all percentages, and the comparison of the two sample rates is performed using the chi-square test. The statistical processing is performed using the GraphPad Prism 8.0 software package, and P<0.05 is considered statistically significant.

[0023] Example 1: After birth, the expression of MFAP2 was downregulated as time went by in newborn mice.

[0024] 1. Experimental animals and their husbandry.

[0025] Experimental animal species, gender, age and source: SD rats, male, female, 8 weeks old. SD rats were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Mice were raised in a specific pathogen free (SPF) animal room with a room temperature of (22±2)℃, a humidity of 45%~70%, a 12-h light cycle, and free access to food and water.

[0026] 2. The Western blot method was used to detect the expression of MFAP2 in the heart tissue of SD rats.

[0027] In order to clarify the role of MFAP2 in the heart tissue of newborn mice, the heart tissues of suckling mice at 1 day, 4 days and 8 days after birth were collected, and the expression of MFAP2 in the heart was detected by Western blot. The hearts of the above-mentioned days of birth were collected, and an appropriate amount of protein lysis buffer was added. The cells were lysed on ice for 30 minutes and mixed every 5 minutes. The supernatant was collected as the total cell protein at 4°C and 12000g for 20 minutes. The protein concentration in the lysate was determined by the BCA colorimetric kit. The protein lysis buffer was used and diluted to a final concentration of 3 mg / mL. Then 5× loading buffer was added in proportion, mixed thoroughly, boiled for 5 minutes in a 100°C water bath, and centrifuged at 12000g for 30 seconds at room temperature. 20 μg of protein sample was added to each sample well, the power was turned on, and electrophoresis was started. The voltage and time were as follows: 100V voltage, 30 minutes, 120V voltage, 60 minutes, and the power was turned off after bromophenol blue electrophoresis reached the bottom of the glass plate. The samples were transferred to PVDF membrane at 90V for 2h. The PVDF membrane was placed in 5% milk blocking solution for 1h and then incubated overnight at 4℃ with primary antibody. 1:1000 anti-MFAP2 antibody (Thermo, USA) and 1:1000 anti-β-actin antibody (Cellsignalling, USA) were used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody (Cellsignalling, USA) was used as secondary antibody for Western blot detection, and ECL kit (Amersham, USA) was used for luminescence development. Protein expression bands of approximately 25KD and 42KD were detected using MFAP2 antibody and β-actin antibody, respectively. ImageJ 1.51 software was used to measure the gray value of the bands and perform statistical analysis.

[0028] The results showed that after the birth of newborn mice, the protein expression level of MFAP2 was downregulated as time went by ( Figure 1 A, B).

[0029] 3. The fluorescence real-time quantitative PCR (Real-time PCR) method was used to detect the expression of MFAP2 in the heart tissue of SD rats.

[0030] 1) Extraction of RNA from cardiac tissue.

[0031] A. Place the heart tissue in an RNase-free EP tube, add 1 mL of Trizol, put in three RNase-free grinding beads, and grind in a tissue grinder homogenizer for 180 s at 50 Hz.

[0032] B. Place at room temperature for 5 minutes, add 1 / 5 of chloroform, then turn it upside down to mix well and let it stand for 15 minutes.

[0033] C. 12000 rpm, 4°C, centrifuge for 15 min; D. Aspirate the supernatant, add an equal volume of isopropanol, invert and mix, and let stand at room temperature for 10 minutes.

[0034] E. Centrifuge at 12000 rpm, 4°C for 15 min and discard the supernatant.

[0035] F. Add 1 mL of 75% ethanol and gently shake the centrifuge tube to resuspend the precipitate.

[0036] G. Centrifuge at 12000 rpm, 4°C for 15 min and discard the supernatant.

[0037] H. Allow to dry at room temperature until transparent.

[0038] I. Add 25 μL of enzyme-free water to dissolve RNA.

[0039] 2) Reverse transcription of RNA into cDNA.

[0040] The Takara reverse transcription kit was used.

[0041] A. Removal of genomic DNA .

[0042] B. Reverse transcription reaction .

[0043] Reaction conditions: 37℃ 15min 85℃ 5s 4℃.

[0044] 3) Primer sequence .

[0045] 4) Fluorescence quantitative PCR .

[0046] According to the volume in the table above, prepare 20 μL qPCR reaction system, and set up 3 replicate wells for each group of samples. Reaction conditions: 95℃5min, (95℃5s, 60℃30s, 72℃30s)×40 cycles, 72℃5s, 95℃15s. －△△Ct The expression of each sample was analyzed.

[0047] The results showed that after the birth of newborn mice, the RNA expression level of MFAP2 was downregulated as the time point increased ( Figure 1 C).

[0048] 4. Immunohistochemical staining.

[0049] Heart tissues of suckling mice at 1, 4, and 8 days after birth were collected for MFAP2 immunohistochemical staining. The specific steps are as follows.

[0050] 1) Preparation of paraffin sections.

[0051] A. Sampling: Place mouse aorta in 4% paraformaldehyde solution overnight.

[0052] B. Dehydration: Dehydration was performed according to different alcohol concentrations: 70% alcohol for 2 hours, 80% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol I for 4 hours, 95% alcohol II overnight, 100% alcohol I for 1.5 hours, and 100% alcohol II for 1.5 hours.

[0053] C. Transparency: Soak the tissue block in xylene I solution for 1 hour, then take it out and soak it in xylene II solution for 1 hour.

[0054] D. Wax immersion: overnight in paraffin I, 1 hour in paraffin II, and 1 hour in paraffin III.

[0055] E. Embedding: Embed the tissue blocks in paraffin and keep at room temperature.

[0056] F. Sectioning: Slice the tissue block with a paraffin slicer to a thickness of 3 μm and mount the sections on glass slides.

[0057] G. Drying and baking the slides: Place the slides in a 60℃ slide dryer for 1 hour, then place the slides in a 65℃ oven for 48 hours.

[0058] 2) Dewaxing of sections: Place the sections in the following reagents according to the steps: xylene I for 20 minutes, xylene II for 20 minutes, 95% alcohol I for 15 minutes, 95% alcohol II for 15 minutes, 90% alcohol for 10 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally place in distilled water for 30 minutes.

[0059] 3) Immunohistochemical staining.

[0060] A. Antigen repair: Soak the slices in antigen repair solution and boil at 100℃ for 40 minutes, then cool to room temperature.

[0061] B. Place the slices in a humidified chamber with the tissue side facing up. Add one drop or 50 μL of peroxidase blocker to each slice. Incubate at room temperature for 10 minutes, and then wash three times with PBS, each time for 10 minutes.

[0062] C. Remove PBS, add one drop or 50 μL of goat serum to each section, and incubate at room temperature for half an hour.

[0063] D. Remove serum and add 50 μL MFAP2 primary antibody (1:100 diluted in PBS) to each section at 4°C overnight.

[0064] E. Rewarm at room temperature for 30 minutes and wash with PBS three times, 10 minutes each time.

[0065] F. After incubation for 10 min, wash three times with PBS, 10 min each time.

[0066] G. Remove PBS and add 50 μL DAB colorimetric solution to each slice. Dilute DAB colorimetric solution 20 times.

[0067] H. Counterstain the cell nuclei with hematoxylin solution.

[0068] I. Place the paraffin sections in 1% hydrochloric acid for 30 seconds for differentiation and rinse with running water.

[0069] J. Place the paraffin sections in ammonia water for 30 seconds to turn the cell nuclei blue, and then rinse with running water.

[0070] K. Dehydrate, transparentize with xylene, and seal with neutral gum.

[0071] L. Observe the staining results under an upright microscope and take photos to save the images.

[0072] The results showed that after the birth of newborn mice, the expression level of MFAP2 decreased with the extension of time. Figure 1 D, E). The above results all show that after the birth of newborn mice, the protein expression level of MFAP2 is downregulated with the extension of time points, suggesting that MFAP2 may be involved in the occurrence and development of cardiomyocyte proliferation.

[0073] Example 2: Systemic overexpression of MFAP2 in mice does not affect body fat percentage, fasting blood glucose, cardiac function, and blood pressure under physiological conditions.

[0074] 1. ApoE - / - Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The feeding conditions were the same as those in Example 1.

[0075] 2. Injection of adeno-associated virus into the tail vein of mice. In order to verify the effect of MFAP2 on cardiac contractile function in vivo, pcAAV-CMV-Mfap2-Linker-ZsGreen1-3xFLAG-WPRE (oe-MFAP2) and its negative control pcAAV-CMV-ZsGreen1-WPRE (oe-NC) were constructed by Shanghai Heyuan Biotechnology Co., Ltd. MFAP2 was overexpressed in vivo by injection into the tail vein of mice for 4 weeks. Figure 2 A).

[0076] 3. Real-time PCR was used to detect the RNA expression of MFAP2 in various tissues of the oe-NC and oe-MFAP2 groups of mice. The results showed that compared with the control group, the expression of MFAP2 RNA in the heart of the overexpression group mice was significantly increased ( Figure 2 B).

[0077] 4. Western blot was used to detect the protein expression of MFAP2 in various tissues of the oe-NC and oe-MFAP2 groups of mice. The results showed that compared with the control group, the expression of MFAP2 in the heart of the overexpression group mice was significantly increased ( Figure 2 C, D).

[0078] 5. The body fat meter was used to detect the body fat percentage of oe-NC and oe-MFAP2 groups of mice.

[0079] First, the body weight of the two groups of mice was measured, and then the fat content was measured through the body composition of the living mice, and the body fat percentage was calculated by the ratio of fat content to body weight.

[0080] The results showed that there was no significant difference in body fat percentage between the oe-NC and oe-MFAP2 groups of mice ( Figure 2 E).

[0081] 6. Use blood glucose meter to detect fasting blood glucose of mice.

[0082] 1) Mouse preparation: The oe-NC and oe-MFAP2 mice were placed in clean cages at 5 pm the day before the experiment and fasted for 16 hours until 9 am the next day. During the fasting period, the mice were given normal drinking water; 2) Determination of fasting basal blood sugar: Take the mouse out of the cage and gently place it on the iron grid. Use scissors to cut off about 1-2 mm of the end of the mouse's tail. Gently squeeze the mouse's tail to allow the blood to accumulate into a drop. Use a blood glucose meter to measure fasting blood sugar. The measured value is considered as the blood sugar value at 0 min. The operation should be as gentle as possible so that the mouse will not be overly frightened.

[0083] The results showed that there was no significant difference in fasting blood glucose between the oe-NC and oe-MFAP2 groups of mice ( Figure 2 F).

[0084] 7. Ultrasound imaging measures changes in cardiac function.

[0085] The changes in cardiac function were detected using the small animal ultrasound system Vevo 2100 apparatus (Visual Sonics, Toronto, Canada). The results showed that there was no significant difference in cardiac function between the oe-NC and oe-MFAP2 groups of mice ( Figure 2 G, H).

[0086] 8. The tail artery pressure measurement system measures the blood pressure of mice.

[0087] 1) Mouse fixation: generally a fixator made of plexiglass is used.

[0088] 2) Determine the initial pulse level: Place the pressurized tail cuff and pulse transducer on the appropriate position of the mouse's tail in sequence.

[0089] 3) Blood pressure measurement: Inflate and pressurize the rubber ball to increase the pressure in the pressurized tail cuff until the pulse disappears completely, then continue to pressurize by about 20 mmHg, and then slowly deflate and decompress until the pulse signal returns to the initial level. At this time, the systolic pressure, diastolic pressure, mean arterial pressure and heart rate can be read from the pressure gauge or recording system. Generally, three consecutive measurements are taken, and the average value is taken as a measurement value.

[0090] The results showed that there was no significant difference in systolic and diastolic blood pressure between the oe-NC and oe-MFAP2 groups of mice ( Figure 2 I, J).

[0091] Example 3: MFAP2 overexpression improves cardiac function damage in mice after long-term MI.

[0092] 1. The myocardial infarction model was established in mice by ligating the left anterior descending coronary artery.

[0093] 1) C57 mice were weighed and anesthetized with 2% chloral hydrate at a dose of 0.2 mL / 10 g.

[0094] 2) After the mouse is fully anesthetized, fix it on the operating table and connect it to an electrocardiogram to monitor its heartbeat.

[0095] 3) Shave the hair on the mouse's chest and neck and apply iodine to disinfect.

[0096] 4) For endotracheal intubation, cut the skin in the middle of the mouse's neck, then use forceps to separate the fat layer and muscle, expose the trachea and thread under the trachea; use surgical scissors to cut a small incision above the trachea, insert the ventilator tube into the trachea, tie it with surgical thread, and turn on the ventilator. The parameters are a respiratory rate of 100 times / min, a tidal volume of 6-10mL, and a respiratory ratio of 1:2.

[0097] 5) Open the chest. Pick up the muscle between the second and third ribs on the left chest of the mouse along the ribs. Then use a skin spreader to spread the ribs to expose the heart. Use forceps to tear open the pericardium.

[0098] 6) Ligation: ligate the left anterior descending coronary artery; the approximate position is as follows: find the left atrial appendage, insert the needle from left to right 0.5-1mm below the lower left corner of the left atrial appendage, and the depth is about 0.5-1mm. Then put a small plastic tube at the ligature to prevent the myocardium from being strangled, and tie a loose knot. At this time, the apex of the heart turns white, and the ST segment in the electrocardiogram is elevated.

[0099] 7) Suture and close the chest; remove the ventilator.

[0100] 8) Sham group experiment, that is, the left anterior descending branch of the coronary artery was not ligated, and the rest of the steps were the same as those of the experimental group.

[0101] 2. Ultrasonic imaging is used to measure changes in cardiac function, as in Example 2.

[0102] The results showed that compared with the control group, the cardiac contractile function of mice in the oe-MFAP2 group was relieved 35 days after myocardial infarction, and MFAP2 overexpression improved the cardiac function damage of mice after long-term MI ( Figure 3 AC).

[0103] Example 4: MFAP2 overexpression promotes cardiomyocyte proliferation.

[0104] 1. Establishment of MFAP2-overexpressing primary fibroblasts.

[0105] 1) Hold a rat suckling baby (within 24 hours of birth), disinfect the skin with 75% ethanol, cut the chest skin, disinfect it again, replace the surgical instruments, extract the heart with curved forceps, and place it in a large dish filled with PBS (1:50 double antibody).

[0106] 2) Cut off the large blood vessels attached to the surface of the heart, cut off the atria, put them into a 5mL sterile centrifuge tube and chop them into a meat paste.

[0107] 3) Add about 3 mL of collagenase and 1.5 mL of 0.05% trypsin, blow well to mix well, digest at 37°C for 8 min, allow to precipitate naturally, discard the supernatant, add about 3 mL of collagenase and 1.5 mL of 0.05% trypsin, blow well to mix well, and digest at 37°C for 10 min.

[0108] 4) Take the supernatant, incubate at 3000 rpm for 5 min, add DMEM culture medium containing 10% fetal bovine serum to the medium dish, add about 3 mL of collagenase and 1.5 mL of 0.5% trypsin to the remaining precipitate, blow thoroughly to mix well, and digest at 37°C for 10 min.

[0109] 5) Repeat step 4 4-5 times until the tissue block is digested.

[0110] 6) Place the cells in the incubator for 2-3 hours and gently blow the culture medium after the fibroblasts attach to the wall. When the cells grow to 70%-80%, add the plasmid containing the MFAP2 overexpression vector (pcDNA3.1-MFAP2-6×his) to the rat fibroblasts for infection. The plasmid with the his tag (pcDNA3.1-6×his) is used as a control. After infection with Lipofectamine 2000 (Invitrogen, USA) for 24 hours, it can be used for experiments. The plasmid was constructed by Genewise Biotechnology Co., Ltd.

[0111] 2. Western blot detection of MFAP2 overexpressing fibroblasts. The specific method is the same as that in Example 1.

[0112] The results showed that compared with pcDNA3.1-6×his, the expression level of MFAP2 protein in the cDNA3.1-MFAP2-6×his group was significantly increased ( Figure 4 A, B).

[0113] 3. Real-time PCR detection of MFAP2 overexpressing fibroblasts, the specific method is the same as Example 1.

[0114] The results showed that compared with pcDNA3.1-6×his, the expression level of MFAP2 RNA in the cDNA3.1-MFAP2-6×his group was significantly increased ( Figure 4 C).

[0115] 4. Primary SD day 1 newborn mouse cardiomyocytes were isolated and cultured in vitro. 24 hours later, the proliferation marker Ki67 and the cardiomyocyte marker α-actinin were used for immunostaining to evaluate the effect of MFAP2 on cardiomyocyte proliferation.

[0116] 1) Hold a rat suckling baby (within 24 hours of birth), disinfect the skin with 75% ethanol, cut the chest skin, disinfect it again, replace the surgical instruments, extract the heart with curved forceps, and place it in a large dish filled with PBS (1:50 double antibody).

[0117] 2) Cut off the large blood vessels attached to the surface of the heart, cut off the atria, put them into a 5mL sterile centrifuge tube and chop them into a meat paste.

[0118] 3) Add about 3 mL of collagenase and 1.5 mL of 0.05% trypsin, blow well to mix well, digest at 37°C for 8 min, allow to precipitate naturally, discard the supernatant, add about 3 mL of collagenase and 1.5 mL of 0.05% trypsin, blow well to mix well, and digest at 37°C for 10 min.

[0119] 4) Take the supernatant, incubate at 3000 rpm for 5 min, add DMEM culture medium containing 10% fetal bovine serum to the medium dish, add about 3 mL of collagenase and 1.5 mL of 0.5% trypsin to the remaining precipitate, blow thoroughly to mix well, and digest at 37°C for 10 min.

[0120] 5) Repeat step 4 4-5 times until the tissue block is digested.

[0121] 6) Place in the incubator for 2-3 hours. After the fibroblasts attach to the wall, gently blow the culture medium. Transfer all the supernatant of the medium dish into a centrifuge tube and centrifuge at 3000 rpm for 5 minutes. Discard the supernatant, add DMEM culture medium containing 10% fetal bovine serum, and spread the medium dish for culture.

[0122] 7) After 48 hours, the medium can be replaced to obtain cardiomyocytes. Observe the morphology and beating status of cardiomyocytes.

[0123] 8) The cells in the culture plate were washed twice with PBS, and the supernatant of the culture medium after transfection of MFAP2-overexpressing fibroblasts was added for treatment. The control group was treated with an equal amount of control culture medium.

[0124] 9) After 24 hours, discard the culture medium, wash the cells twice with PBS, and fix them with 4% paraformaldehyde for 15 minutes.

[0125] 10) Wash twice with PBS, add blocking solution (1 mL / well) containing 0.3% TritonX-100 prepared by adding goat serum to TritonX-100, and block for 1 hour at room temperature.

[0126] 11) Discard the blocking solution, dilute the primary antibody Ki67 (dilution ratio 1:100) and α-actinin (dilution ratio 1:400) with PBS for immunostaining, and add them to the culture plate. Incubate at 4°C overnight.

[0127] 12) Place the cell culture plate on a shaker and wash with PBS three times, 5 min each time.

[0128] 13) Dilute AlexaFluor488 donkey anti-rabbit immunofluorescent antibody (1:400) and AlexaFluor594 donkey anti-mouse immunofluorescent antibody (1:400) in PBS and add to the culture plate. Incubate at room temperature in the dark for 1 hour.

[0129] 14) Protect the cell culture plate from light and place it on a shaker. Wash with PBS three times, 5 min each time.

[0130] 15) Dilute DAPI with PBS (dilution ratio 1:100) and react for 5 min.

[0131] 16) The slides were placed on a Zeiss laser confocal microscope (Zeiss LSM800, Germany) for imaging. The co-localization analysis of the proliferation marker Ki67 was performed using the confocal microscope Z-axis scanning.

[0132] The results showed that compared with pcDNA3.1-6×his, cDNA3.1-MFAP2-6×his group had a higher expression of Ki67 in cardiomyocytes + Increased cell number ( Figure 4 D, E).

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug for treating acute myocardial infarction, characterized in that: The medicament includes an agent that increases the expression of MFAP2 protein.

2. The drug according to claim 1, characterized in that The reagent for increasing the expression of MFAP2 protein includes MFAP2 gene, or plasmid containing MFAP2 gene coding region, or recombinant vector containing MFAP2 gene coding region, or host bacteria containing the recombinant vector, or cells containing the recombinant vector.

3. The drug according to claim 1, characterized in that The drug also includes a pharmaceutically acceptable carrier; the dosage form of the drug is a pharmaceutically acceptable dosage form.

4. A reagent for promoting cardiomyocyte proliferation, characterized in that: The agents include agents that increase the expression of MFAP2 protein.

5. The drug according to claim 4, characterized in that The reagent for increasing the expression of MFAP2 protein includes MFAP2 gene, or plasmid containing MFAP2 gene coding region, or recombinant vector containing MFAP2 gene coding region, or host bacteria containing the recombinant vector, or cells containing the recombinant vector.

6. Use of a detection reagent for the expression level of the MFAP2 gene or its encoded protein in the preparation of an auxiliary diagnosis product for acute myocardial infarction.

7. Use of a detection reagent for the expression level of the MFAP2 gene or its encoded protein in the preparation of a product for diagnosing cardiomyocyte proliferation.

8. The use according to claim 6 or claim 7, characterized in that: The product detects the expression level of the MFAP2 gene or its encoded protein in a sample by reverse transcription PCR, real-time quantitative PCR, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

9. The use according to claim 8, characterized in that: The product contains specific primers for amplifying the MFAP2 gene, a probe hybridizing with the MFAP2 gene nucleotide sequence, or an antibody specifically binding to the MFAP2 protein; the specific primer sequence of the MFAP2 gene is as shown in SEQ ID NO.1-2.

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