Application of Slc25a51 gene in preparation of medicine for preventing myocardial infarction

Abstract

The invention discloses application of an Slc25a51 gene in preparation of a medicine for preventing myocardial infarction, and belongs to the technical field of biological medicines. According to the application of the Slc25a51 gene in preparation of the medicine for preventing myocardial infarction, a reagent for improving the expression quantity of the Slc25a51 gene is used as an active ingredient, especially a carrier for overexpressing the Slc25a51 gene, and the carrier is an adenovirus carrier or an adeno-associated virus carrier. The AAV9 virus carrying the Slc25a51 gene is injected into the caudal vein of a mouse, myocardial infarction treatment is carried out after 14 days, and the result shows that the myocardial infarction prevention medicine prepared from the Slc25a51 can remarkably improve the prognosis of the myocardial infarction model mouse, the prognosis shows that the cardiac function is improved, the inflammation infiltration is relieved, the fibrosis is relieved and the like, and the myocardial infarction prevention medicine can be used for preventing myocardial infarction. Therefore, the heart failure prognosis can be improved by directly increasing the content of NAD + in mitochondria, so that a good treatment effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of Slc25a51 gene in preparing a drug for preventing myocardial infarction. Background Art

[0002] AMI (Acute Myocardial Infarction) is a common and life-threatening serious heart disease. Although the current treatment methods for myocardial infarction (MI) are relatively mature, it is still an important public health problem worldwide, and myocardial infarction is still the leading cause of death worldwide. Although the application of effective vascular reconstruction methods such as percutaneous transluminal coronary angioplasty, coronary artery bypass grafting, and laser myocardial revascularization has improved the survival rate and quality of life of patients, the postoperative restenosis rate is still as high as 20%-30%, and the effect is not good for patients with diffuse coronary artery lesions and poor heart function due to old myocardial infarction. There is currently no solution. This forces people to explore a better treatment method. In recent years, cell transplantation and gene therapy for coronary heart disease have become research hotspots, aiming to improve myocardial function by changing the gene expression of cardiomyocytes. Among them, improving the energy metabolism and antioxidant capacity of cardiomyocytes by modifying or regulating the expression of genes related to mitochondria and oxidative stress has become an important treatment method. AAV is often used for in vivo or systemic treatment, that is, direct administration by intravenous injection or injection into the patient's target tissue. Since in vivo treatment is simpler than in vitro cell manipulation, it is theoretically safer than autologous stem cell transplantation. In recent years, studies have continuously found that NAD+ has the effects of enhancing mitochondrial energy supply, anti-aging, promoting DNA repair, increasing chromosome stability, and anti-inflammatory. It has become a star molecule that is expected to improve human health. NAD+ in the cytoplasm is transported to the mitochondria through its transporter, thereby playing a corresponding biological role in mitochondrial metabolism. Previous studies on NAD+ transporters in yeast and plants have been relatively in-depth, and their NAD+ transporters have been identified. However, research on mammalian mitochondrial transporters has been controversial. The Slc25 family genes function as mitochondrial transporters. It can transport inorganic ions, co-transcription factors, nucleotides, amino acids, etc. to the inner membrane of mitochondria and play a variety of biological functions. In December 2020, a study confirmed for the first time in a variety of human cell models that Slc25a51 (formerly known as Mcart1) plays the role of an NAD+ transporter in the mitochondria of human cells. Summary of the invention

[0003] In view of the above problems existing in the prior art, the present invention provides the use of Slc25a51 gene in the preparation of drugs for preventing myocardial infarction, that is, a gene therapy program for delivering Slc25a51 using AAV9 for the treatment after myocardial infarction.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0005] The invention relates to the use of the Slc25a51 gene in the preparation of a drug for preventing myocardial infarction, wherein the drug for preventing myocardial infarction uses a reagent for increasing the expression amount of the Slc25a51 gene as an active ingredient.

[0006] In some embodiments of the present invention, the agent for increasing the expression level of Slc25a51 is a vector for overexpressing the Slc25a51 gene.

[0007] In some embodiments of the present invention, the vector is an adenovirus vector or an adeno-associated virus vector.

[0008] In some embodiments of the present invention, the Slc25a51 gene sequence is shown as SEQ ID NO.1.

[0009] In some embodiments of the present invention, the drug for preventing myocardial infarction is administered by intravenous injection.

[0010] A drug for preventing myocardial infarction, comprising a vector for overexpressing the Slc25a51 gene.

[0011] In some embodiments of the present invention, the vector is an adenovirus vector or an adeno-associated virus vector.

[0012] In some embodiments of the present invention, the vector is specifically AAV9 virus.

[0013] In some embodiments of the present invention, the drug is in the form of an injection.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The application of Slc25a51 provided by the present invention in the preparation of a drug for preventing myocardial infarction, i.e., a gene therapy scheme for delivering Slc25a51 with AAV9, is used for the treatment of myocardial infarction. In the embodiment of the present invention, the mouse was given a tail vein injection of Slc25a51-AAV9 virus 14 days before the establishment of the mouse myocardial infarction model. 28 days after myocardial infarction surgery, the results of cardiac color ultrasound and tissue sections showed that Slc25a51 overexpression can significantly improve the prognosis of myocardial infarction model mice, including improved cardiac function, reduced inflammatory infiltration and reduced fibrosis, etc. The experimental results suggest that Slc25a51 is expected to become a new therapeutic target for heart failure. The present invention has experimentally confirmed that Slc25a51-AAV9 overexpression can improve the prognosis of heart failure by directly increasing the NAD+ content in myocardial mitochondria, thereby achieving better therapeutic effects, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the GV571 plasmid spectrum;

[0017] Figure 2 This is the sequencing result of the positive clone;

[0018] Figure 3 Experimental flow chart of the present invention;

[0019] Figure 4A This is the ultrasound test result showing that cardiac function is significantly improved after overexpression of Slc25a51;

[0020] Figure 4B This is a statistical graph of ultrasound detection results showing that cardiac function was significantly improved after overexpression of Slc25a51 (3 days and 1 week after surgery);

[0021] Figure 4C This is a statistical graph of ultrasound detection results showing that cardiac function was significantly improved after overexpression of Slc25a51 (2 weeks and 4 weeks after surgery);

[0022] Figure 5 This is the fluorescence detection diagram of successful overexpression of Slc25a51;

[0023] Figure 6 This is the FLAG electrophoresis detection diagram;

[0024] Figure 7 This is a diagram for detecting the successful overexpression of Slc25a51;

[0025] Fig. 8A HE staining of tissue sections taken 28 days after surgery;

[0026] Figure 8B for Fig. 8A Masson statistical diagram;

[0027] Figure 8C This is the mRNA statistical chart of the fibrosis-related gene Col1a1. DETAILED DESCRIPTION

[0028] In order to make the technical problems solved by the present invention, the technical solutions adopted and the technical effects achieved clearer, the technical solutions of the present invention are further described below in conjunction with the accompanying drawings and through specific implementation methods. The reagents and instruments used in the examples can be purchased from the market unless otherwise specified.

[0029] Example 1: Construction of an AAV vector for cardiomyocyte-specific expression of the mouse Slc25a51 gene

[0030] The AAV packaging process is as follows: First, clone the exogenous gene (Slc25a51) into the adeno-associated virus vector, and then co-transfect the recombinant plasmid carrying the exogenous gene with the auxiliary plasmid Ad Helper Vector and pAAV-rep / capVector into the packaging cell HEK293T. A large amount of recombinant virus will be produced in the cells 72 hours after transfection. Harvest the culture supernatant and cell precipitate, precipitate the virus in the culture supernatant with PEG8000, lyse the cell precipitate to collect the virus, and combine the virus obtained from the cell precipitate and supernatant. Finally, purify and concentrate the collected virus, and test the titer and purity for later use.

[0031] 1. Preparation of Overexpression Adeno-associated Virus Clone

[0032] 1. Target gene and tool vector information

[0033] Target gene name: Slc25a51 (NM_001009949); vector name: GV571; cloning site: NheI / NheI; GV571 vector map can be found at Figure 1 (Purchased from Shanghai Jikai Gene Chemistry Technology Co., Ltd.).

[0034] 2. Vector digestion

[0035] Prepare the enzyme digestion system: ddH2O 42μl, 10x CutSmart Buffer 5μl, purified plasmid DNA (1μg / μl) 2μl, NheI restriction endonuclease (10U / μl) 1μl. Use a pipette to gently blow and mix the enzyme digestion system, centrifuge and place at 37℃ for 3h or overnight. Perform agarose gel electrophoresis on the vector digestion product and recover the target band.

[0036] 3. Obtaining target gene fragments

[0037] (1) Design and synthesize Slc25a51 gene primers as shown in Table 1 below:

[0038] Table 1 Primers

[0039]

[0040] Primer description: Contains exchange pairing bases, restriction sites, and partial sequence at the 5' end of the target gene for PCR fishing of the target gene.

[0041] (2) PCR amplification of target gene fragments: According to the instruction manual of PrimeSTAR HSDNA polymerase, the reaction system was prepared and placed in a PCR instrument for reaction. After the reaction was completed, the PCR product was recovered by electrophoresis.

[0042] 4. Exchange PCR products with vectors

[0043] Use the homologous recombination kit to prepare the following reaction system in an ice water bath: ddH2O 3.5μl, 5×CE II Buffer 2μl, vector DNA 2.5 2μl after enzyme digestion, 1μl of purified PCR product fragment, Exnase TM II 1μl. After preparation, gently pipette to mix, centrifuge briefly to avoid bubbles. React at 37℃ for 30min, then cool in an ice water bath for 5min and transform immediately.

[0044] 5. Conversion

[0045] Add 10 μl of the exchange reaction product to 100 μl of competent cells, flick the tube wall several times to mix, and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds, and incubate in an ice water bath for 2 minutes. Add 500 μL of antibiotic-free LB medium and place on a shaker at 37°C for 1 hour. Take an appropriate amount of bacterial solution and evenly spread it on a plate containing the corresponding antibiotics, and invert and culture it in a constant temperature incubator for 12-16 hours.

[0046] 6. Colony PCR identification

[0047] Prepare the identification reaction system: ddH2O 9μl, 2×Taq Plus Master Mix 10μl, upstream and downstream primers 0.5μl (10μM) each. Vortex to mix, centrifuge briefly. Pick a single colony into 20μl identification system, pipette to mix, and place in PCR instrument for reaction.

[0048] Table 2 PCR identification primers

[0049]

[0050] The identified positive clone transformants were inoculated into an appropriate amount of LB liquid medium containing the corresponding antibiotics, cultured at 37°C for 12-16 hours, and an appropriate amount of bacterial liquid was taken for sequencing. The sequencing results were compared with the target gene sequence. The sequencing splicing results are shown in Figure 2 As shown, the sequencing and splicing results show that the sequencing results are completely consistent with the target sequence.

[0051] 2. Plasmid transfection and AAV virus harvest

[0052] 1. 24 h before transfection, digest the AAV-293 cells in the logarithmic growth phase with trypsin and adjust the cell density to about 5×10 in a medium containing 10% serum. 6 cells / 15mL, re-inoculate into 10cm cell culture dish, and culture in 37℃, 5% CO2 incubator. When the cell density reaches 70%-80% in 24h, it can be used for transfection;

[0053] 2. 2 h before transfection, replace the medium with 2% serum;

[0054] 3. Add the prepared DNA solutions (5 μg of GV vector plasmid, 5 μg of pHelper vector plasmid, 5 μg of AAV-RC plasmid) to a sterilized centrifuge tube, mix evenly with the corresponding volume of GeneCare transfection reagent, adjust the total volume to 1 mL, and incubate at room temperature for 15 min.

[0055] 4. Slowly add the mixed solution to the culture medium of AAV-293 cells, mix well, and culture in a cell culture incubator at 37°C and 5% CO2;

[0056] 5. After 6 hours of culture, discard the culture medium containing the transfection mixture, add 10 mL of PBS solution to wash once, gently shake the culture dish to wash the remaining transfection mixture and then discard;

[0057] 6. Slowly add 10 mL of cell culture medium containing 5% serum and continue culturing in an incubator at 37°C and 5% CO2 for 48-72 hours.

[0058] 3. AAV Virus Concentration and Purification

[0059] 1. According to the cell status, collect AAV-293 cell supernatant and cells 72h after transfection (transfection can be counted from 0h);

[0060] 2. Centrifuge at 3000 g for 5 min at 4°C to separate cells and supernatant;

[0061] 3. Add GeneCare AAV virus concentration kit to the supernatant to obtain the virus in the supernatant, add resuspension solution to the cell pellet and resuspend it, freeze and thaw it repeatedly in liquid nitrogen / 37°C for 4 times, and centrifuge it. Then combine the two parts of the virus;

[0062] 4. Balance the samples separately, and place the ultracentrifuge tubes with virus supernatant into the Beckman ultracentrifuge one by one. Set the centrifugation parameters to 63000rpm, the centrifugation time to 2h, and the centrifugation temperature to 18℃;

[0063] 5. Absorb the separation layer where the virus is located. Place it into an ultrafiltration column for ultrafiltration until a colorless or light pink clear liquid is obtained;

[0064] 6. Sterilize the obtained virus solution through a 0.22 μm filter membrane and package as required;

[0065] 7. Prepare samples for testing.

[0066] AAV virus titer detection method: The AAV virus titer is detected by QPCR, and the result is the number of packaged virus particles. The principle is to use adeno-associated virus genome-specific primers for real-time quantitative PCR. Within the linear range of the quantitative curve, the ratio of the Ct value to the Ct value of the plasmid with a known copy number is the initial copy number of the viral genome.

[0067] AAV virus purity detection method: It can be detected by sensitive silver staining method. AAV capsid protein contains three subunits VP1 (82kDa), VP2 (72kDa), VP3 (62kDa), and the proportion is VP1: VP2: VP3 = 1:1:10. Therefore, a high-purity AAV only shows three bands in SDS-PAGE analysis.

[0068] Example 2: Application of recombinant adeno-associated virus (AAV9:cTNT-Slc25a51) in improving myocardial infarction in mice

[0069] Mouse experimental procedures Figure 3 As shown, on day 0, the AAV9 virus carrying the Slc25a51 gene (OE-Slc25a51) was injected into the tail vein of the experimental group mice, and the AAV9 virus without any gene (Control AAV9) was injected into the tail vein of the control group mice. The number of injected viruses was 1*10 11 PFU.

[0070] On the 14th day of the virus, according to the literature method (Kolk, MV, Meyberg, D., Deuse, T., Tang-Quan, KR, Robbins, RC, Reichenspurner, H., & Schrepfer, S. (2009). LAD-ligation: A murine model of myocardial infarction. Journal of Visualized Experiments, 32, 1438.), a part of the experimental group mice and the control group mice were ligated by the left anterior descending branch of the mouse coronary artery to construct the mouse myocardial infarction model group (MI group), and another part of the experimental group mice and the control group mice were opened by abdominal cavity without ligating the mouse coronary artery to construct the mouse sham operation group (Sham group). On the 3rd day, 1, 2, and 4 weeks after surgery, the four types of mice were tested by echocardiography. The results are as follows Figure 4A As shown: The systolic function of the MI group decreased. Figure 4B , Figure 4C yes Figure 4AStatistical charts showed that the cardiac function EF (left ventricular ejection fraction) and FS (left ventricular shortening fraction) after slc25a51 overexpression were significantly improved compared with the MI group.

[0071] On the 28th day after injection, the hearts of mice in the control group and the experimental group were taken and the expression of Slc25a51 in cardiomyocytes was detected by immunofluorescence staining. Figure 5 As shown, the control group only showed the fluorescent staining DAPI of the cell nucleus, while the entire cardiomyocytes in the experimental group showed the green FLAG fluorescent labeling, indicating that exogenous Slc25a51 was successfully expressed in cardiomyocytes in myocardial tissue. Figure 6 , Figure 7 The Western Blot method was used to detect the expression of Slc25a51 in the cardiomyocytes of the control group and the experimental group mice. Figure 6 This indicates that the virus carrying the Slc25a51 gene successfully transfected the experimental group mice. Figure 7 This showed that the expression of Slc25a51 in the cardiomyocytes of the experimental group of mice was significantly higher than that in the control group.

[0072] On the 28th day after surgery, the mouse hearts were dissected and myocardial tissue sections were prepared. Fig. 8A As shown, HE staining of tissue sections showed that the myocardial fibers in the MI group and the control virus group were disordered, the myometrium was thinned, and the myofibers were dissolved or even disappeared. Inflammatory cell infiltration was visible. The sham operation group and the Slc25a51 overexpression group were neatly arranged, without breakage, degeneration and necrosis. Masson staining showed obvious fibrosis in the MI group and the control virus group. Figure 8B The statistics of the results of the 8A experiment showed that the fibrosis level in the Slc25a51 overexpression group was significantly lower than that in the MI group. Figure 8C The mRNA expression of fibrosis-related gene Col1a1 in Slc25a51 group was significantly decreased compared with that in MI group.

[0073] The above results indicate that Slc25a51-AAV9 can improve the prognosis of heart failure by directly increasing the NAD+ content in mouse myocardial mitochondria, thereby achieving better therapeutic effects. The present invention has good application prospects.

Claims

1. Application of Slc25a51 gene in the preparation of drugs for preventing myocardial infarction, It is characterized in that The drug for preventing myocardial infarction uses an agent for increasing the expression level of the Slc25a51 gene as an active ingredient.

2. The use according to claim 1, It is characterized in that The reagent for increasing the expression level of Slc25a51 is a vector for overexpressing the Slc25a51 gene.

3. The use according to claim 2, It is characterized in that The vector is an adenovirus vector or an adeno-associated virus vector.

4. The use according to claim 2, It is characterized in that The Slc25a51 gene sequence is shown in SEQ ID NO.

1.

5. The use according to claim 2, It is characterized in that The administration method of the drug for preventing myocardial infarction is specifically through intravenous injection.

6. A drug for preventing myocardial infarction, It is characterized in that The drug contains a vector for overexpressing the Slc25a51 gene.

7. The drug according to claim 6, It is characterized in that The vector is an adenovirus vector or an adeno-associated virus vector.

8. The drug according to claim 7, It is characterized in that The vector is specifically AAV9 virus.

9. The drug according to claim 8, It is characterized in that The dosage form of the drug is injection.

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