Viral vector expressing cyclin D1 gene and its application

Abstract

The present invention discloses a viral vector expressing the cyclin D1 gene and its application. The present invention constructs a vector encoding the cyclin D1 gene or a truncated gene fragment, inserting the SP-C1 protein expression cassette, and uses it in the preparation of a drug for treating myocardial infarction by packaging a lentivirus. We have discovered for the first time that macrophages expressing CCND1 or a truncated sequence of CCND1 that does not exert a proliferation function can improve cardiac function after myocardial infarction. Mechanistically, the truncated sequence of CCND1 that does not exert a proliferation function can regulate the metabolic reprogramming of macrophages, prompting macrophages with a pro-inflammatory phenotype to transform into a repair phenotype, reducing the inflammatory response and promoting cardiac repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a viral vector for expressing cyclin D1 gene and application thereof. Background Art

[0002] Coronary heart disease (CAD), also known as coronary atherosclerotic heart disease (CAD), and sometimes also called ischemic heart disease, refers to heart disease caused by myocardial ischemia and hypoxia due to coronary atherosclerosis. CAD presents diverse clinical manifestations due to differences in pathoanatomy and pathophysiology. Acute myocardial infarction (MI) is the leading cause of death from CAD. Current drug treatments for MI primarily target inflammatory pathways (such as NLRP3 / caspase-1, TLR4 / MyD88 / NF-κB) and fibrotic pathways (such as TGF-β / SMADs, Wnt / β-catenin). Drugs (such as canakinumab and colchicine) have shown some efficacy in clinical trials, but their long-term safety remains to be determined. For example, canakinumab may increase the risk of fatal infections, while colchicine may cause pneumonia. Other treatments, such as cell therapy, exosome therapy, and gene therapy, are still in the research stage and have not yet become standard treatments worldwide. However, due to their highly personalized nature, they are becoming a trend in MI treatment. Gene therapy offers unique advantages in the treatment of myocardial infarction. Gene therapy can deliver therapeutic genes (such as VEGF and SERCA2a) to cardiomyocytes or vascular endothelial cells via vectors (e.g., adeno-associated virus (AAV) and lentivirus), precisely regulating key pathways (e.g., PI3K / Akt and Notch) to promote angiogenesis or inhibit inflammatory responses. Furthermore, viral vectors enable long-term expression of therapeutic genes, avoiding frequent medication use. Furthermore, treatment plans designed based on a patient's gene expression profile can provide personalized treatment for myocardial infarction. Clinical trials (e.g., NCT01174095) have demonstrated the safety and preliminary efficacy of gene therapy. With advances in delivery technology and in-depth clinical research, gene therapy is expected to become a key pillar of cardiovascular disease treatment.

[0003] Current research on gene therapy for myocardial infarction focuses on targeted regulation of cardiomyocytes (anti-apoptosis, regeneration) and endothelial cells (angiogenesis, anti-inflammation), while research on intervention of macrophages is seriously insufficient.

[0004] Cardiomyocyte gene therapy: Targeting the PI3K / Akt signaling pathway through overexpression of noncoding protein sequences, such as small interfering RNA (miRNA-21) and long noncoding RNA (Snhg1), is difficult to reverse myocardial loss after large-scale infarction. Targeting the Notch signaling pathway (miRNA-133a, miRNA-124a, miRNA-199b) may have a protective effect in cardiomyocytes.

[0005] Endothelial cell gene therapy: focuses on pro-angiogenic pathways such as VEGF and FGF, but has limited regulatory effects on the inflammatory microenvironment.

[0006] Macrophages account for over 50% of infiltrating immune cells after myocardial infarction and are key regulators of the inflammatory response and repair process. A PubMed search reveals that over the past five years, only approximately 7% of "myocardial infarction + gene therapy" studies have targeted macrophages, compared to 62% for cardiomyocytes and 31% for endothelial cells. Therefore, research on macrophage gene therapy is urgently needed. Summary of the Invention

[0007] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a mutated CCND1 gene fragment that does not play a proliferation function, a CCND1 gene truncated sequence, and applications thereof.

[0008] Another object of the present invention is to provide a viral vector for expressing the CCND1 gene and its application.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] A mutated CCND1 gene fragment that does not play a proliferation function, wherein the CCND1 gene fragment is a gene sequence encoding a mutation in amino acids 56-84 of cyclin D1 (i.e., encoding a mutation in the cyclin box of the proliferation function domain).

[0011] The preferred nucleotide sequence of the CCND1 gene fragment is shown in SEQ ID NO.3.

[0012] A truncated sequence of the CCND1 gene that does not play a proliferation function is a cyclin D1 gene sequence encoding a cyclin box domain that lacks the proliferation function domain.

[0013] The preferred nucleotide sequence of the CCND1 gene truncated sequence is shown in SEQ ID NO.4.

[0014] Use of the gene CCND1 encoding the full length of cyclin D1, the mutated CCND1 gene fragment that does not play a proliferation function, or the CCND1 gene truncated sequence that does not play a proliferation function in preparing a drug for treating myocardial infarction.

[0015] The cyclin D1 gene CCND1 sequence is preferably as shown in SEQ ID NO.1.

[0016] A recombinant expression plasmid contains the gene CCND1 encoding the full length of cyclin D1, the mutated CCND1 gene fragment that does not play a proliferation function, or the CCND1 gene truncated sequence that does not play a proliferation function.

[0017] As a preferred embodiment of the present invention, the recombinant expression plasmid further contains the macrophage-targeting promoter sequence SP-C1 shown in SEQ ID NO.5, and the SP-C1 promoter is used to drive the gene CCND1 encoding the full-length cyclin D1, the mutated CCND1 gene fragment that does not play a proliferation function, or the CCND1 gene truncated sequence that does not play a proliferation function.

[0018] As a preferred embodiment of the present invention, the target gene fragments (CCND1, CCND1-mut 56-84, CCND1-delt52-152) were cloned into the pLVX-Sp promoter-ZsGreen vector (Viraltherapy technologies) containing the SP-C1 promoter.

[0019] A CCND1 gene-related virus contains the CCND1 gene encoding the full-length cyclin D1, the mutated CCND1 gene fragment that does not play a proliferation function, or the CCND1 gene truncated sequence that does not play a proliferation function.

[0020] As a preferred embodiment of the present invention, the CCND1 gene-related virus is obtained by viral packaging the recombinant expression plasmid.

[0021] The invention relates to the use of the recombinant expression plasmid and the CCND1 gene-related virus in the preparation of a drug for treating myocardial infarction.

[0022] The virus of the present invention can be used to prepare therapeutic gene drugs, such as preparing freeze-dried viral vector preparations (such as AAV9-STAT3 or lentivirus-PPARγ), which are reconstituted with physiological saline for clinical use.

[0023] The drug for treating myocardial infarction can be injected intravenously to regulate macrophages systemically and is suitable for diffuse myocardial injury. It can also be injected intrapericardially or intramyocardially (catheter intervention): delivering high concentrations locally and reducing the risk of systemic exposure.

[0024] The drug for treating myocardial infarction described in this invention can also be used in combination with existing cardiovascular drugs, such as anti-heart failure drugs: gene therapy improves the microenvironment, while drugs maintain cardiac function, creating a synergistic effect. Alternatively, it can be used to prepare biomaterial scaffolds: injectable hydrogels loaded with gene vectors and exosomes provide sustained release and structural support for the myocardium.

[0025] Beneficial effects

[0026] This invention constructs a vector encoding the cyclin D1 gene or a truncated gene fragment, inserting an SP-C1 protein expression cassette, and uses it in the preparation of a drug for treating myocardial infarction via lentiviral packaging. We have discovered for the first time that macrophage expression of CCND1 or a truncated sequence of CCND1 that does not contribute to proliferation can improve cardiac function after myocardial infarction. Mechanistically, this truncated sequence of CCND1 can regulate the metabolic reprogramming of macrophages, prompting the transition of macrophages from a proinflammatory phenotype to a reparative phenotype, alleviating the inflammatory response and promoting cardiac repair.

[0027] 1. This invention offers the advantage of precision in treating myocardial infarction: gene therapy directly regulates macrophage phenotype rather than globally suppressing inflammation.

[0028] 2. Long-term efficacy: Lentiviral vectors can integrate into the genome, enabling long-term, sustained expression. A specific promoter (SP-C1) ensures gene expression only in macrophages.

[0029] 3. Synergy: repairing the microenvironment while reducing scar formation and fibrosis area ( Figure 7 ), the expression of inflammatory factors decreased ( Figure 6 ). Improve heart function and break through the limitations of existing treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 . CCND1 full-length amino acid sequence and mutant composition.

[0031] Figure 2 . Plasmid map constructed by the present invention.

[0032] Figure 3 . Strategy for constructing mouse lentiviral overexpression myocardial infarction model.

[0033] Figure 4 Left ventricular ejection fraction in mice on day 0, day 3, day 7, and day 14 after myocardial infarction.

[0034] Figure 5 . Results of virus infection efficiency test in mice.

[0035] Figure 6 . Expression of inflammatory factors Il-1β, Il6, and Tnf-α in the mouse heart after viral treatment.

[0036] Figure 7 . HE and Masson staining of mouse heart.

[0037] Figure 8 Macrophages were infected with NC virus, ① virus, ② virus, ③ virus, and ④ virus, and the changes in proliferation function were detected by BrdU test and Ki-67 time.

[0038] Figure 9 Macrophages were infected with NC virus, ① virus, ② virus, ③ virus, and ④ virus, and the levels of lactate and acetyl-CoA metabolites were detected. DETAILED DESCRIPTION

[0039] Example 1

[0040] We have found a key gene, CCND1, that effectively regulates macrophage functional transformation. Its truncated sequence, which does not contain the proliferation domain, can alleviate macrophage inflammatory response through metabolic reprogramming, thereby improving adverse cardiac remodeling. We constructed a lentivirus expressing the CCND1 gene with a macrophage-targeting promoter sequence, SP-C1. Figure 1 The full-length and mutant CCND1 amino acid sequences are shown. ① is the full-length amino acid sequence of CCND1, and its coding gene sequence is shown in SEQ ID NO.1; ② is a plasmid expressing the CCND1 amino acid mutation sequence at positions 222-224 (related to the regulation of metabolic function), and the coding gene sequence of the CCND1 amino acid mutation sequence at positions 222-224 is shown in SEQ ID NO.2; ③ is a plasmid expressing the CCND1 mutation sequence that enters the cell nucleus to exert the proliferation function, and the coding gene sequence of the mutation sequence is shown in SEQ ID NO.3; ④ is a plasmid expressing the CCND1 cyclin box amino acid sequence that lacks the proliferation function domain (i.e., lacks CCND1 amino acids 52-152), and the nucleotide sequence of the truncated gene is shown in SEQ ID NO.4. The four plasmid maps constructed are shown in FIG. Figure 2 shown.

[0041] 1.1 Plasmid construction

[0042] The target gene fragments (NC, CCND1, CCND1-mut222-224, CCND1-mut 56-84, CCND1-delt52-152) were cloned into the pLVX-Sp promoter-ZsGreen vector to obtain the NC plasmid and plasmids ① ② ③ ④, respectively. The plasmids were packaged using the viral packaging kit described in Example 1 of CN 107384872 B.

[0043] The vector plasmid pLVX-Sp promoter-ZsGreen was digested with NotI and BamHI. The digestion reaction was carried out in a 37°C water bath for 3 h. The vector digestion system is shown in Table 1:

[0044] Table 1

[0045]

[0046] The large NotI+BamHI fragment of pLVX-Sp promoter-ZsGreen was recovered by 1% agarose gel electrophoresis. The target gene plasmids (pUC57-NC, pUC57-CCND1, pUC57-CCND1-mut222-224, pUC57-CCND1-mut 56-84, and pUC57-CCND1-delt 52-152) were digested with NotI and BamHI. The digestion reaction was incubated at 37°C in a water bath for 3 h. The digestion system is shown in Table 2:

[0047] Table 2

[0048]

[0049] Small NotI+BamHI fragments of pUC57-NC, pUC57-CCND1, pUC57-CCND1-mut222-224, pUC57-CCND1-mut 56-84, and pUC57-CCND1-delt 52-152 were recovered by 1% agarose gel electrophoresis. Large fragments recovered from plasmid pLVX-Sp promoter-ZsGreen were ligated with the NC, CCND1, CCND1-mut222-224, CCND1-mut56-84, and CCND1-delt 52-152 fragments, respectively. The ligation reactions were incubated at 22°C for 3 hours. The ligation reaction system is shown in Table 3:

[0050] Table 3

[0051]

[0052] Transform the ligation product. Mix 10 μl of the ligation product with 100 μl of JM109 competent bacteria, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and immediately place on ice for 2 minutes. Add 400 μl of LB medium preheated to room temperature, incubate at 37°C on a shaker for 1 hour, centrifuge at 4000 rpm for 1 minute, discard the 400 μl supernatant, and mix the remaining 100 μl with a pipette. Spread the remaining 100 μl evenly on an LB plate containing 100 μg / ml Ampicillin-resistant bacteria. Incubate in an incubator at 37°C overnight.

[0053] Three single colonies were picked and inoculated into 5 ml of LB culture medium containing 100 μg / ml Ampicillin resistance, and cultured in a constant temperature shaker at 250 rpm and 37°C overnight. The plasmid was extracted using a small amount of plasmid extraction kit and identified by enzyme digestion with NotI+BamHI. The positive clones identified by enzyme digestion were picked for sequencing verification. The sequencing results showed that the vector was successfully constructed. The sequencing primers were: ZsGreen-R: 5'-CTCCACCACGCACAGGTTGA-3' (SEQ ID NO.6), Sp-F: 5'-TCGGGTTTATTACAGGGACAGCAGAG-3' (SEQ ID NO.7).

[0054] The control NC plasmid and the positive clone plasmid were packaged with the kit and method disclosed in CN107384872B to obtain the lentivirus packaged with the NC plasmid and ① ② ③ ④ plasmids ( Figure 2 ).

[0055] Example 2

[0056] Gene model construction: The lentivirus constructed in Example 1 and packaged from plasmids ① ② ③ ④ was injected into the tail vein of mice. The mice were simply fixed, the tail was fully exposed, and the tail was wiped with 75% alcohol to dilate the blood vessels. 200 μL of a 10 11 AU virus suspension was injected. The mice rested for one week after injection, and myocardial infarction model was established in the mice. The cardiac function of the mice was detected by small animal ultrasound.

[0057] Myocardial infarction model: The experiment used 8-10 week old male mice, which were kept in a constant temperature (22-24°C), constant humidity, and a standard light cycle (12 hours light, 12 hours dark). The mice had free access to sterilized water and standard feed. Ccnd1 Knockout mice were treated with five intraperitoneal injections of equal doses of tamoxifen (100 mg / kg body weight each), while control mice were injected with an equal volume of corn oil. Injections were administered daily starting five days before MI surgery and every other day thereafter. Mice underwent permanent ligation of the left anterior descending artery (LAD) or a sham surgery without ligation. Mice were anesthetized with fluoxetine (0.2 g / kg body weight per mouse) and mechanically ventilated via a small animal ventilator. A thoracotomy was performed between the third and fourth intercostal spaces to expose the heart, and the left anterior descending coronary artery was ligated 2 mm below the atrial appendage with a 7-0 monofilament nylon suture. The thorax was closed after confirming whiteness of the left ventricular anterior wall. Mice were placed on a 37°C heating pad for recovery until fully awake and resumed spontaneous activity.

[0058] Echocardiography: Mice were anesthetized with 3% isoflurane, and transthoracic echocardiography was performed using a VEVO2100 biomicroscope (Visualsonics) to assess left ventricular function. Cardiac function was assessed before MI and on days 3, 7, and 14 after MI. Mice were placed in the supine position and secured to a testing platform, with all four limbs resting on gel-coated electrodes. After securing the mice, an air cannula was placed at the nose and mouth, and 1-1.5% isoflurane was continuously delivered through the cannula. Before performing echocardiography, hair was removed from the left chest and abdomen to ensure close contact between the ultrasound probe and the skin, ensuring clear images. Throughout the echocardiography procedure, the mouse's body temperature was strictly controlled within the range of 37 ± 0.5°C to simulate normal physiological conditions. The heart rate was also maintained within the normal range of 415-460 beats per minute to avoid arrhythmias or tachycardia that could affect the accuracy of the test results.

[0059] During the test, B-Mode (two-dimensional echocardiography) and M-Mode (M-mode echocardiography) images of the mouse heart were collected. B-Mode images allow for intuitive observation of the overall morphology of the mouse heart, including ventricular cavity size, ventricular wall thickness, and wall motion. M-Mode images, on the other hand, provide more detailed temporal information on cardiac motion, such as ventricular contraction and relaxation. During the test, special attention was paid to indicators such as normal heart morphology, normal ventricular cavity size, uniform ventricular wall thickness, and normal wall motion amplitude. These indicators are important for assessing the functional status of the mouse heart. The left ventricular ejection fraction (LVEF) was measured, and data were taken as the average of three cardiac cycles.

[0060] Mouse cardiac function Figure 4 As shown, treatment with virus ①, ③, and ④ was highly effective, significantly improving cardiac function in mice, whereas treatment with virus ② did not significantly improve cardiac function. These results suggest that overexpression of full-length CCND1 in macrophages improves cardiac function after myocardial infarction, and that CCND1 mutations and deletions of the proliferation domain, the cyclin box, can also improve cardiac function.

[0061] Detection of viral infection efficiency in mice: The heart tissue of mice with myocardial infarction 14 days ago was fixed, embedded and immunofluorescence stained. For the immunofluorescence experiment, paraffin sections of the heart cross section were selected, and the sections were dewaxed and hydrated. Antigen retrieval was then performed, and the antigen retrieval solution was heated in a microwave oven on high for 5 minutes. The paraffin sections were placed in the antigen retrieval solution and continued to be heated in a microwave oven on low for 15 minutes, and then naturally cooled to room temperature. The cooled paraffin sections were blocked in 1% BSA diluted in TBST for 20 minutes. After blocking, the paraffin sections were directly incubated with the primary antibody (CD68, Flag) at 4°C overnight. After overnight, the paraffin sections were washed 3 times with TBST for 5 minutes each time, incubated with the secondary antibody at room temperature for 1 hour, and sealed with a fluorescent mounting medium. The results are as follows Figure 5 As shown, green fluorescently labeled Flag and red fluorescently labeled CD68 co-localized in multiple locations, indicating that the virus was successfully overexpressed in mice.

[0062] RNA extraction, reverse transcription, and real-time quantitative PCR (RT-qPCR) analysis

[0063] RNA extraction: Add an appropriate amount of Trizol reagent (0.5-1 mL) to the sample, resuspend repeatedly to fully lyse the cells, and let stand for 5 minutes. Next, add 200 µL of chloroform per 1 mL of Trizol reagent, shake vigorously for 15 seconds, and let stand for another 2-3 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. The solution will separate into three layers: an upper aqueous phase (containing RNA), a middle protein phase, and a lower organic phase (containing DNA). Transfer the upper aqueous phase to a new RNase-free centrifuge tube, add an equal volume of isopropanol, mix gently, and let stand at -20°C for 10-15 minutes. Next, centrifuge at 12,000 rpm for 10 minutes at 4°C to precipitate the RNA. Discard the supernatant and wash the RNA pellet with 75% ethanol, gently inverting the tube several times to ensure the RNA pellet is completely submerged in the ethanol. Finally, dissolve the RNA pellet in an appropriate amount of RNase-free or DEPC-treated water.

[0064] Subsequently, the extracted RNA was converted into cDNA by reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA fragments were amplified in a real-time quantitative PCR instrument using the AceQ qPCR SYBRGreen Master Mix. Primers are listed in Table 4. Amplification was performed using the following thermal cycling conditions: enzyme activation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. The mRNA level of each target gene was normalized to endogenous GAPDH or 18S rRNA, and relative mRNA expression or fold change was calculated using the 2-ΔΔCt method.

[0065] Table 4

[0066]

[0067] The results are as follows Figure 6 As shown, the release of inflammatory factors (Il-1β, Il6, Tnf-α) in cardiac tissue of mice treated with ① ③ ④ viruses was reduced compared with that of mice treated with ② virus, and the level of cardiac tissue inflammation was reduced.

[0068] Hematoxylin and eosin (H&E) staining

[0069] (1) Dewaxing and hydration: Place the paraffin-embedded sections in xylene to remove the paraffin on the surface of the sections, and then gradually hydrate them through a gradient ethanol solution (100%, 95%, 80%) to restore the sections to a state suitable for staining.

[0070] (2) Hematoxylin staining: Immerse the hydrated sections in hematoxylin staining solution. Adjust the staining time according to the thickness of the sections so that the cell nuclei are stained dark blue.

[0071] (3) Bluing treatment: After acid alcohol differentiation to remove excess dye, the sections are bluing in a weak alkaline solution to enhance the staining effect of the cell nucleus.

[0072] (4) Eosin staining: Immerse the sections in eosin staining solution, so that the cytoplasm and matrix are stained pink, forming a sharp contrast with the dark blue cell nucleus.

[0073] (5) Sealing: Finally, use DAPI anti-fading mounting medium to seal the slices to protect the staining results and facilitate subsequent observation.

[0074] Masson's trichrome staining

[0075] (1) Dewaxing and hydration: Similar to H&E staining, the sections are first dewaxed with xylene and hydrated with graded ethanol until they are completely immersed in water.

[0076] (2) Nuclear staining: Use Weigert iron hematoxylin stain to stain the cell nucleus, making the cell nucleus appear dark blue.

[0077] (3) Differentiation and anti-blueing: Remove excess dye through acid alcohol differentiation, and then perform anti-blueing treatment in a weak alkaline solution to make the color of the cell nucleus more vivid.

[0078] (4) Staining of muscle fibers and collagen fibers: Immerse the sections in Masson trichrome staining solution so that the collagen fibers are stained green or blue, and the muscle fibers are stained red, thereby clearly distinguishing the fibrotic areas.

[0079] (5) Dehydration and clearing: After staining, the sections are dehydrated with gradient ethanol and cleared with xylene to enhance the transparency of the sections.

[0080] (6) Sealing: Finally, the sections were sealed with neutral resin mounting medium to protect the staining results and facilitate subsequent observation. H&E and Masson staining were performed to determine the overall structure of the cardiac tissue and the degree of fibrosis. Image J software was used to calculate the percentage of the infarct area to the total left ventricular (LV) area.

[0081] The results are as follows Figure 7 As shown, mice treated with the ①, ③, and ④ viruses showed increased ventricular wall thickness and enlarged cardiac chamber cross-sectional area compared to mice treated with the ② virus. These results suggest that overexpression of full-length CCND1 in macrophages attenuates cardiac pathological remodeling after myocardial infarction, and that CCND1 mutations and deletions of the proliferation domain, the cyclin box, can also ameliorate pathological remodeling.

[0082] Example 3

[0083] Flow cytometry was used to assess cell proliferation: the cell suspension was filtered through a 0.22 μm filter, and the antibody was added to the single-cell suspension. The cells were incubated at 4°C in the dark for 0.5–1 hour. The cells were washed three times with PBS buffer for 5 minutes each. To measure cytoplasmic and nuclear proteins, the cells were processed using the Cytofix / Cytoperm kit. The cells were then incubated with the antibody for 0.5 hour and washed three times with PBS buffer. Flow cytometric data were acquired on a FACS Verse flow cytometer and processed and analyzed using FlowJo software.

[0084] The results are as follows Figure 8 As shown in the figure, we can observe that the proliferation capacity of macrophages overexpressing ① and ② is significantly increased, while the proliferation capacity of macrophages overexpressed by viruses ③ and ④ does not show an upward trend. These results indicate that mutations in ③ and ④, or deletion of the cyclin box domain involved in proliferation function, result in the loss of their ability to promote cell proliferation.

[0085] Example 4

[0086] Lactic acid detection kit was used to detect lactic acid content in cell culture medium: 0.02 mL of culture medium was diluted with normal saline at a ratio of 1:4 and prepared according to Table 5. After terminating the reaction, the OD value of each tube was measured at 530 nm and a light path of 1 cm.

[0087] Table 5

[0088]

[0089] Lactic acid content (mmol / L) = (test tube - blank tube) × 3 × 5 / (standard tube - blank tube)

[0090] Acetyl-CoA assay kit: Before beginning the experiment, remove the kit from the refrigerator and allow it to stand at room temperature for at least 20 minutes to allow all components to equilibrate. Add 50 μL of each standard concentration to the standard wells. Add 50 μL of the sample to be tested to the sample wells; leave blank wells empty. If sample dilution is required, follow the kit instructions and use the included diluent. Add 50 μL of biotinylated antibody to each well, seal the wells with sealing film, and incubate in a 37°C waterbath or incubator for 30 minutes. After incubation, discard the liquid from the wells and pat dry with absorbent paper. Next, add 350 μL of wash buffer to each well, let it sit for 1 minute, discard the wash buffer, and pat dry again with absorbent paper. Repeat this wash step a total of five times. This process can be performed using a microplate washer. Add 100 μL of horseradish peroxidase (HRP)-conjugated detection antibody to each standard and sample well, except for the blank well. Block the wells with sealing film and incubate in a 37°C water bath or incubator for 30 minutes. Add 50 μL of stop solution to each well. Within 15 minutes of adding the stop solution, measure the absorbance (OD) of each well at 450 nm.

[0091] The results are as follows Figure 9 As shown, macrophages overexpressing proteins ①, ③, and ④ exhibited decreased lactate synthesis and increased acetyl-CoA production. These results suggest that mutations in proteins ③ and ④, or deletion of the cyclin box domain associated with proliferation, do not affect the regulation of cellular metabolism. Overexpression of proteins ①, ③, and ④ promotes aerobic oxidation and inhibits glycolysis, thereby causing metabolic reprogramming in macrophages and alleviating their inflammatory response.

Claims

1. A mutated CCND1 gene fragment that does not play a proliferation function, characterized in that: The CCND1 gene fragment is a gene sequence encoding amino acid mutations at positions 56-84 of cyclin D1, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. Use of the mutated CCND1 gene fragment that does not play a proliferation function according to claim 1 in the preparation of a drug for treating myocardial infarction.

3. A truncated sequence of the CCND1 gene that does not play a proliferation function, characterized in that: The gene sequence encoding cyclin D1 lacking the proliferation function domain cyclin box, the nucleotide sequence of which is shown in SEQ ID NO.

4.

4. Use of the CCND1 gene truncated sequence that does not exert proliferation function according to claim 3 in the preparation of a drug for treating myocardial infarction.

5. A recombinant expression plasmid, characterized in that: A CCND1 gene fragment containing the mutation described in claim 1 that does not play a proliferation function or a CCND1 gene truncated sequence that does not play a proliferation function as described in claim 3.

6. The recombinant expression plasmid according to claim 5, characterized in that The recombinant expression plasmid also contains the macrophage-targeting promoter sequence SP-C1 shown in SEQ ID NO.5, and uses the SP-C1 promoter to drive the mutated CCND1 gene fragment that does not play a proliferation function or the CCND1 gene truncated sequence that does not play a proliferation function.

7. Use of the recombinant expression plasmid according to claim 5 or 6 in the preparation of a drug for treating myocardial infarction.

8. A CCND1 gene-associated virus, characterized in that: A CCND1 gene fragment containing the mutation described in claim 1 that does not play a proliferation function or a CCND1 gene truncated sequence that does not play a proliferation function as described in claim 3.

9. Use of the CCND1 gene-related virus according to claim 8 in the preparation of a drug for treating myocardial infarction.

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