Virus vector for expressing cyclin D1 gene and application thereof

Abstract

The invention discloses a viral vector for expressing a cyclin D1 gene and application of the viral vector. The invention constructs and encodes a periodic protein D1 gene or a truncated gene segment, inserts a vector of an SP-C1 protein expression cassette, and packages lentivirus to prepare the medicine for treating myocardial infarction. It is found for the first time that the truncated sequence of CCND1 expressed by macrophages or CCND1 not playing a proliferation function can improve the cardiac function after myocardial infarction. In mechanism, the truncated sequence of CCND1 which does not exert a proliferation function can regulate and control metabolic reprogramming of macrophages, promote transformation of pro-inflammatory phenotype macrophages to repair phenotype, alleviate inflammatory response and promote heart repair.

Description

Technical Field

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

[0002] Coronary heart disease, the full name of which is coronary atherosclerotic heart disease, sometimes also called ischemic heart disease, refers to heart disease caused by myocardial ischemia and hypoxia due to coronary atherosclerosis. Due to different pathological anatomy and pathophysiological changes, coronary heart disease has different clinical manifestations. Among them, acute myocardial infarction (MI) constitutes the leading cause of death in coronary heart disease. At present, drug treatments for myocardial infarction mainly target drugs for inflammatory pathways (such as NLRP3 / caspase-1, TLR4 / MyD88 / NF-κB) and fibrotic pathways (such as TGF-β / SMADs, Wnt / β-catenin) (such as canakinumab, colchicine), which have shown certain effects in clinical trials, but their long-term safety still needs to be verified. For example, canakinumab may increase the risk of fatal infections, and colchicine may cause pneumonia. Other treatment methods such as cell therapy, exosome therapy, and gene therapy are still in the research stage and have not become standard treatment means globally. However, due to its highly personalized treatment characteristics, it is becoming a trend in the treatment of myocardial infarction. Gene therapy has shown unique advantages in the treatment of myocardial infarction. Gene therapy can deliver therapeutic genes (such as VEGF, SERCA2a) to cardiomyocytes or vascular endothelial cells through vectors (such as adeno-associated virus AAV, lentivirus), precisely regulate key pathways (such as PI3K / Akt, Notch), and promote angiogenesis or inhibit inflammatory responses. In addition, viral vectors can achieve long-term expression of therapeutic genes and avoid frequent use of drugs. And designing treatment plans based on the patient's gene expression profile can achieve personalized treatment of the patient's myocardial infarction. Clinical trials (such as NCT01174095) have confirmed the safety and preliminary efficacy of gene therapy. With the progress of delivery technology and the in-depth clinical research, gene therapy is expected to become an important pillar in the treatment of cardiovascular diseases.

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

[0004] Gene therapy for cardiomyocytes: Targeting the PI3K / Akt signaling pathway by overexpressing non-coding protein sequences, such as small interfering RNA (miRNA-21), long non-coding RNA (Snhg1), but it is difficult to reverse myocardial loss after large-area infarction. Targeting the Notch signaling pathway (miRNA-133a, miRNA-124a, miRNA-199b) may play a protective role in cardiomyocytes.

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

[0006] Macrophages account for more than 50% of infiltrating immune cells after myocardial infarction and are the core regulators of the inflammatory response and repair process. PubMed search shows that among the studies related to "myocardial infarction + gene therapy" in the past 5 years, compared with the 62% of cardiomyocyte research and 31% of endothelial cell research, only about 7% involve targeting macrophages. Therefore, the research on gene therapy for macrophages is very urgent. Summary of the Invention

[0007] The object of the present invention is to provide a mutant CCND1 gene fragment that does not exert a proliferative function, a truncated CCND1 gene sequence, and their applications in view of the above deficiencies of the prior art.

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

[0009] The object of the present invention can be achieved by the following technical solutions: A mutant CCND1 gene fragment that does not exert a proliferative function, and the CCND1 gene fragment is a gene sequence encoding a mutation at amino acids 56 - 84 of cyclin D1 (i.e., a mutation in the cyclin box of the proliferative functional domain).

[0010] The CCND1 gene fragment preferably has a nucleotide sequence as shown in SEQ ID NO.3.

[0011] A truncated CCND1 gene sequence that does not exert a proliferative function, which is a cyclin D1 gene sequence encoding the deletion of the cyclin box of the proliferative functional domain.

[0012] The CCND1 gene truncated sequence preferably has a nucleotide sequence as shown in SEQ ID NO.4.

[0013] The application of the gene CCND1 encoding the full-length cyclin D1, the mutant CCND1 gene fragment that does not exert a proliferative function, or the truncated CCND1 gene sequence that does not exert a proliferative function in the preparation of drugs for treating myocardial infarction.

[0014] The CCND1 gene sequence of the cyclin D1 gene preferably is as shown in SEQ ID NO.1.

[0015] A recombinant expression plasmid containing the gene CCND1 encoding the full-length cyclin D1, the mutant CCND1 gene fragment that does not exert a proliferative function, or the truncated CCND1 gene sequence that does not exert a proliferative function.

[0016] As a preference of the present invention, the recombinant expression plasmid further contains a macrophage-targeting promoter sequence SP-C1 shown in SEQ ID NO.5, and the gene CCND1 encoding the full-length cyclin D1, a mutant CCND1 gene fragment that does not exert a proliferative function, or a truncated sequence of the CCND1 gene that does not exert a proliferative function is driven by the SP-C1 promoter.

[0017] As a preference of the present invention, the target gene fragments (CCND1, CCND1-mut 56-84, CCND1-delt52-152) are cloned into the pLVX-Sp promoter-ZsGreen vector (Viraltherapytechnologies) containing the SP-C1 promoter.

[0018] A CCND1 gene-related virus contains the gene CCND1 encoding the full-length cyclin D1, the mutant CCND1 gene fragment that does not exert a proliferative function, or the truncated sequence of the CCND1 gene that does not exert a proliferative function.

[0019] As a preference of the present invention, the CCND1 gene-related virus is obtained by virus packaging of the recombinant expression plasmid.

[0020] Use of the recombinant expression plasmid and the CCND1 gene-related virus of the present invention in the preparation of a medicament for treating myocardial infarction.

[0021] The virus of the present invention can be used to prepare a therapeutic gene medicament, such as being prepared into a freeze-dried virus carrier preparation (such as AAV9-STAT3 or lentivirus-PPARγ), and is reconstituted with physiological saline during clinical use.

[0022] The medicament for treating myocardial infarction can be intravenously injected for systemic regulation of macrophages and is applicable to diffuse myocardial injury. The medicament for treating myocardial infarction can also be injected into the pericardium or into the myocardium (catheter intervention): for local high-concentration delivery to reduce the risk of systemic exposure.

[0023] The medicament for treating myocardial infarction of the present invention can also be used in combination with existing cardiovascular drugs, such as in combination with anti-heart failure drugs: gene therapy improves the microenvironment, and the drug maintains heart function to form a synergistic effect. Or it can be used to prepare a biomaterial scaffold: an injectable hydrogel loads gene carriers and exosomes for slow-release delivery and provides myocardial structural support.

[0024] Beneficial effects

[0025] The present invention relates to the application of a vector constructed by encoding cyclin D1 gene or truncated gene fragment and inserting an SP-C1 protein expression cassette in the preparation of drugs for treating myocardial infarction. We first discovered that the expression of CCND1 or the truncated sequence of CCND1 that does not exert proliferative function in macrophages can improve cardiac function after myocardial infarction. Mechanistically, the truncated sequence of CCND1 that does not exert proliferative function can regulate the metabolic reprogramming of macrophages, promote the transformation of pro-inflammatory phenotype macrophages into repair phenotype, reduce the inflammatory response, and promote cardiac repair.

[0026] 1. The present invention has the advantage of precision in treating myocardial infarction: gene therapy directly regulates macrophage phenotype instead of globally inhibiting inflammation.

[0027] 2. Long-acting: The lentiviral vector can be integrated into the genome to achieve long-term and continuous expression. The specific promoter (SP-C1) ensures that the gene is only expressed in macrophages.

[0028] 3. Synergy: While repairing the microenvironment, it reduces scar formation, with a reduction in the fibrotic area ( Figure 7 ), and a reduction in the expression of inflammatory factors ( Figure 6 ). It improves cardiac function and breaks through the limitations of existing treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 . The full-length amino acid sequence of CCND1 and the composition of mutants.

[0030] Figure 2 . The plasmid map constructed by the present invention.

[0031] Figure 3 . The construction strategy of a mouse lentiviral overexpression myocardial infarction model.

[0032] Figure 4 . Left ventricular ejection fraction of mice on day 0, day 3, day 7, and day 14 after myocardial infarction.

[0033] Figure 5 . Detection results of virus infection efficiency in mice.

[0034] Figure 6 . Expression of cardiac inflammatory factors Il-1β, Il6, and Tnf-α in mice after virus treatment.

[0035] Figure 7 . HE and Masson staining of mouse hearts.

[0036] Figure 8 . Brdu experiment and Ki-67 time detection of proliferation function changes after NC virus, virus ①, virus ②, virus ③, and virus ④ infect macrophages.

[0037] Figure 9 . NC virus, virus ①, virus ②, virus ③, and virus ④ infect macrophages, and the contents of lactic acid and acetyl coenzyme A metabolites are detected. Detailed implementation manners

[0038] Example 1

[0039] We found the key gene CCND1 that effectively regulates the functional transformation of macrophages. The truncated sequence of CCND1 without the proliferation domain can reduce the inflammatory response of macrophages through metabolic reprogramming, thereby improving the adverse remodeling of the heart. We constructed a lentivirus expressing the CCND1 gene with the macrophage-targeting promoter sequence SP-C1. Figure 1 It consists of the full-length amino acid sequence of CCND1 and mutants shown. ① is the full-length amino acid sequence of CCND1, and its coding gene sequence is as shown in SEQ ID NO.1; ② is a plasmid expressing the mutant sequence of amino acids 222-224 of CCND1 (related to regulating metabolic function), and the coding gene sequence of the mutant sequence of amino acids 222-224 of CCND1 is as shown in SEQ ID NO.2; ③ is a plasmid expressing the mutant sequence of the sequence where CCND1 enters the nucleus to exert its proliferation function, and the coding gene sequence of this mutant sequence is as shown in SEQ ID NO.3; ④ is a plasmid expressing the deletion of the cyclin box amino acid sequence of the proliferation function domain of CCND1 (i.e., the deletion of amino acids 52-152 of CCND1), and the nucleotide sequence of this truncated gene is as shown in SEQ ID NO.4. The four constructed plasmid maps are as Figure 2 shown.

[0040] 1.1 Plasmid construction Clone the target gene fragments (NC, CCND1, CCND1-mut222-224, CCND1-mut 56-84, CCND1-delt52-152) into the pLVX-Sp promoter-ZsGreen vector to obtain the NC plasmid and plasmids ①, ②, ③, and ④ respectively. The plasmids are packaged into viruses using the virus packaging kit in Example 1 of CN 107384872 B.

[0041] Digest the vector plasmid pLVX-Sp promoter-ZsGreen with NotI and BamHI. The digestion reaction is carried out in a 37°C water bath for 3 h. The vector digestion system is shown in Table 1: Table 1

[0042] Recover the large fragment of pLVX-Sp promoter-ZsGreen digested with NotI+BamHI by 1% agarose gel electrophoresis. Digest the target gene plasmids pUC57-NC, pUC57-CCND1, pUC57-CCND1-mut222-224, pUC57-CCND1-mut 56-84, pUC57-CCND1-delt 52-152 with NotI and BamHI. The digestion reaction is carried out in a 37°C water bath for 3 h. The digestion system is shown in Table 2: Table 2

[0043] Recover the small fragments of pUC57-NC, pUC57-CCND1, pUC57-CCND1-mut222-224, pUC57-CCND1-mut 56-84, pUC57-CCND1-delt 52-152 digested with NotI+BamHI by 1% agarose gel electrophoresis. Ligate the large fragment recovered from plasmid pLVX-Sp promoter-ZsGreen with the fragments of NC, CCND1, CCND1-mut222-224, CCND1-mut56-84, CCND1-delt 52-152 respectively. The ligation reaction is carried out at 22°C for 3 h. The ligation reaction system is shown in Table 3: Table 3

[0044] Transformation of the ligation product. Mix 10 μl of the ligation product with 100 μl of JM109 competent bacteria and incubate on ice for 30 min, heat shock at 42°C for 45 s, immediately place on ice for 2 min, add 400 μl of LB medium preheated to room temperature, culture in a 37°C constant temperature shaker for 1 h, centrifuge at 4000 rpm for 1 min, discard 400 μl of the culture supernatant, mix the remaining 100 μl with a pipette and evenly spread it on an LB plate containing 100 μg / ml Ampicillin resistance, and incubate upside down in a 37°C constant temperature incubator overnight.

[0045] Pick 3 single colonies and inoculate them into 5 ml LB culture medium containing 100 μg / ml Ampicillin resistance, and culture them in a constant temperature shaker at 250 rpm and 37°C overnight. Extract the plasmid with a small amount of plasmid extraction kit, and perform restriction digestion identification with NotI+BamHI. Pick the positive clones with correct restriction digestion identification for sequencing verification. The sequencing results showed that the vector was successfully constructed. Sequencing primers: ZsGreen-R: 5'-CTCCACCACGCACAGGTTGA-3' (SEQ ID NO.6), Sp-F: 5'-TCGGGTTTATTACAGGGACAGCAGAG-3' (SEQ ID NO.7).

[0046] The control NC plasmid and the positive clone plasmid were packaged with the kit and method disclosed in CN107384872B to obtain the lentivirus ( Figure 2 ).

[0047] Example 2 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 10 11 AU virus suspension was injected, and the mice rested for one week. Myocardial infarction model was established in the mice, and the cardiac function of the mice was detected by small animal ultrasound.

[0048] Myocardial infarction modeling: 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 of light, 12 hours of darkness). The mice had free access to sterilized water and standard feed. Ccnd1 For knockout mice, we used an intraperitoneal injection of five equal doses of tamoxifen (100 mg / kg body weight each time), and control mice were injected with an equal volume of corn oil. The injection time started five days before MI surgery, once a day, and once every other day after MI surgery. Mice underwent permanent ligation of the left anterior descending artery (LAD) or sham surgery without ligation. Mice were anesthetized with fluoxetine (0.2 g / kg body weight each) and mechanically ventilated by a small animal ventilator. 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 auricle with a 7-0 monofilament nylon suture, and the chest was closed after confirming that the anterior wall of the left ventricle was white. After surgery, the mice were placed on a 37°C heating pad to recover until they were fully awake and resumed spontaneous activities.

[0049] Echocardiography: Mice were anesthetized with 3% isoflurane, and transthoracic echocardiography was performed using a VEVO 2100 biomicroscope (Visualsonics) to evaluate left ventricular function. Cardiac function was evaluated in mice before MI and on days 3, 7, and 14 after MI. The mice to be tested were placed in the supine position and fixed on the detection platform, ensuring that the four limbs of the mice were on the electrode pads coated with gel. After fixing the mice, an air catheter was placed at the mouth and nose of the mice, and 1-1.5% isoflurane was continuously delivered into the catheter. Before performing cardiac ultrasound detection, the hair on the left chest and abdomen of the mice was removed to ensure that the ultrasound probe could be in close contact with the skin to obtain clear images. During the entire cardiac ultrasound detection process, the body temperature of the mice was strictly controlled to keep it within the range of 37 ± 0.5 °C to simulate the normal physiological state of the mice. At the same time, it was ensured that the heart rate of the mice was stable, maintained within the normal range of 415-460 beats per minute, to avoid affecting the accuracy of the detection results due to arrhythmia or too fast / slow heart rate.

[0050] During the detection, B-Mode (two-dimensional echocardiogram) images and M-Mode (M-mode echocardiogram) images of the mouse heart were collected respectively. Through the B-Mode images, the overall morphology of the mouse heart can be visually observed, including the size of the ventricular cavity, the thickness of the ventricular wall, and the movement of the ventricular wall, etc. The M-Mode images can provide more detailed information on the cardiac motion time, such as the systolic and diastolic functions of the ventricle. During the detection process, key attention was paid to indicators such as whether the cardiac morphology was normal, whether the size of the ventricular cavity was within the normal range, whether the ventricular wall thickness was uniform, and whether the amplitude of the ventricular wall movement was normal. These indicators are of great significance for evaluating the functional status of the mouse heart. The left ventricular ejection fraction (LVEF) was measured, and the data were taken as the average of 3 cardiac cycles.

[0051] The cardiac function of the mice was as Figure 4 shown. The therapeutic effects of viruses ①, ③, and ④ were obvious, and the cardiac function of the mice was significantly improved, while the cardiac function was not significantly improved after the treatment with virus ②. The above results indicate that overexpression of full-length CCND1 in macrophages improves cardiac function after myocardial infarction. After the CCND1 mutation and deletion of the cyclin box, the proliferative functional domain, cardiac function can still be improved.

[0052] Detection of viral infection efficiency in mice: The heart tissues of mice with myocardial infarction for 14 days were fixed, embedded, and immunofluorescently stained. For the immunofluorescent experiment, paraffin sections of heart cross-sections were selected, and the sections were dewaxed and hydrated. Subsequently, antigen retrieval was performed. The antigen retrieval solution was heated in a microwave oven at high power for 5 minutes, and the paraffin sections were placed in the antigen retrieval solution and continued to be heated in the microwave oven at low power for 15 minutes, and then naturally cooled to room temperature. The cooled paraffin sections were blocked in 1% BSA diluted with TBST for 20 minutes. After blocking, the paraffin sections were directly incubated with primary antibodies (CD68, Flag) at 4°C overnight. After overnight incubation, the paraffin sections were washed 3 times with TBST for 5 minutes each time, incubated with secondary antibodies for 1 hour at room temperature, and sealed with a fluorescent mounting medium. The results are as Figure 5 shown. There are multiple co-localizations between Flag labeled with green fluorescence and CD68 labeled with red fluorescence, indicating that the virus was successfully overexpressed in mice.

[0053] RNA extraction, reverse transcription, and real-time quantitative PCR (RT-qPCR) analysis RNA extraction: An appropriate amount of Trizol reagent (0.5 - 1 mL) was added to the samples, and the samples were resuspended repeatedly to fully lyse the cells, and then left standing for 5 minutes. Then, 200 μL of chloroform was added to every 1 mL of Trizol reagent, shaken vigorously for 15 seconds, and then left standing for 2 - 3 minutes. Centrifugation was performed at 12,000 rpm for 15 minutes at 4°C. At this time, the solution was divided into three layers: the upper aqueous phase (containing RNA), the middle protein layer, and the lower organic phase (containing DNA). The upper aqueous phase was transferred to a new RNase-free centrifuge tube, an equal volume of isopropanol was added, gently mixed, and left standing at -20°C for 10 - 15 minutes. Subsequently, centrifugation was performed at 12,000 rpm for 10 minutes at 4°C to precipitate RNA. The supernatant was discarded, and the RNA precipitate was washed with 75% ethanol. The centrifuge tube was gently inverted several times to ensure that the RNA precipitate was completely immersed in ethanol. Finally, the RNA precipitate was dissolved with an appropriate amount of RNase-free water or DEPC-treated water.

[0054] Subsequently, a reverse transcription reaction was carried out using the iScript cDNA Synthesis Kit (Bio-Rad) to convert the extracted RNA into cDNA. The obtained cDNA fragments were amplified in a real-time quantitative PCR instrument, and the reagent used was AceQ qPCR SYBR Green Master Mix, and the primers are shown in Table 4. The following thermal cycling conditions were used for the amplification process: First, the enzyme was activated at 95°C for 5 minutes, and then 40 cycles were carried out. Each cycle included 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 by endogenous GAPDH or 18S rRNA, and the relative mRNA expression or fold change was calculated using the 2⁻ΔΔCt method.

[0055] Table 4

[0056]

[0057] The results are as Figure 6 shown. ①③④ virus-treated mice had a lower release of inflammatory factors (Il-1β, Il6, Tnf-α) in the heart tissue compared with ② virus-treated mice, and the inflammatory level in the heart tissue was reduced.

[0058] Hematoxylin-eosin (H&E) staining (1) Deparaffinization and hydration: The paraffin-embedded sections were placed in xylene to remove the paraffin on the surface of the sections, and then gradually hydrated through a gradient ethanol solution (100%, 95%, 80%) to restore the sections to a state suitable for staining.

[0059] (2) Hematoxylin staining: The hydrated sections were immersed in hematoxylin staining solution, and the staining time was adjusted according to the section thickness to stain the cell nuclei dark blue.

[0060] (3) Bluing treatment: After removing the excess dye by acid alcohol differentiation, the sections were blued in a weakly alkaline solution to enhance the staining effect of the cell nuclei.

[0061] (4) Eosin staining: The sections were immersed in eosin staining solution to stain the cytoplasm and matrix pink, forming a sharp contrast with the dark blue cell nuclei.

[0062] (5) Sealing: Finally, the sections were sealed with DAPI anti-quenching mounting medium to protect the staining results and facilitate subsequent observation.

[0063] Masson trichrome staining (1) Deparaffinization and hydration: Similar to H&E staining, the sections were first deparaffinized with xylene and hydrated through a gradient ethanol solution until completely immersed in water.

[0064] (2) Nucleus staining: The cell nuclei were stained with Weigert iron hematoxylin staining solution to make the cell nuclei show dark blue.

[0065] (3) Differentiation and bluing: The excess dye was removed by acid alcohol differentiation, and then bluing treatment was carried out in a weakly alkaline solution to make the color of the cell nuclei more distinct.

[0066] (4) Muscle fiber and collagen fiber staining: The sections were immersed in Masson trichrome staining solution to stain the collagen fibers green or blue and the muscle fibers red, so as to clearly distinguish the fibrotic areas.

[0067] (5) Dehydration and clearing: After staining, the sections were dehydrated through a gradient of ethanol and cleared with xylene to enhance the transparency of the sections.

[0068] (6) Mounting: Finally, the sections were mounted with a neutral resin mounting medium to protect the staining results and facilitate subsequent observation. H&E and Masson staining were used to determine the overall structure of the cardiac tissue and the degree of fibrosis. Image J software was applied to calculate the percentage of the infarcted area in the total area of the left ventricle (LV).

[0069] The results are as Figure 7 shown. ①③④ Compared with the mice treated with virus ②, the ventricular wall thickness increased and the cross-sectional area of the cardiac chamber expanded in the mice treated with virus. The above results indicate that overexpression of full-length CCND1 in macrophages alleviates pathological remodeling after myocardial infarction, and after mutation and deletion of the proliferative functional domain cyclin box of CCND1, pathological remodeling can still be improved.

[0070] Example 3 Detection of cell proliferation ability by flow cytometry: The cell suspension was filtered through a 0.22 μm filter, and antibodies were added to the single-cell suspension, which was incubated at 4 °C in the dark for 0.5 - 1 hour. It was washed 3 times with PBS buffer for 5 minutes each time. To measure cytoplasmic and nuclear proteins, we continued the treatment using the Cytofix / Cytoperm kit. Subsequently, the cells were incubated with antibodies for 0.5 hour, washed 3 times with PBS buffer, and flow cytometry data were collected on a FACS Verse flow cytometer, and the data were processed and analyzed using FlowJo software.

[0071] The results are as Figure 8 shown. As can be observed from the figure, the proliferation ability of macrophages overexpressed in ① and ② was significantly upregulated, while there was no upward trend in the proliferation ability of macrophages overexpressed by virus ③ and ④. The above results indicate that after mutation and deletion of the proliferative functional domain cyclin box related to ③ and ④, the promoting function of cell proliferation is lost.

[0072] Example 4 Detection of lactic acid content in cell culture medium using a lactic acid detection kit: 0.02 mL of the culture medium was diluted 1:4 with normal saline and prepared according to Table 5. After terminating the reaction, the OD values of each tube were measured at 530 nm with a 1 cm light path.

[0073] Table 5

[0074]

[0075] Lactic acid content (mmol / L) = (measured tube - blank tube) × 3 × 5 / (standard tube - blank tube) Kit for detecting the content of acetyl coenzyme A: Before starting the experiment, take the kit out of the refrigerator and place it at room temperature for at least 20 minutes to ensure that all components in the kit reach room temperature equilibrium. Add different concentrations of standards to the standard wells, 50 μL to each well. Add the sample to be tested to the sample wells, also 50 μL to each well; do not add any sample to the blank wells. If the sample needs to be diluted, dilute it according to the instructions provided in the kit using the accompanying diluent. Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with a sealing film, and then incubate in a water bath or incubator at 37 °C for 30 minutes. After incubation, discard the liquid in the wells and pat dry with absorbent paper. Then, add 350 μL of washing solution to each well, let it stand for 1 minute, pour out the washing solution, and pat dry again with absorbent paper. Repeat this washing step 5 times in total. This process can be completed using a plate washer. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard wells and sample wells. After sealing the reaction wells with a sealing film, incubate in a water bath or incubator at 37 °C for 30 minutes. Add 50 μL of stop solution to each well. Measure the absorbance (OD value) of each well at a wavelength of 450 nm within 15 minutes after adding the stop solution.

[0076] The results are as Figure 9 shown. It can be observed that the lactic acid synthesis of macrophages overexpressing ①, ③, and ④ decreases, and the generation of acetyl coenzyme A increases. The above results indicate that after the cyclin box, the domain related to the proliferation function of ③ and ④ is mutated or deleted, it does not affect the regulation of cell metabolism. Overexpression of ①, ③, and ④ promotes aerobic oxidation reactions and inhibits glycolysis, thereby causing metabolic reprogramming of macrophages and reducing macrophage inflammatory responses.

Claims

1. A mutant CCND1 gene fragment that does not exhibit proliferative function, characterized in that, The CCND1 gene fragment described above is a gene sequence encoding the mutation of amino acids 56-84 of cyclin D1, and its nucleotide sequence is as shown in SEQ ID NO.

3.

2. Use of the mutated CCND1 gene fragment that does not exert a proliferative function described in claim 1 in the preparation of a medicament for treating myocardial infarction.

3. A truncated CCND1 gene sequence that does not exhibit proliferative function, characterized in that, It is a gene sequence encoding cyclin D1 lacking the proliferative functional domain cyclin box, and its nucleotide sequence is as shown in SEQ ID NO.

4.

4. Use of the truncated CCND1 gene sequence that does not exert a proliferative function described in claim 3 in the preparation of a medicament for treating myocardial infarction.

5. Use of cyclin D1 gene CCND1 in the preparation of a medicament for treating myocardial infarction, characterized in that, The cyclin D1 gene CCND1 sequence described above is as shown in SEQ ID NO.

1.

6. A recombinant expression plasmid, characterized in that, Containing the gene CCND1 encoding the full length of cyclin D1, the mutated CCND1 gene fragment that does not exert a proliferative function described in claim 1, or the truncated CCND1 gene sequence that does not exert a proliferative function described in claim 3.

7. The recombinant expression plasmid according to claim 6, wherein The recombinant expression plasmid described above also contains the macrophage-targeting promoter sequence SP-C1 shown in SEQID NO.5, and uses the SP-C1 promoter to initiate the gene CCND1 encoding the full length of cyclin D1, the mutated CCND1 gene fragment that does not exert a proliferative function, or the truncated CCND1 gene sequence that does not exert a proliferative function.

8. Use of the recombinant expression plasmid described in claim 6 or 7 in the preparation of a medicament for treating myocardial infarction.

9. A virus related to the CCND1 gene, characterized in that, Containing the gene CCND1 encoding the full length of cyclin D1, the mutated CCND1 gene fragment that does not exert a proliferative function described in claim 1, or the truncated CCND1 gene sequence that does not exert a proliferative function described in claim 3.

10. Use of the CCND1 gene-related virus described in claim 9 in the preparation of a medicament for treating myocardial infarction.

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