Construction method and application of anti-fibrosis mesenchymal stem cells

Abstract

The application belongs to the technical field of genetic engineering and cell modification, and discloses a construction method and application of anti-fibrosis mesenchymal stem cells. The application finds and verifies the key role of AHNAK gene in the regulation of MSC anti-fibrosis function, and AHNAK knockdown can significantly improve the survival rate of MSC under hypoxic conditions. The application innovatively associates AHNAK with the fibrosis microenvironment adaptation ability of MSC, providing a new target for MSC function modification. The AF-MSC constructed by the application can inhibit the transformation of fibroblasts into myofibroblasts, increase the secretion of anti-fibrosis paracrine factors, significantly reduce collagen deposition, significantly enhance the anti-fibrosis effect, improve the treatment efficiency of single administration, reduce the need for repeated administration, and reduce the overall treatment cost.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and cell modification technology, and to a method for constructing mesenchymal stem cells, specifically to a method for constructing and applying anti-fibrotic mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells that possess all the common characteristics of stem cells, namely self-renewal and multi-lineage differentiation capabilities. They are most widely used clinically, and when combined with hematopoietic stem cells, they can improve transplant success rates and accelerate hematopoietic reconstitution. When patients receive high-dose chemotherapy, infusing mesenchymal stem cells along with hematopoietic stem cells can significantly accelerate the recovery time of blood cells, and is safe with no adverse reactions.

[0003] The existing MSC intravenous infusion therapy for pulmonary fibrosis faces three major technological bottlenecks that urgently need to be overcome:

[0004] 1) Low survival rate in the fibrotic microenvironment: The hypoxic environment of pulmonary fibrosis tissue leads to accelerated apoptosis of exogenous MSCs, and the duration of their activity is extremely limited;

[0005] 2) The anti-fibrotic function is limited and unstable: MSCs exhibit significant batch-to-batch variability in key functions such as inhibiting fibroblast activation and regulating the TGF-β signaling pathway, making it difficult to achieve stable therapeutic effects;

[0006] 3) Insufficient duration of efficacy: Due to the short survival time and weak functional strength of MSCs, the efficacy maintenance period after a single administration is short, usually requiring multiple repeated infusions, which greatly increases the treatment burden on patients.

[0007] Therefore, this invention provides a method for constructing and applying anti-fibrotic mesenchymal stem cells. Summary of the Invention

[0008] This invention aims to provide a method for constructing and applying anti-fibrotic mesenchymal stem cells. By targeting and knocking down the AHNAK gene in MSCs, an anti-fibrotic functionally enhanced mesenchymal stem cell is constructed, which improves the metabolic adaptability and functional stability of MSCs in the hypoxic microenvironment of fibrosis. This results in a stronger effect in inhibiting abnormal fibroblast activation and reducing collagen deposition, prolonging the duration of action, and enhancing the sustainability of therapeutic efficacy, thus providing a more precise, efficient, and safe technical solution for cell therapy of pulmonary fibrosis.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides the application of human mesenchymal stem cells with silenced AHNAK gene expression in the preparation of antifibrotic drugs, wherein the nucleotide sequence of the AHNAK gene is shown in SEQ ID NO.1, and the human mesenchymal stem cells are derived from bone marrow.

[0011] Preferably, the drug comprises any pharmaceutically acceptable dosage form made from human mesenchymal stem cells with silenced AHNAK gene expression and pharmaceutically acceptable carriers and / or excipients.

[0012] Preferably, the dosage form of the drug is an injection.

[0013] Preferably, when the drug is administered to rats via intravenous infusion, the number of human mesenchymal stem cells with silenced AHNAK gene expression in the drug is not less than 1 × 10⁻⁶. 6 cells.

[0014] Preferably, the method for constructing human mesenchymal stem cells with silenced AHNAK gene expression includes: transfecting human mesenchymal stem cells with AHNAK knockdown virus, changing the medium after transfection, and adding puromycin for continuous screening.

[0015] Preferably, the construction method further includes: measuring AHNAK knockdown virus, Polybrene enhancer, and DMEM complete culture medium in a volume ratio of 10:1:48 to prepare a transfection mixture, adding 2 ml / well to human mesenchymal stem cells cultured in a multi-well plate for transfection, changing the medium after transfection, and adding 1-2 μg / mL of puromycin for continuous screening to obtain the desired result.

[0016] Preferably, the transfection time is not less than 48 hours, the screening time is 7 to 14 days, and the MOI value of the AHNAK knockdown virus is 10.

[0017] Preferably, the human mesenchymal stem cells are P3 generation human mesenchymal stem cells, and the human mesenchymal stem cells are expressed at a ratio of 1×10⁻⁶. 5 Cells / holes are laid in a porous plate.

[0018] Preferably, the method for constructing the AHNAK knockdown slow virus includes the following steps:

[0019] S1. Design and synthesize shRNA targeting AHNAK with the sequence shown in SEQ ID NO.2;

[0020] S2. The shRNA was inserted into the GV492 vector by double enzyme digestion, transformed into competent bacteria, and the AHNAK lentiviral vector plasmid was extracted.

[0021] S3. The AHNAK lentiviral vector plasmid and two helper packaging plasmids were co-transfected into cells. Virus-containing serum was collected, centrifuged, and filtered to obtain AHNAK knockdown lentivirus.

[0022] Preferably, the GV492 vector contains a GFP reporter gene and a puromycin resistance gene; the competent bacteria are Stbl3; the cells are HEK293T; and the packaging plasmids are psPAX2 and pMD2.G.

[0023] Secondly, the present invention provides anti-fibrotic mesenchymal stem cells constructed using the above-described method for constructing human mesenchymal stem cells with silenced AHNAK gene expression.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. AHNAK is a giant skeletal protein with a molecular weight of about 700 kDa. Previous studies have focused on its role in myocardium, nerves and tumors. This invention is the first to discover and verify the key role of the AHNAK gene in the regulation of MSC antifibrotic function, and innovatively associates it with the fibrotic microenvironment adaptability of MSCs, providing a new target for MSC functional modification.

[0026] 2. Compared with ordinary MSCs, the AF-MSCs constructed in this invention can inhibit the transformation of fibroblasts into myofibroblasts; they can also increase the secretion of anti-fibrotic paracrine factors and significantly reduce collagen deposition (reduced Collagen I and Fibronectin), thus significantly enhancing the anti-fibrotic effect; secondly, AHNAK knockdown can significantly improve the survival rate of MSCs under hypoxic conditions, enhance the survival ability and functional stability of MSCs under hypoxic conditions, prolong the effective duration of action, improve the therapeutic efficiency of a single dose, reduce the need for repeated dosing, and reduce the overall treatment cost; in addition, the functional phenotype of MSCs can be stably modified through genetic engineering, reducing batch-to-batch differences and improving the controllability of product quality. Attached Figure Description

[0027] Figure 1 These are the verification results of AHNAK knockdown efficiency in AF-MSC;

[0028] Figure 2 It is an ELISA assay used to detect the RNA quantitative levels of α-SMA, HGF, PGE2, and TSG-6 factors in AF-MSCs;

[0029] Figure 3 These are the results of bright-field light microscopy observations of lung organoids (scale bar = 100 μm).

[0030] Figure 4These are paraffin sections of lung organoids stained with hematoxylin and eosin (HE) and Masson staining (scale bar = 50 μm).

[0031] Figure 5 These are the results of gross observation of rat lung tissue;

[0032] Figure 6 HE and Masson staining of rat lung tissue (scale bar = 200 μm);

[0033] Figure 7 These are Western Blot results for lung tissue protein fibrosis markers (Tublin is the internal reference protein).

[0034] Figure 8 This is the result of mitochondrial TMRE flow cytometry quantification;

[0035] Figure 9 This is the quantitative result of ECAR from glycolysis. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Example 1: Extraction and Identification of Human Mesenchymal Stem Cells

[0040] 1. Isolation of MSCs from bone marrow

[0041] Bone marrow of 30 mL was extracted from young, healthy volunteers (aged 18-35, excluding those with a history of hereditary diseases, infectious diseases, or cancer) via posterior superior iliac spine aspiration. Heparin was added to a final concentration of 20 U / mL for anticoagulation and used to isolate MSCs. The specific steps are as follows:

[0042] 1) Mix the bone marrow with an equal volume of PBS buffer, centrifuge at 1500 rpm for 5 min, and remove the supernatant and the fat layer floating on the top.

[0043] 2) Slowly add the cell suspension to an equal volume of human lymphocyte separation medium with a density of 1.077 g / mL, centrifuge at 2000 rpm for 20 min, and carefully aspirate the white membrane layer containing MSCs and monocytes at the interface.

[0044] 3) Wash twice with PBS buffer, centrifuging at 300×g for 10 min after each wash, count the samples, resuspend, and centrifuge at 1×10⁻⁶. 5 cells / cm 2 The samples were inoculated at high density into a low-glucose Duchenne modified Eagle (DMEM) medium containing 15% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0045] 4) Incubate at 37℃ and 5% CO2. After 48 hours, perform a half-volume medium change to remove non-adherent cells.

[0046] 5) When the cells reach 80-90% confluence, passage them using 0.25% trypsin-EDTA digestion solution and passage them to P2 at a ratio of 1:3. After passage from P2 to P3, the cells are used for subsequent experiments.

[0047] The MSCs extracted in this embodiment were identified as conforming to the ISCT 2006 standard.

[0048] 2. MSC Qualification Criteria (compliant with ISCT 2006 standards)

[0049] 1) Morphology: Adherent growth, appearing as fibroblast-like spindle or polygonal shapes;

[0050] 2) Surface markers (flow cytometry): CD73(+), CD90(+), CD105(+); CD34(-), CD45(-), CD11b(-), CD19(-), HLA-DR(- / low);

[0051] 3) Multi-directional differentiation potential: It can differentiate into osteoblasts (positive for Alizarin Red staining), adipocytes (positive for Oil Red O staining), and chondrocytes (positive for Safranin O staining) under specific induction culture conditions.

[0052] Example 2: Construction of AHNAK knockdown slow virus

[0053] 1. Target gene and shRNA design

[0054] Based on the human AHNAK gene, referencing sequence number NC_000011.10 (chromosome 11q12.1, approximately 17.5 kb in length, encoding a giant skeletal protein of 5890 amino acids, amino acid sequence as shown in SEQ ID NO.1), a shRNA target sequence (SEQ ID NO.2) was designed, with the sequence: 5'-TTCGGAGCATGCATGTTTATA-3'.

[0055] This sequence, verified by BLAST alignment, showed no significant homology with other regions of the human genome (E value < 0.01), effectively reducing the risk of off-target effects. A negative control scramble sequence was also designed: 5'-TTCTCCGAACGTGTCACGT-3'.

[0056] The amino acid sequence of AHNAK is shown in SEQ ID NO.1.

[0057] 2. Construction of lentiviral vector plasmids

[0058] The lentiviral vector plasmid GV492 (pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin, purchased from Shanghai Jikai Gene Medical Technology Co., Ltd.) was selected. This vector contains a GFP reporter gene for easy detection of transfection efficiency and a puromycin resistance gene for stable cell selection.

[0059] Cloning: The GV492 vector and the shRNA target sequence of the target gene AHNAK were double-digested with AgeI and NheI restriction endonucleases, respectively, and incubated at 37℃ for 2 h; the target fragment was recovered by 1% agarose gel electrophoresis; ligation was completed by incubation at 50℃ for 15 min using the In-Fusion seamless cloning kit, and the resulting fragments were transformed into Stbl3 competent bacteria. Single colonies were picked and cultured; plasmids were extracted, and the sequence correctness was verified by PCR and Sanger sequencing.

[0060] 3. Lentiviral production and titer detection

[0061] Lentiviral vectors were packaged using a three-plasmid system: the constructed lentiviral vector plasmids were co-transfected with two helper packaging plasmids, psPAX2 (encoding Gag-Pol, viral structure and replicase gene) and pMD2.G (encoding VSV-G envelope protein, conferring broad host spectrum), at a mass ratio of 4:3:1 using cationic liposome transfection reagent (Lipofectamine 2000, purchased from Invitrogen) into HEK293T cells (HEK293T cells were plated and cultured to 70% confluence 24 h before transfection).

[0062] Virus harvesting and processing: Change the medium 12 h after transfection to reduce the toxicity of Lipofectamine. Collect the virus-containing supernatant at 48 h and 72 h after transfection. Centrifuge at 1000×g for 5 min to remove cell debris. Filter the virus through a 0.45μm low protein-bound cellulose acetate membrane (Millipore) for sterilization. Handle on ice, aliquot and store at -80℃ to avoid repeated freeze-thaw cycles.

[0063] Titer determination (functional titer): HEK293T cells were infected with serially diluted virus, and the proportion of GFP-positive cells was detected by flow cytometry after 48 hours to calculate the functional titer.

[0064] The results showed that the titer of the AHNAK knockdown lenvirus constructed in this embodiment was 1×10⁻⁶. 12 TU / mL, satisfying a titer ≥1×10 9 The TU / mL requirement can be used for subsequent experiments.

[0065] Example 3: Construction of Anti-fibrotic Mesenchymal Stem Cells

[0066] 1. Lentiviral transfection and stable strain screening

[0067] 1) Lamination: The P3 generation MSCs obtained in Example 1 were laid in a 1×10- ratio. 5 Cells / wells were seeded into 12-well plates and incubated at 37°C for about 24 hours until 50-60% confluence was achieved.

[0068] 2) Transfection: Remove the supernatant from the DMEM complete medium, prepare the transfection mixture according to the volume ratio of AHNAK knockdown virus: Polybrene enhancer: DMEM complete medium = 10:1:48, mix well and add 2 mL / well. Set the infection index (MOI value) to 10 to ensure that >80% of cells are transfected.

[0069] 3) Post-transfection culture: Change the medium 48 hours after transfection and observe GFP expression under a fluorescence microscope. The GFP positivity rate should be >80%.

[0070] 4) Stable strain screening: After changing the medium, add 1-2 μg / mL of puromycin and continue screening for 7-14 days to remove cells that have not integrated the virus; after screening, maintain culture in normal medium to obtain anti-fibrotic mesenchymal stem cells, denoted as AF-MSC;

[0071] 2. Efficiency Verification

[0072] Observation revealed that the AF-MSCs constructed in this embodiment showed no significant morphological abnormalities compared to the parental MSCs obtained in Example 1. Further analysis using RT-qPCR (forward primer: 5'-ATGGCCGTTGC) confirmed the presence of these abnormalities.

[0073] (AGAGAAGAT-3', reverse primer: 5'-TGGCTCTTGTCCACAGTCCT-3') to detect the expression level of AHNAK mRNA in AF-MSC cells and parental MSC cells.

[0074] The results showed that, compared with the parental MSCs, the AHNAK knockdown efficiency in the AF-MSCs constructed in this embodiment was ≥70% (see Figure 1 ).

[0075] 3. Molecular level detection

[0076] The levels of cytokines such as α-SMA, PEG2, HGF, and TSG-6 in parental MSCs and AF-MSCs were detected by ELISA.

[0077] The results showed that, compared with the parental MSCs, the expression of α-SMA in the AF-MSCs constructed in this embodiment was reduced, indicating that AF-MSCs can inhibit the transformation of fibroblasts into myofibroblasts; while the secretion of antifibrotic paracrine factors (HGF, PGE2, TSG-6) was increased, indicating that the antifibrotic effect of AF-MSCs was enhanced (see...). Figure 2 ).

[0078] Therefore, this embodiment can establish a stable AF-MSC cell bank based on stable lentiviral transfection. Combined with the puromycin screening system, it can ensure that the AHNAK knockdown efficiency of each batch of products is consistent (>70%), greatly reducing the batch-to-batch differences and meeting the requirements for large-scale preparation.

[0079] Example 4: Validation of the antifibrotic activity of AF-MSCs – In vitro experiments

[0080] 1. Establishment of lung organoid models

[0081] A lung organoid model was constructed using human alveolar type II epithelial cells (AT2 cells) and divided into a control group, a fibrosis-induced group (denoted as TGF-β group), and a treatment group (denoted as AF-MSC group).

[0082] Control group: AT2 cells were administered at a rate of 1-2 × 10⁻⁶. 4 Cells / 50μL were seeded in Matrigel drops in Advanced DMEM / F12 culture medium supplemented with B27, N2, EGF 50ng / mL, FGF10 100ng / mL, Noggin 100ng / mL, and R-spondin 1 500 ng / mL. Mature lung organoids were formed after 7-10 days of culture.

[0083] Fibrosis induction group: AT2 cells were seeded under the same conditions as the control group, with an additional 5 ng / mL of TGF-β1 added to induce fibrosis, and cultured for 7-10 days.

[0084] Treatment group: AT2 cells and AF-MSCs were mixed at a ratio of 1:3 and seeded in matrix gel at the same density. 5 ng / mL TGF-β1 was added, and the cells were cultured for 7-10 days.

[0085] 2. In vitro observation indicators

[0086] 2.1 Morphological assessment of organoids

[0087] Inverted optical microscopes were used to perform bright-field light microscopy on each group of lung organoid models to assess the diameter, number, morphological integrity, and fragmentation ratio of each group of lung organoids.

[0088] The results showed that organoid growth was slowed and debris increased after TGF-β1 induction; however, organoid growth was significantly restored and debris decreased after co-culture with AF-MSCs (see [link]). Figure 3 ).

[0089] 2.2 Histopathological evaluation

[0090] Immunofluorescence after sectioning, HE staining and Masson staining of paraffin sections were used to assess the number of alveoli, interstitial area and degree of collagen deposition in lung organoids of each group.

[0091] The results showed that TGF-β1 induction reduced the number of alveoli and increased the interstitial tissue; while co-culture with AF-MSCs restored the number of alveoli, reduced the interstitial tissue, and significantly decreased collagen deposition (see [link to article]). Figure 4 ).

[0092] In summary, this embodiment uses an in vitro model of human AT2 lung organoids to simulate the pulmonary fibrosis microenvironment for verification, demonstrating the anti-fibrotic ability of the AF-MSCs constructed in Example 3.

[0093] Example 5: Validation of the antifibrotic activity of AF-MSCs – Animal Experiments

[0094] 1. Bleomycin-induced rat pulmonary fibrosis model

[0095] 1.1 Laboratory Animals

[0096] SPF-grade male SD rats (male rats are more sensitive to bleomycin and have a more stable model), weighing 200-230g, aged 6-8 weeks; acclimatized for 1 week before the experiment, with free access to food and water, temperature 22-25℃, humidity 50%-60%, circadian rhythm 12 / 12h, in accordance with IACUC ethical guidelines.

[0097] 1.2 Modeling Method

[0098] In this embodiment, the rat model was established by intratracheal instillation. Before modeling, the rats were accurately weighed and the dosage of bleomycin was calculated. 10% chloral hydrate was injected intraperitoneally at a dose of 0.3 ml per 100 g of rat body weight, or isoflurane was administered for inhalation anesthesia. The rats were fixed supine on a 45° inclined surgical board. Ophthalmic forceps were used to retract the tongue base to expose the glottis, and a 16G lumbar puncture needle was inserted into the trachea through the oral-glottis. Bleomycin saline solution was slowly injected at a calculated dose of 5 mg / kg. After injection, 0.3 mL of sterile air was added to ensure the drug entered the lungs. After administration, the rats were upright and rotated from side to side to ensure even distribution of the drug solution. They were placed in an oxygen chamber for 1 hour to reduce mortality.

[0099] 1.3 Experimental Group Design

[0100] The experimental group design in this embodiment is shown in the table below:

[0101]

[0102] 1.4 Cell infusion procedure

[0103] Before injection, place the rat on a 37℃ warming table or immerse its tail in warm water for 2-3 minutes to allow the tail vein to fully dilate. Disinfect the tail with a 70% alcohol swab and select the lateral tail vein (the thickest veins on both sides). Using a 1 mL syringe fitted with a 30G needle, insert the needle at a 10-15° angle to the vein, bevel facing upwards. Once blood return is observed, fix the needle in place. Slowly inject the cell suspension (1×10⁻⁶). 6 Cells dissolved in 0.5-1 mL PBS, infusion rate ≤0.2 mL / min (to prevent pulmonary embolism). After injection, apply pressure with cotton balls for 1-2 min to stop bleeding. Infuse once on day 7 and once on day 14 (2 times in total); AF-MSCs are pre-labeled with CM-DiI fluorescent dye for in vivo cell homing tracking (IVIS in vivo imaging or frozen section fluorescence).

[0104] 2. Animal experimental observation indicators

[0105] 2.1 Gross pathological observation

[0106] After the infusion of rat cells into the control group, model group, and AF-MSC treatment group, the rats in each group were grossly dissected, and the changes in the size, color, and texture of the lung tissue were observed.

[0107] The results showed that, compared with the control group, the lung tissue in the model group was whiter, had reduced blood supply, and was harder; the lung tissue color, blood supply, and hardness in the AF-MSC treatment group were restored to some extent compared with the model group (see...). Figure 5 ).

[0108] 2.2 Histopathology

[0109] Lung tissues from rats in each group in step 2.1 were collected, fixed, dehydrated, embedded in paraffin, and sectioned. HE staining was used to assess inflammatory infiltration and alveolar structure; Masson staining was used to assess the degree of fibrosis.

[0110] The results showed that, compared with the control group, the model group had thicker alveolar interstitium, increased fiber staining, and smaller alveolar volume; the AF-MSC treatment group showed some recovery in alveolar interstitium thickness, fiber staining depth, and alveolar volume compared with the model group (see...). Figure 6 ).

[0111] 2.3 Protein Indicators

[0112] Western blotting was used to detect protein fibrosis markers in the lung tissue of rats in step 2.1, such as Collagen I and Fibronectin.

[0113] The results showed that the pulmonary tissue protein fibrosis index was higher in the model group compared with the control group; the pulmonary tissue protein fibrosis index in the AF-MSC treatment group recovered to some extent compared with the model group (see...). Figure 7 ).

[0114] 2.4 Mitochondrial TMRE staining

[0115] After the rat cells infusion was completed in the control group, parental MSC treatment group, and AF-MSC treatment group, they were cultured in a hypoxic incubator with conditions of 5% O2 + 5% CO2 + 90% N2 and were designated as the control group, hypoxia + MSC group, and hypoxia + AF-MSC group, respectively. After the culture was completed, the rats in each group were grossly dissected, and the lung tissue mesenchymal stem cells were stained with mitochondrial TMRE dye. The fluorescence intensity was used to determine the mitochondrial membrane potential difference and reflect mitochondrial function.

[0116] The results showed that in the hypoxia + MSC group (MSCs extracted from the lung tissue of rats treated with conventional MSC injection and cultured under hypoxia), the mitochondrial membrane potential difference of mesenchymal stem cells in the lung tissue was reduced compared to the control group; in the hypoxia + AF-MSC group (MSCs extracted from the lung tissue of rats treated with AF-MSC injection and cultured under hypoxia), the mitochondrial membrane potential difference of mesenchymal stem cells in the hypoxia + MSC group was restored to some extent compared to the hypoxia + MSC group (see...). Figure 8 This indicates that AF-MSCs can still restore mitochondrial membrane potential and improve pulmonary fibrosis under hypoxic conditions.

[0117] 2.5 Quantitative analysis of extracellular acidification rate (ECAR) in glycolysis

[0118] The glycolytic function of lung mesenchymal stem cells was detected using ECARs from glycolysis. Lung mesenchymal stem cells extracted from rats in step 2.4 were plated and adherent in buffer-free Seahorse basal medium and equilibrated at 37°C in a CO2-free incubator for at least 1 hour to ensure the accuracy of pH measurements. Drug administration sequence and detection indicators: Glucose injection: Cells utilize glucose for basal glycolysis, resulting in an increase in ECAR values. The ECAR value at this point reflects the cell's basal glycolytic capacity. Oligomycin injection: Oligomycin inhibits mitochondrial ATP synthesis, forcing cells to rely entirely on glycolysis for energy, further peaking the ECAR value. This peak reflects the cell's maximum glycolytic capacity. 2-Deoxyglucose (2-DG) injection: 2-DG is a glycolysis inhibitor that completely blocks the glycolytic pathway, significantly reducing the ECAR value to baseline. This verifies that the ECAR signal indeed originates from glycolysis. Data Interpretation: By calculating the differences between the above stages, we can also derive the glycolytic reserve (Glycolytic reserve = maximum glycolytic capacity - basal glycolytic capacity), which reflects the metabolic elasticity of cells.

[0119] The results showed that the glycolytic capacity of lung tissue mesenchymal stem cells in the hypoxia + MSC group was decreased compared with the control group; the glycolytic capacity of mesenchymal stem cells in the hypoxia + AF-MSC group was restored to some extent compared with the hypoxia + MSC group (see...). Figure 9 ).

[0120] In summary, the animal experimental results of this embodiment show that intravenous infusion of AF-MSCs significantly reduced various pulmonary fibrosis indicators, decreased the degree of pulmonary fibrosis, restored mitochondrial glycolysis function, and demonstrated good therapeutic effects.

[0121] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. The application of human mesenchymal stem cells with silenced AHNAK gene expression in the preparation of antifibrotic drugs, characterized in that, The amino acid sequence of the AHNAK gene is shown in SEQ ID NO.1, and the human mesenchymal stem cells are derived from bone marrow.

2. The application according to claim 1, characterized in that, The drug comprises any pharmaceutically acceptable dosage form made from human mesenchymal stem cells with silenced AHNAK gene expression and pharmaceutically acceptable carriers and / or excipients.

3. The application according to claim 2, characterized in that, The drug is in the form of an injection.

4. The application according to claim 3, characterized in that, When the drug is administered to rats via intravenous infusion, the number of human mesenchymal stem cells with silenced AHNAK gene expression in the drug is not less than 1 × 10⁻⁶. 6 cells.

5. The method for constructing human mesenchymal stem cells with silenced AHNAK gene expression according to claim 1 includes: Human mesenchymal stem cells were transfected with AHNAK knockdown virus, and the medium was changed after transfection. Puromycin was added and the cells were continuously screened to obtain the desired results.

6. The application according to claim 5, characterized in that, The construction method further includes: measuring AHNAK knockdown virus, Polybrene enhancer, and DMEM complete culture medium in a volume ratio of 10:1:48 to prepare a transfection mixture, adding 2 ml / well to human mesenchymal stem cells cultured in a multi-well plate for transfection, changing the medium after transfection, and adding 1-2 μg / mL of puromycin for continuous screening.

7. The application according to claim 5, characterized in that, The transfection time is no less than 48 hours, the screening time is 7 to 14 days, and the MOI value of the AHNAK knockdown virus is 10.

8. The application according to claim 5, characterized in that, The human mesenchymal stem cells are P3 generation human mesenchymal stem cells, and the human mesenchymal stem cells are expressed at a ratio of 1×10⁻⁶. 5 Cells / holes are laid in a porous plate.

9. The application according to claim 5, characterized in that, The method for constructing the AHNAK knockdown slow virus includes the following steps: S1. Design and synthesize shRNA with the sequence shown in SEQ ID NO.2 that targets AHNAK; S2. The shRNA was inserted into the GV492 vector by double enzyme digestion, transformed into competent bacteria, and the AHNAK lentiviral vector plasmid was extracted. S3. Co-transfect cells with AHNAK lentiviral vector plasmid and two helper packaging plasmids, collect virus-containing liquid, centrifuge and filter to obtain AHNAK knockdown lentivirus.

10. The application according to claim 9, characterized in that, The GV492 vector contains a GFP reporter gene and a puromycin resistance gene; the competent bacteria are Stbl3; the cells are HEK293T; and the packaging plasmids are psPAX2 and pMD2.G.

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