Application of mesenchymal stem cells overexpressing hepatocyte growth factor in the preparation of asthma drugs

Abstract

The present invention provides an application of mesenchymal stem cells overexpressing hepatocyte growth factor in the preparation of asthma drugs, and relates to the field of biotechnology. Mesenchymal stem cells overexpressing hepatocyte growth factor can alleviate the asthma symptoms of subjects, including reducing the airway resistance stimulated by methacholine, reducing the number of nucleated cells in alveolar lavage fluid, and alleviating inflammatory cell infiltration in tissues, thus enriching the treatment methods for asthma.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the use of mesenchymal stem cells overexpressing hepatocyte growth factor in preparing asthma drugs. Background Art

[0002] Asthma is a common chronic respiratory disease with high morbidity and mortality. Asthma is a heterogeneous disease characterized by varying degrees of airflow limitation, bronchial hyperresponsiveness (BHR), excessive mucus secretion, and airway inflammation, leading to airway narrowing, which causes symptoms such as wheezing, dyspnea, and chest tightness in patients. Persistent or recurrent airway inflammation can lead to changes in airway structure, such as epithelial hyperplasia and degeneration, changes in mucus-secreting cells, subepithelial fibrosis, myocyte hyperplasia, and angiogenesis. These pathological changes in the airways may cause changes in the composition, distribution, thickness, mass or volume, and / or number of structural components of the airway wall. This is believed to be the cause of the gradual and irreversible loss of lung function, known as airway remodeling. Depending on the underlying pathological changes in the airways and the corresponding physiological consequences, patients may experience mild, moderate, severe, or refractory asthma.

[0003] Inhaled corticosteroids (ICS), introduced in the 1980s, are generally effective in reducing asthma symptoms, preventing exacerbations, and improving lung function in patients with mild to moderate asthma, but their ability to change the natural history of asthma and their efficacy on disease progression remain questionable. Similarly, the combination of inhaled ICS and long-acting β2-agonists (LABAs) has further improved the treatment of asthma, but their efficacy on disease progression remains to be evaluated. At the same time, the efficacy of inhaled corticosteroids in asthma disappears rapidly after discontinuation of the drug. In addition, current asthma drugs are generally unable to reverse or slow most of the long-term remodeling changes that occur in various cell types in the airways. This may be partly due to the inability of inhaled corticosteroids to inhibit IL-33, which is thought to be a mediator that plays a role in the remodeling process.

[0004] Initially, asthma was divided into high Th2 type and low Th2 type based on T helper cells (Th), but later it was discovered that natural lymphocytes and the like can also secrete Th2 cytokines, so asthma was divided into type 2 and non-type 2 based on whether Th2 cytokines are involved. In type 2 asthma, cytokines that cause type 2 inflammatory responses play a major role, while in non-type 2 asthma, Th1 or Th17 participates in the inflammatory polarization process.

[0005] A variety of cytokines are involved in the pathological process of type 2 asthma, including interleukin (IL)-4, IL-5, IL-9 and IL-13. Among them, IL-4 stimulates the synthesis of immunoglobulin E (IgE) and activates mast cells; IL-5 is a key cytokine for the synthesis, activation, maturation and survival of eosinophils. IL-13 participates in epithelial cell-mediated inflammatory responses, is related to bronchial hyperresponsiveness, mucus secretion and nitric oxide release, and can stimulate the release of chemical factors, thereby leading to the mobilization and recruitment of eosinophils in airway inflammatory tissues and their persistence; its receptors are expressed in airway smooth muscle cells and airway epithelial cells. IL-9 is secreted by mast cells, and its content can reflect the proliferation level of activated T cells and mast cells; at the same time, IL-9 can also stimulate mucus hypersecretion and regulate the gene transcription level of airway epithelial cells through IL-13. Type 2 asthma can be further divided into three subtypes based on the dominant cytokines: high IgE type, high IL-5 type, and high IL-13 type. Non-type 2 immune responses are induced by stimulants, pollutants, or infectious factors that can activate IL-33, IL-23, CXCL8, transforming growth factor-β, and IL-6. IL-33 activates Th17 to synthesize IL-6, IL-17, and IL-8, which can cause the activation of immune responses dominated by neutrophils. Among them, increased levels of IL-17 are associated with hormone resistance and can lead to increased mucus secretion and aggravated airway obstruction. Therefore, asthma is related to multiple physiological processes, and it is necessary to explore innovative, effective, and safe treatment methods to enrich the treatment of asthma.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide the use of mesenchymal stem cells overexpressing hepatocyte growth factor in the preparation of a drug for treating and / or alleviating asthma.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] The present invention provides use of mesenchymal stem cells overexpressing hepatocyte growth factor in preparing a medicine for treating and / or alleviating asthma.

[0010] In an optional embodiment, the mesenchymal stem cells include dental pulp-derived mesenchymal stem cells.

[0011] In an optional embodiment, the hepatocyte growth factor comprises hepatocyte growth factor of human origin.

[0012] In an optional embodiment, the mesenchymal stem cells overexpressing hepatocyte growth factor include mesenchymal stem cells modified using a recombinant adenovirus, wherein the recombinant adenovirus expresses hepatocyte growth factor.

[0013] In an optional embodiment, the concentration of hepatocyte growth factor in the culture supernatant of the mesenchymal stem cells overexpressing hepatocyte growth factor is at least 100 ng / mL.

[0014] In an optional embodiment, the use for treating and / or alleviating asthma comprises reducing airway resistance in a subject.

[0015] In an optional embodiment, the method for treating and / or alleviating asthma comprises reducing the level of inflammatory cells in the lungs of the subject.

[0016] In an optional embodiment, the inflammatory cells include at least one of neutrophils, lymphocytes, macrophages and basophils.

[0017] In an optional embodiment, the method for treating and / or alleviating asthma comprises reducing lymphocyte infiltration in lung tissue.

[0018] In an optional embodiment, the lymphocytes include T cells and / or B cells.

[0019] In an optional embodiment, the T cells include CD4+T cells and / or CD8+T cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention finds that overexpression of hepatocyte growth factor in mesenchymal stem cells does not affect the distribution of mesenchymal stem cells in subjects, and administration of mesenchymal stem cells overexpressing hepatocyte growth factor to subjects can alleviate the asthma symptoms of subjects, including reducing airway resistance stimulated by methacholine, reducing the number of nucleated cells in alveolar lavage fluid, and alleviating inflammatory cell infiltration in tissues. Based on the above findings, the use of mesenchymal stem cells overexpressing hepatocyte growth factor provided by the present invention in the preparation of drugs for treating and / or alleviating asthma enriches the treatment methods for asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1The morphology of MSCs isolated in Example 1 of the present invention under a 10x microscope;

[0024] Figure 2 The morphology of hHGF-MSC prepared in Example 1 of the present invention under a 10x microscope;

[0025] Figure 3 The HGF concentration in the culture supernatant of hHGF-MSC prepared in Example 1 of the present invention, each group represents CN (untransfected with Ad-HGF), 80MOI (MSC transfected with 80MOI Ad-HGF) and 100MOI (MSC transfected with 100MOI Ad-HGF) from left to right;

[0026] Figure 4 The HGF concentration in the culture supernatant of hHGF-MSC prepared in Example 1 of the present invention, each group represents CN (untransfected with Ad-HGF) and 100 MOI (MSC transfected with 100 MOI Ad-HGF) from left to right;

[0027] Figure 5 The distribution of hHGF-MSC in mice in Example 2 of the present invention;

[0028] Figure 6 The distribution of MSCs in mice in Example 2 of the present invention;

[0029] Figure 7 is a curve showing the change of airway resistance of mice in each experimental group as the dosage of methacholine increases in Example 3 of the present invention;

[0030] Figure 8 The nucleated cell infiltration in BALF fluid of mice in each experimental group in Example 3, each group from left to right is as follows: PBS, HDM+PBS, HDM+HGF, HDM+MSC, HDM+hHGF-MSC;

[0031] Fig. 9 The following are the lymphocyte infiltration and cell types in the lung tissue of mice in each experimental group in Example 3. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In a first aspect, a use of mesenchymal stem cells overexpressing hepatocyte growth factor in the preparation of a drug for treating and / or alleviating asthma is provided.

[0034] Mesenchymal stem cells (MSC) can be selected from conventional mesenchymal stem cells in the art. The tissue sources of the mesenchymal stem cells include, but are not limited to, bone marrow, dental pulp, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue, preferably including mesenchymal stem cells derived from dental pulp. In an optional embodiment, the mesenchymal stem cells are from mammals, including but not limited to humans, pigs, cattle, horses, monkeys, rats, mice, guinea pigs, sheep, sheep or goats.

[0035] Hepatocyte growth factor (HGF) is a multifunctional cytokine, which was originally called tissue plasminogen (HGF / SF) when it was first discovered. It is produced by hepatocytes and other cell types. In addition to playing a key role in liver regeneration, repair and functional regulation, it can also regulate a variety of biological processes through its c-MET receptor activation signaling pathway. In an optional embodiment, the HGF includes human-derived HGF.

[0036] In the present invention, mesenchymal stem cells overexpressing HGF refer to mesenchymal stem cells that can express a higher level of HGF than mesenchymal stem cells without artificial intervention. Those skilled in the art can make mesenchymal stem cells overexpress HGF according to conventional techniques in the art.

[0037] In an optional embodiment, mesenchymal stem cells are made to carry a vector capable of expressing HGF. The vector refers to a nucleic acid carrier into which a polynucleotide is inserted, and the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it are expressed in the host cell. Examples of vectors include plasmids, and may also include animal viruses that can be used as vectors, including but not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In an optional embodiment, a person skilled in the art may use any gene editing system to integrate the polynucleotide encoding HGF into the genome of mesenchymal stem cells, increase the copy number of the HGF gene in the genome of mesenchymal stem cells, and overexpress HGF.

[0038] In an optional embodiment, the mesenchymal stem cells overexpressing HGF include mesenchymal stem cells modified with a recombinant adenovirus, wherein the recombinant adenovirus expresses HGF. The recombinant adenovirus carries a polynucleotide encoding HGF and is transfected into mesenchymal stem cells to overexpress HGF.

[0039] In an optional embodiment, the concentration of HGF in the culture supernatant of the mesenchymal stem cells overexpressing HGF is at least 100 ng / mL.

[0040] In an optional embodiment, the drug further comprises an optional acceptable auxiliary material in the art, including but not limited to one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, fillers, osmotic pressure regulators, stabilizers, glidants, flavoring agents, antibacterial agents, suspending agents, aromatics, antioxidants, chelating agents, pH regulators, adsorbents, surfactants, protective agents, humectants, absorbents, diluents, release regulators, hardeners, hollow capsules, matrix and drug carrier materials. The drug may also contain an acceptable amount of residues in the cell culture process, such as culture medium.

[0041] In an optional embodiment, the dosage form of the drug can be selected from any acceptable dosage form in the art, including but not limited to injection, nasal preparation, inhalation preparation, spray or oral preparation. The appropriate formulation depends on the selected route of administration, can be manufactured according to common sense known in the art, and suitable excipients can be selected.

[0042] In an optional embodiment, the dosage form of the drug is an injection, and the drug further comprises a solvent and a buffer component. In an optional embodiment, the buffer component comprises phosphate. In an optional embodiment, the injection contains PBS buffer.

[0043] In an optional embodiment, the subject of the drug includes a mammal, including but not limited to humans, pigs, cattle, horses, monkeys, rats, mice, guinea pigs, sheep, sheep or goats. The subject can be an asthma patient or an animal model obtained by artificial modeling. In an optional embodiment, the subject includes a BALB / c mouse asthma animal model caused by house dust mites.

[0044] In an optional embodiment, when the subject is a BALB / c mouse asthma animal model caused by house dust mites, the dosage of mesenchymal stem cells overexpressing HGF is 1 to 5×10 6 / cell / body, preferably 2×10 6 Pieces / cells / pieces.

[0045] In an optional embodiment, the use for treating and / or alleviating asthma comprises reducing airway resistance in a subject.

[0046] In an optional embodiment, the method for treating and / or alleviating asthma comprises reducing the level of inflammatory cells in the lungs of the subject.

[0047] In an optional embodiment, the inflammatory cells include at least one of neutrophils, lymphocytes, macrophages and basophils.

[0048] In an optional embodiment, the method for treating and / or alleviating asthma comprises reducing lymphocyte infiltration in lung tissue.

[0049] In an optional embodiment, the lymphocytes include T cells and / or B cells. T cells and B cells include cell subtypes that meet any definition of T cells and B cells in the art, for example, T cells include but are not limited to at least one of CD4+T cells, CD8+T cells and CD4-CD8-T cells. In an optional embodiment, the T cells include CD4+T cells and / or CD8+T cells.

[0050] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0051] Example 1

[0052] A method for preparing mesenchymal stem cells overexpressing hHGF.

[0053] 1. Experimental methods

[0054] 1. Experimental materials: Human dental pulp-derived mesenchymal stem cells, extracted from the pulp of surgically extracted teeth.

[0055] 2. Construction method:

[0056] (1) Isolation and purification of mesenchymal stem cells from human dental pulp:

[0057] Dental pulp was isolated by tissue block culture or enzyme digestion and cultured to the third generation. Cell surface markers and osteogenic and adipogenic differentiation of the obtained human dental pulp-derived mesenchymal stem cells were detected and confirmed to be mesenchymal stem cells.

[0058] The specific isolation and culture method is as follows: Under anesthesia, human impacted third molars or teeth that need to be extracted for orthodontics are aseptically extracted. Freshly extracted teeth are immediately placed in a centrifuge tube containing sterile PBS and antibiotics, and dental pulp stem cells are isolated and cultured within 12 hours. The pulp tissue is taken after the extracted tooth is split, repeatedly washed with PBS, cut as much as possible, placed in a solution containing 3mg / ml type I collagenase and 4mg / ml Dispase, digested in a 37℃ water bath for 0.5-1 hour, and the cells are collected through a 70μm cell sieve, centrifuged at 1000rpm for 10min, and resuspended into a single cell suspension with an appropriate amount of culture medium. The cells are inoculated in a 10cm culture dish and cultured in α-MEM culture medium (containing 10% fetal bovine serum, 2mmol / L glutamine) at 37℃ and 5% CO2, and the medium is changed every 3-5 days. The cell growth status is observed under an inverted microscope every day. After 1-2 weeks, the cloned cells are taken and digested with 0.25% trypsin for passage. The human dental pulp-derived mesenchymal stem cells isolated and cultured according to the above method were tested for cell surface markers and osteogenic and adipogenic differentiation, respectively, and proved to be mesenchymal stem cells. (See Perry BC, Zhou D, Wu X, Yang FC, Byers MA, Chu TM, Hockema JJ, Woods EJ, Goebel WS. Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods. 2008; 14(2): 149–156; Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.)

[0059] (2) In vitro modification of human dental pulp-derived mesenchymal stem cells:

[0060] 10,000 human dental pulp-derived mesenchymal stem cells were inoculated per square centimeter of culture area. After culturing for 24 hours, Ad-HGF was added at 100 MOI (the preparation method of Ad-HGF refers to the application number CN01129209.1, the invention name is "A recombinant adenovirus and its use in the treatment of myocardial ischemia" Chinese patent application, publication number CN1392254A text, and the preparation method of Ad-HGF described in the examples) to modify the human dental pulp-derived mesenchymal stem cells. After 48 hours, HGF-modified human dental pulp-derived mesenchymal stem cells were obtained. The amino acid sequence of HGF expressed by Ad-HGF is shown in SEQ ID NO.1, and the nucleotide sequence encoding it is shown in SEQ ID NO.2. The human dental pulp-derived mesenchymal stem cells and in vitro modified human dental pulp-derived mesenchymal stem cells prepared above are used in the following examples.

[0061] (II) Experimental results:

[0062] 1. Build results: Figure 1 and Figure 2 As shown, under the microscope, after adenovirus transfection, the morphology and number of mesenchymal stem cells overexpressing HGF were normal, and there was no significant change compared with those not transfected with Ad-HGF.

[0063] 2. Post-construction testing: ELISA results Figure 3 and Figure 4 The results showed that the expression level of HGF in mesenchymal stem cells after Ad-HGF transfection was significantly increased, indicating that the in vitro validation experiment was in line with experimental expectations.

[0064] In the following examples, MSC refers to the mesenchymal stem cells separated in Example 1, and hHGF-MSC or HGF-MSC refers to the mesenchymal stem cells overexpressing hHGF prepared in Example 1.

[0065] Example 2

[0066] Evaluation of the in vivo distribution of MSC and hHGF-MSC

[0067] 1. Experimental methods

[0068] 1. Cell transfer animal model:

[0069] Experimental animals: BALB / c mice, female, 6-8 weeks old.

[0070] Experimental groups: MSC and hHGF-MSC groups, both groups were treated with 2×10 6 Equal volume of PBS as control.

[0071] Adoptive transfer method: tail vein injection.

[0072] Adoptive transfer volume: 200 μl / mouse.

[0073] Adoptive transfer protocol: One injection via tail vein (day 0).

[0074] The mice were killed in the first week after adoptive transfer, and the number of MSC and hHGF-MSC colonization in the lung, liver, and kidney tissues of the mice was detected, and their distribution in the body and retention time were analyzed.

[0075] 2. Detection of MSC and hHGF-MSC colonization in mouse tissues and organs:

[0076] The number of MSC and hHGF-MSC colonization in the lung, liver and kidney tissues of mice was detected by immunofluorescence method.

[0077] (1) After the mice were euthanized, blood was collected from their orbits and the hearts were perfused with PBS.

[0078] (2) Slowly perfuse the heart with 4% paraformaldehyde for 5-10 minutes.

[0079] (3) Take the lungs, liver, and kidneys and soak them in 4% paraformaldehyde for 48 hours.

[0080] (4) Remove the lungs, liver, and kidneys and embed them in paraffin.

[0081] (5) After embedding, cut 5 μm sections with a microtome, spread the sections with sterile water, and place them on glass slides.

[0082] (6) Dewaxing and antigen retrieval.

[0083] (7) Permeabilization with 0.5% Triton X-100 at room temperature for 20 min.

[0084] (8) After permeabilization, wash the slides three times with PBS, each time for 3 min. Dry the PBS with absorbent paper, add goat serum to the slides, and block at room temperature for 30 min.

[0085] (9) Dilute the primary antibody with the primary antibody diluent according to the instructions of the primary antibody in an appropriate ratio. Absorb the blocking solution with absorbent paper, add the diluted primary antibody to each slide and place in a humidified box. Incubate overnight at 4°C. Recover the primary antibody, add PBST, and wash slowly on a shaker for 5 minutes. Wash 3 times in total.

[0086] (10) After absorbing all the washing solution with absorbent paper, add the diluted fluorescent secondary antibody dropwise and incubate in a wet box for 1 hour. Then, recover the secondary antibody and wash the plate three times with PBST, each time for 5 minutes.

[0087] (11) Add DAPI to the slide and incubate in the dark for 5 min to stain the nucleus. Then wash with PBST three times, 5 min each time, to remove excess DAPI.

[0088] (12) Use absorbent paper to absorb the liquid on the slide, seal the slide with sealing solution containing anti-fluorescence quenching agent, and then observe under a fluorescence microscope.

[0089] The antibodies used for immunofluorescence are listed in the following table.

[0090] Table 1

[0091]

[0092]

[0093] (II) Experimental results:

[0094] The experimental results are as follows Figure 5 and Figure 6 As shown, hHGF-MSC and MSC are mainly distributed in the lungs of mice and secondly in the kidneys. Overexpression of hHGF has no adverse effect on the distribution of MSC in vivo, and more MSCs are accumulated in the lungs.

[0095] Example 3

[0096] Evaluation of the protective effects of MSC and hHGF-MSC on asthma model mice:

[0097] 1. Experimental methods

[0098] 1. Cell transfer and asthma animal models:

[0099] Experimental animals: BALB / c mice, female, 6-8 weeks old.

[0100] Groups: PBS, HDM+PBS, HDM+MSC, HDM+hHGF-MSC and HDM+hHGF. MSC / hHGF-MSC, 2×10 6 10 ng / cell / mouse. HGF, 10 ng / mouse. HDM (house dust mite), 50 μg / mouse. The details are shown in the following table.

[0101] Table 2

[0102]

[0103] Administration: MSC, hHGF-MSC and HGF were injected into tail vein; HDM was injected into nose.

[0104] Administration volume: MSC, hHGF-MSC and HGF, 200 μl / mouse; HDM, 20 μl / mouse.

[0105] Dosage regimen: MSC, hHGF-MSC and HGF were injected into the tail vein once (day 15).

[0106] The mice were sensitized once with HDM nasal drops on day 0 and day 7, and challenged once with HDM nasal drops on days 14, 16, 18, 20, 22, 24, and 26. Lung function test was performed after anesthesia on day 27.

[0107] 2. Mouse lung function test

[0108] (1) Mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital.

[0109] (2) Place the mouse in supine position, glue its front legs to the surgical board, and fix the maxillary incisor with a rubber band.

[0110] (3) A high-intensity fiber optic flexible illuminator is placed 1 cm away from the ventral side of the sternum, and the fiber optic cable is illuminated through the trachea to facilitate the distinction between the esophageal cavity and the tracheal cavity.

[0111] (4) Use a serrated blunt-nosed (20° angle) stainless steel steep forceps to pull the mouse tongue outward to the side of the mouth to allow observation of the epiglottis and larynx. The tongue remains in this position throughout the surgery.

[0112] (5) Insert the catheter into the trachea to the position before the tracheal bifurcation.

[0113] (6) Use a syringe to inject 10 μL of 0 mg / ml methacholine through the catheter.

[0114] (7) Test 12 breathing cycles and check airway resistance.

[0115] (8) Use a syringe to inject 10 μL of 6 mg / ml methacholine through the catheter.

[0116] (9) Test 12 breathing cycles and check airway resistance.

[0117] (10) Use a syringe to inject 10 μL of 12 mg / ml methacholine through the catheter.

[0118] (11) Test 12 breathing cycles and check airway resistance.

[0119] (12) Use a syringe to inject 10 μL of 24 mg / ml methacholine through the catheter.

[0120] (13) Test 12 breathing cycles and check airway resistance.

[0121] (14) Use a syringe to inject 10 μL of 48 mg / ml methacholine through the catheter.

[0122] (15) Test 12 breathing cycles and check airway resistance.

[0123] 3. Infiltration of nucleated cells in mouse BALF fluid (alveolar lavage fluid):

[0124] (1) Lavage the lungs with 0.8 ml PBS through the endotracheal tube 3-5 times.

[0125] (2) The recovered lavage fluid was centrifuged at 1500 r / min at 4°C for 10 min.

[0126] (3) Resuspend the cell pellet in 200 μl of PBS containing 1% BSA and take 10 μl of the resuspended solution for cell counting.

[0127] 4. Inflammatory cell infiltration and cell types in mouse lung tissue

[0128] Inflammatory cell infiltration and cell types in mouse lung tissue were detected by IHC.

[0129] (1) After the mice were euthanized, blood was collected from their orbits and the hearts were perfused with PBS.

[0130] (2) Slowly perfuse the heart with 4% paraformaldehyde for 5-10 minutes.

[0131] (3) Soak the lungs in 4% paraformaldehyde for 48 hours.

[0132] (4) Remove the lungs and embed them in paraffin.

[0133] (5) After embedding, cut 5 μm sections with a microtome, spread the sections with sterile water, and place them on glass slides.

[0134] (6) Dewaxing and antigen retrieval.

[0135] (7) Permeabilization with 0.5% Triton X-100 at room temperature for 20 min.

[0136] (8) After permeabilization, wash the slides three times with PBS, each time for 3 min. Dry the PBS with absorbent paper, add goat serum to the slides, and block at room temperature for 30 min.

[0137] (9) Dilute the primary antibody with the primary antibody diluent according to the instructions of the primary antibody in an appropriate ratio. Absorb the blocking solution with absorbent paper, add the diluted primary antibody to each slide and place in a humidified box. Incubate overnight at 4°C. Recover the primary antibody, add PBST, and wash slowly on a shaker for 5 minutes. Wash 3 times in total.

[0138] (10) After absorbing all the washing solution with absorbent paper, add the diluted secondary antibody dropwise and incubate in a wet box for 1 hour. Then, recover the secondary antibody and wash the plate three times with PBST, each time for 5 minutes.

[0139] (11) Add one drop or 50 μL of streptavidin-peroxidase solution (Secondary Antibody Kit D) to each slide, incubate at room temperature for 10 minutes, wash with PBS for three minutes × 3 times, and wash with distilled water for three minutes.

[0140] (12) Add two drops or 100 μl of DAB solution to each slide and observe under a microscope for 3-10 minutes. Stop staining when staining is appropriate and rinse with tap water.

[0141] (13) Counterstain with hematoxylin for 1-2 minutes until the cell nuclei turn blue. Rinse with tap water or PBS to reverse the blueing (0.5% ammonia solution can be used to reverse the blueing).

[0142] (14) Differentiate with 1% hydrochloric acid alcohol for 3 seconds, rinse with tap water for 3 minutes, and after the slide is dried, add an appropriate amount of neutral gum to seal the slide, cover with a cover glass, and be careful of bubbles. Let it dry for half a day. Take pictures under a microscope.

[0143] (II) Experimental results:

[0144] The results of mouse lung function test were as follows Figure 7 As shown, the results showed that the single administration of HDM significantly increased the airway resistance after acetylcholine stimulation, indicating that the model was successfully established; the separate administration of HGF and MSC improved the airway resistance of mice, but the improvement effect was not as good as that of hHGF-MSC, indicating that the delivery of MSC enhanced the improvement effect of HGF on the airway resistance of mice.

[0145] The infiltration of nucleated cells in mouse BALF fluid is as follows Figure 8 As shown, the results showed that HDM significantly increased the number of neutrophils, lymphocytes, macrophages and basophils in the alveolar lavage fluid of mice, and HGF and MSC could reduce the number of nucleated cells in the alveolar lavage fluid, but both were inferior to the ability of hHGF-MSC to reduce the number of nucleated cells in the alveolar lavage fluid, especially for neutrophils and lymphocytes.

[0146] Lymphocyte infiltration and cell types in mouse lung tissue Fig. 9 As shown, the results showed that the administration of hHGF-MSC can reduce CD3+ (total T cells), CD19+ (B cells), CD4+ (CD4 T cells) and CD8+ (CD8 T) lymphocytes in the lung tissue of mice. In summary, the above experimental results show that the administration of hHGF-MSC to mice can alleviate the asthma symptoms of mice.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of mesenchymal stem cells overexpressing hepatocyte growth factor in the preparation of a drug for treating and / or alleviating asthma, wherein the asthma is asthma caused by house dust mites, the mesenchymal stem cells are dental pulp-derived mesenchymal stem cells, and the hepatocyte growth factor is human-derived hepatocyte growth factor.

2. The use according to claim 1, characterized in that: The hepatocyte growth factor-overexpressing mesenchymal stem cells include mesenchymal stem cells modified by a recombinant adenovirus, wherein the recombinant adenovirus expresses the hepatocyte growth factor.

3. The use according to claim 1, characterized in that: In the culture supernatant of the mesenchymal stem cells overexpressing hepatocyte growth factor, the concentration of hepatocyte growth factor is at least 100 ng / mL.

4. The use according to any one of claims 1 to 3, characterized in that: The use for treating and / or alleviating asthma includes reducing the airway resistance of the subject.

5. The use according to any one of claims 1 to 3, characterized in that: The method for treating and / or alleviating asthma comprises reducing the level of inflammatory cells in the lungs of the subject.

6. The use according to claim 5, characterized in that: The inflammatory cells include at least one of neutrophils, lymphocytes, macrophages and basophils.

7. The use according to any one of claims 1 to 3, characterized in that: The use for treating and / or alleviating asthma includes reducing lymphocyte infiltration in lung tissue.

8. The use according to claim 7, characterized in that: The lymphocytes include T cells and / or B cells.

9. The use according to claim 8, characterized in that The T cells include CD4+ T cells and / or CD8+ T cells.

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