Mesenchymal stem cell culture and detection method for treating respiratory system diseases and therapeutic effect

Abstract

The invention provides a mesenchymal stem cell culture and detection method for treating respiratory system diseases and a treatment effect, and belongs to the technical field of biological medicines. The invention provides a mesenchymal stem cell culture medium with a good therapeutic effect on respiratory system diseases, a DMEM / F12 culture medium is used as a basic culture medium, the culture medium further comprises FBS, bFGF and glutamine, and hydrogen and sulfobutyl-beta-cyclodextrin (SBE-beta-CD) can also be added into a re-optimization system. The culture medium provided by the invention can be used for carrying out culture and passage on mesenchymal stem cells, and the cells obtained after culture and passage have adipogenesis and osteogenesis differentiation capacities and can express and secrete HGF, sTNFR1 and KGF. The mesenchymal stem cells prepared by the method have a good treatment effect on respiratory system diseases, so that the mesenchymal stem cells prepared by the method can be used for preparing injections and medicines with a treatment effect on the respiratory system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a mesenchymal stem cell culture and detection method for treating respiratory diseases and the therapeutic effects thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with the potential for self-renewal and multidirectional differentiation. They are found in a variety of tissues, such as bone marrow, fat, umbilical cord, and placenta. They exhibit adherent growth in vitro, similar in morphology to fibroblasts, and possess the ability to differentiate into various interstitial tissues, including bone, cartilage, and fat. They possess anti-inflammatory, immunomodulatory, and tissue repair properties, along with low immunogenicity, and are widely used clinically, particularly in respiratory diseases.

[0003] The production process and quality inspection of MSCs still restrict their drug development. Because they need to be separated from tissues such as the umbilical cord, they may encounter problems such as low separation efficiency, slow growth, easy aging or differentiation during the separation and culture process. Therefore, their culture conditions and preparation process need to be optimized.

[0004] Basic fibroblast growth factor (bFGF) can promote the growth, proliferation and maintenance of stem cell stemness; recent research results show that hydrogen can prolong the replication life of cells in vitro and protect the cells' antioxidant capacity, while sulfobutyl-β-cyclodextrin (SBE-β-CD) can regulate cell membrane signal transduction and cell membrane fluidity and permeability, and has the potential to increase factor secretion levels; the combined use of the above factors and H2 can optimize the proliferation and differentiation ability of MSCs cells, maintain their stemness and multipotency, and at the same time promote the cell's secretion level, allowing them to maintain stem cell characteristics while proliferating.

[0005] Chinese patent CN106177918A discloses a mesenchymal stem cell injection, its preparation method and application. The mesenchymal stem cell injection comprises mesenchymal stem cells, human serum albumin, low molecular weight heparin calcium, vitamin C, lentinan, dimethyl sulfoxide and a solvent. Each 100 mL of the mesenchymal stem cell injection comprises: mesenchymal stem cells (0.5-1) × 10 8 The invention comprises the following ingredients: 1-5 g human serum albumin, 0.5 g low molecular weight heparin calcium, 0.3-0.7 g vitamin C, 0.2-0.6 g lentinan and 5-9 mL dimethyl sulfoxide; the mesenchymal stem cell injection provided by the invention can effectively maintain the vitality of stem cells and increase the dopamine content in the striatum.

[0006] Currently, there are few studies on mesenchymal stem cell injections, and there is an urgent need to provide a mesenchymal stem cell injection that has better effects in treating respiratory diseases. Summary of the Invention

[0007] The present invention provides a mesenchymal stem cell culture and detection method for treating respiratory diseases and a therapeutic effect thereof. The prepared mesenchymal stem cells have a good therapeutic effect on respiratory diseases.

[0008] The invention provides a culture medium for mesenchymal stem cells with the function of treating respiratory diseases. The culture medium uses DMEM / F12 culture medium as a basic culture medium and further comprises 7.5-10% v / v FBS, 2.5-5 μg / L bFGF and 2-5 μg / L glutamine.

[0009] In a preferred embodiment of the present invention, the culture medium further comprises hydrogen and sulfobutyl-β-cyclodextrin (SBE-β-CD), the amount of hydrogen is 0.5-1.0 μg / ml, and the amount of SBE-β-CD is 0.1-0.3 mmol / L.

[0010] The present invention also provides a method for preparing mesenchymal stem cells having therapeutic effects on respiratory diseases, comprising the following steps: placing tissue containing mesenchymal stem cells in the above-mentioned culture medium for culturing, and digesting the tissue after the cell confluence reaches 70-90%, thereby obtaining P0 mesenchymal stem cells;

[0011] The P0 generation mesenchymal stem cells were collected after centrifugation, and subcultured twice using the above culture medium to the P2 generation and frozen to obtain mesenchymal stem cell seed bank cells;

[0012] After the mesenchymal stem cell seed bank cells are revived, they are serially passaged using the above-mentioned medium containing hydrogen and SBE-β-CD, cultured to the P5 generation and then frozen to obtain mesenchymal stem cells with therapeutic effects on respiratory diseases.

[0013] In a preferred embodiment of the present invention, the types of mesenchymal stem cells include any one of the following: adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells.

[0014] In a preferred embodiment of the present invention, the culture temperature is 38-39° C., the CO 2 concentration is 4-6%, and the O 2 concentration is 2-20%.

[0015] In a preferred embodiment of the present invention, the temperature of the subculture and the continuous subculture is 36-37° C., the CO 2 concentration is 4-6%, and the O 2 concentration is 2-20%.

[0016] The present invention also provides an injection of mesenchymal stem cells having a therapeutic effect on respiratory diseases, comprising the mesenchymal stem cells having a therapeutic effect on respiratory diseases prepared by the above preparation method and a cell preservation solution;

[0017] The cell preservation solution includes 5-10 g / L human serum albumin, 60-70 IU / ml heparin, 0.5-1 mmol / L potassium chloride, 2.5-5 mmol / L sodium lactate, 2-5 mmol / L sodium gluconate and 5-10 mmol / L sodium chloride.

[0018] The present invention also provides the use of mesenchymal stem cells with respiratory disease therapeutic effects prepared by the above preparation method in the preparation of drugs for treating respiratory diseases.

[0019] The present invention also provides a stem cell medicine for treating respiratory diseases, comprising mesenchymal stem cells with respiratory disease therapeutic effects prepared by the above preparation method and pharmaceutically acceptable excipients.

[0020] The present invention also provides a method for detecting the biological efficacy of mesenchymal stem cells in treating respiratory diseases, comprising detecting the ability of mesenchymal stem cells to secrete HGF, KGF and / or TNFR1;

[0021] Every 1×10 6 When the HGF secretion of mesenchymal stem cells is 100-300 ng at 48 hours, the cell activity is considered to be high.

[0022] Every 1×10 6 When the secretion of KGF by mesenchymal stem cells is 1000-2000 pg within 48 hours, the cell activity is considered to be high.

[0023] Every 1×10 6 When the secretion of TNFR1 by mesenchymal stem cells is 250-800 pg at 48 hours, the cell activity is considered to be high.

[0024] The sources of the mesenchymal stem cells include any one of the following: mesenchymal stem cells with respiratory disease therapeutic effects prepared by the above preparation method, the above injection solution, and the above stem cell medicine.

[0025] Beneficial Effects: The present invention provides a mesenchymal stem cell culture medium with a promising therapeutic effect on respiratory diseases. The culture medium comprises DMEM / F12 medium as a basal medium and also includes 8-20% v / v FBS, 3-12 μg / L bFGF, and 0.6-4.2 μg / L glutamine. The culture medium can be used to culture and passage mesenchymal stem cells. The cells obtained after culture and passage exhibit adipogenic and osteogenic differentiation capabilities and can express and secrete HGF, sTNFR1, and KGF.

[0026] The mesenchymal stem cells prepared by the present invention have a good therapeutic effect on respiratory diseases. For acute lung injury combined with acute respiratory distress syndrome, mesenchymal stem cells can improve the survival rate, increase arterial oxygen partial pressure and blood oxygen saturation, reduce carbon dioxide partial pressure, improve lung ventilation function, and inhibit the release of inflammatory factors; for pulmonary fibrosis, mesenchymal stem cells can reduce lung weight, lung index and mortality, and reduce the content of hydroxyproline and inflammatory factors, reducing the degree of fibrosis; for chronic obstructive pulmonary disease, mesenchymal stem cells can increase body weight, increase FEV2 and FEV2 / FVC, improve lung function, and reduce lung inflammation. Therefore, the mesenchymal stem cells prepared by the present invention can be used to prepare injections and medicines with therapeutic effects on respiratory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The cell morphology of the mesenchymal stem cell working bank cells;

[0028] Figure 2 This is the result of mesenchymal stem cell proliferation;

[0029] Figure 3 This is the result of mesenchymal stem cell differentiation;

[0030] Figure 4 is the survival curve of rats in each group;

[0031] Figure 5 Figure 2 is the result of blood oxygen partial pressure, compared with the model control group: *P<0.05;

[0032] Figure 6 The results of partial pressure of carbon dioxide are shown in the figure. Compared with the model control group: *P<0.05, **P<0.01;

[0033] Figure 7 Figure 2 is the blood oxygen saturation result. Compared with the sham operation group, ##P<0.01; compared with the model control group: **P<0.01, ***P<0.001;

[0034] Figure 8The effect of hUC-MSC on IL-6 in LPS-induced acute lung injury and multiple organ failure, compared with the sham operation group, ###P<0.001; compared with the model control group, ***P<0.001;

[0035] Figure 9 Effects of hUC-MSC on TNF-α in acute lung injury and multiple organ failure induced by LPS, compared with the sham operation group, ###P<0.001; compared with the model control group, ***P<0.001;

[0036] Figure 10 is the effect of animal body weight during the experimental period (± sd);

[0037] Figure 11 The experimental endpoint is the rat lung weight (± sd), compared with the sham operation group, ###P < 0.001; compared with the model control group, ***P < 0.001;

[0038] Figure 12 The organ coefficients at the experimental endpoint (± sd) were compared with the sham operation group, ###P < 0.001; compared with the model control group, **P < 0.01, ***P < 0.001;

[0039] Figure 13 is the rat survival curve;

[0040] Figure 14 The hydroxyproline content in the lung tissue of rats at the experimental endpoint (± sd) was compared with the sham operation group, ###P < 0.001; compared with the model control group, *P < 0.05, **P < 0.01;

[0041] Figure 15 The content of IL-1β in the lung lavage fluid of rats at the experimental endpoint (± sd) was compared with the sham operation group, ###P < 0.001; compared with the model control group, ***P < 0.001;

[0042] Figure 16 The TGF-β1 content in the lung lavage fluid of rats at the experimental endpoint (± sd) was compared with the sham operation group, ###P < 0.001; compared with the model control group, ***P < 0.001;

[0043] Figure 17 The Ashcroft score of the rat lung tissue pathological section at the end of the experiment was compared with the sham operation group, ###P<0.001; compared with the model control group, *P<0.05, **P<0.01, ***P<0.001;

[0044] Figure 18The effect of hUC-MSCs treatment on the body weight of mice (x±SD), compared with the sham operation group, ##P<0.01; compared with the model control group, **P<0.01;

[0045] Figure 19 Effects of hUC-MSCs treatment on FEV2 (x±SD), compared with the sham operation group, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01;

[0046] Figure 20 The effect of hUC-MSCs treatment on FEV2 / FVC% (x±SD), compared with the sham operation group, ###P<0.001; compared with the model control group, **P<0.01, ***P<0.001;

[0047] Figure 21 Effects of hUC-MSCs treatment on lung lavage fluid cells in mice (x±SD), compared with model control, **P<0.01, ***P<0.001;

[0048] Figure 22 The effect of hUC-MSCs treatment on lung inflammation in mice (x±SD), compared with the sham operation group, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01;

[0049] Figure 23 The effect of hUC-MSCs treatment on the alveolar lining interval index of mice x±SD, compared with the sham operation group, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01;

[0050] Figure 24 The secretion of HGF, KGF, and TNFR1 by hUC-MSCs after siRNA treatment;

[0051] Figure 25 is the inhibition level of HGF, KGF, and TNFR1 expression in hUC-MSCs after siRNA treatment;

[0052] Figure 26 The effect of hUC-MSCs treatment on rat body weight is x±SD;

[0053] Figure 27 is the mortality and survival curve of rats;

[0054] Figure 28 The rat lung weight (± sd) and organ coefficient (± sd) were the experimental endpoints. Compared with the sham operation group, ###P < 0.001; compared with the model control group, ***P < 0.001; compared with the hUC-MSC treatment group, $$P < 0.01;

[0055] Figure 29 The content of hydroxyproline in the lung tissue of rats (± sd) is the experimental endpoint. Compared with the sham operation group, ###P < 0.001; compared with the model control group, ***P < 0.001; compared with the hUC-MSC treatment group, $$P < 0.01;

[0056] Figure 30 The levels of TNF-α and IL-1β in the lung lavage fluid of rats at the end point of the experiment (±sd) were compared with the sham operation group, ##P<0.01; compared with the model control group, ***P<0.001;

[0057] Figure 31 The pathological section scores of rat lung tissue were compared with the sham operation group, ###P<0.001; compared with the model control group, ***P<0.001; compared with the hUC-MSC treatment group, $$P<0.01. DETAILED DESCRIPTION

[0058] The invention provides a culture medium for mesenchymal stem cells with the function of treating respiratory diseases. The culture medium uses DMEM / F12 culture medium as a basic culture medium and further comprises 7.5-10% v / v FBS, 2.5-5 μg / L bFGF and 2-5 μg / L glutamine.

[0059] The culture medium of the present invention uses DMEM / F12 culture medium as the basal culture medium, and there is no special limitation on the source of the basal culture medium. Conventional commercially available DMEM / F12 culture medium in the art can be used.

[0060] The culture medium of the present invention contains FBS, and the content of the FBS is 7.5-10% v / v, which can be 7.5% v / v, 8% v / v, 8.5% v / v, 9% v / v, 9.5% v / v, or 10% v / v. In one embodiment, 10% v / v is used as an example for illustration, but this alone cannot be considered the entire scope of protection of the present invention. In the present invention, the content can be a listed number or a decimal value between any two values.

[0061] The culture medium of the present invention contains bFGF, and the concentration of bFGF is 2.5 to 5 μg / L, such as 2.5 μg / L, 3 μg / L, 3.5 μg / L, 4 μg / L, 4.5 μg / L, or 5 μg / L. In one embodiment, 5 μg / L is used as an example for illustration, but this should not be considered the only concentration to cover the entire scope of the present invention.

[0062] The culture medium of the present invention contains glutamine at a concentration of 2 to 5 μg / L, such as 2 μg / L, 3 μg / L, 4 μg / L, or 5 μg / L. In one embodiment, 4 μg / L is used as an example for illustration, but this alone cannot be considered the entire scope of protection of the present invention.

[0063] The culture medium of the present invention may further comprise SBE-β-CD and hydrogen, wherein the concentration of SBE-β-CD is 0.1 to 0.3 mmol / L, such as 0.1 mmol / L, 0.2 mmol / L, or 0.3 mmol / L; the concentration of hydrogen is 0.5 to 1.0 μg / mL, such as 0.5 μg / L, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, or 1.0 μg / mL. In one embodiment, 0.2 mmol / L of SBE-β-CD and 1.0 μg / mL of hydrogen are used as an example for illustration, but this should not be considered the only example to constitute the entire scope of protection of the present invention.

[0064] In the culture medium of the present invention, FBS can provide basic nutrients for the growth and proliferation of mesenchymal stem cells; bFGF can bind to specific receptors on the cell surface to promote proliferation and maintain stemness; glutamine is one of the important energy substances in cells and plays a key role in the energy metabolism of cells; SBE-β-CD is a cyclodextrin derivative with good water solubility and strong solubilization ability, which can affect the fluidity and permeability of cell membranes; hydrogen can neutralize hydroxyl free radicals and regulate cell signaling pathways, has antioxidant and anti-apoptotic effects, and can reduce the damage of oxidative stress to cells; the combination of the above factors and hydrogen can synergistically enhance cell proliferation efficiency, maintain stemness, and promote factor secretion, thereby improving the ability to treat respiratory diseases.

[0065] The present invention also provides a method for preparing mesenchymal stem cells having therapeutic effects on respiratory diseases, comprising the following steps: placing tissue containing mesenchymal stem cells in the above-mentioned culture medium for culturing, and digesting the tissue after the cell confluence reaches 70-90%, thereby obtaining P0 mesenchymal stem cells;

[0066] The P0 generation mesenchymal stem cells were collected after centrifugation, and subcultured twice using the above culture medium to the P2 generation and frozen to obtain mesenchymal stem cell seed bank cells;

[0067] After the mesenchymal stem cell seed bank cells are revived, they are serially passaged using the above-mentioned medium containing hydrogen and SBE-β-CD, cultured to the P5 generation and then frozen to obtain mesenchymal stem cells with therapeutic effects on respiratory diseases.

[0068] The types of mesenchymal stem cells that can be cultured using the methods of the present invention include any of the following: adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells. The tissue containing these mesenchymal stem cells can be adipose tissue, umbilical cord, placenta, umbilical cord blood, bone marrow, or dental pulp. In one embodiment, umbilical cord-derived mesenchymal stem cells were cultured as an example.

[0069] The present invention minces the tissue into blocks and then plates them into culture bottles. For example, in the embodiment, each umbilical cord tissue block is plated into a T75 culture bottle and 15 ml of the culture medium is added for culture. The culture temperature is 38-39°C, the CO2 concentration is 4-6%, and the O2 concentration is 2-20%. For example, in one embodiment, the culture is carried out at 38°C, 5% CO2, and 20% O2. In one embodiment, the culture is carried out at 38°C, 5% CO2, and 5% O2. During the culture period, the culture medium is replaced every 3-5 days. After the cell confluence reaches 80%, the cells are digested with trypsin to obtain P0 mesenchymal stem cells.

[0070] The present invention is to centrifuge the P0 generation mesenchymal stem cells at low speed, discard the supernatant, add the culture medium, and perform two subcultures. The cells can be passed to the P2 generation and frozen to obtain mesenchymal stem cell seed bank cells. The centrifugal force of the low-speed centrifugation of the present invention is 300-400g, such as 300g, 310g, 320g, 330g, 340g, 350g, 360g, 370g, 380g, 390g or 400g, and the centrifugation time is 5 minutes. The subculture temperature from the P0 generation to the P1 generation of the present invention is 36-37°C, the CO2 concentration is 4-6%, and the O2 concentration is 2-20%. In one embodiment, the subculture is carried out at 37°C, 5% CO2 and 20% O2, and the subculture is carried out at 37°C, 5% CO2 and 5% O2 in a culture medium, and the subculture time is 72 hours.

[0071] The present invention revives the mesenchymal stem cell seed bank cells, performs continuous passage using the above-mentioned medium containing hydrogen and SBE-β-CD, cultures them to the P5 generation and then freezes them to obtain mesenchymal stem cells with therapeutic effects on respiratory diseases. The continuous passage temperature is 36-37°C, the CO2 concentration is 4-6%, and the passage time for each generation is 72 hours.

[0072] The present invention also provides an injection of mesenchymal stem cells having a therapeutic effect on respiratory diseases, comprising the mesenchymal stem cells having a therapeutic effect on respiratory diseases prepared by the above preparation method and a cell preservation solution;

[0073] The cell preservation solution includes 5-10 g / L human serum albumin, 60-70 IU / ml heparin, 0.5-1 mmol / L potassium chloride, 2.5-5 mmol / L sodium lactate, 2-5 mmol / L sodium gluconate and 5-10 mmol / L sodium chloride.

[0074] The cell preservation solution of the present invention contains 5-10 g / L human serum albumin, such as 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L.

[0075] The cell preservation solution of the present invention is a compound electrolyte injection containing 60-70 IU / ml of heparin, such as 60 IU / ml, 61 IU / ml, 62 IU / ml, 63 IU / ml, 64 IU / ml, 65 IU / ml, 66 IU / ml, 67 IU / ml, 68 IU / ml, 69 IU / ml, and 70 IU / ml.

[0076] The cell preservation solution of the present invention contains 0.5-1 mmol / L potassium chloride, such as 0.5 mmol / L, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L or 1 mmol / L.

[0077] The cell preservation solution of the present invention contains 2.5-5 mmol / L sodium lactate, such as 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L or 5 mmol / L.

[0078] The cell preservation solution of the present invention contains 2-5 mmol / L sodium gluconate, such as 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L or 5 mmol / L.

[0079] The cell preservation solution of the present invention contains 5-10 mmol / L sodium chloride, such as 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L or 10 mmol / L.

[0080] In the cell preservation solution of the present invention, human serum albumin provides cells with necessary nutrients and stabilizes the structure and function of cell membranes; the addition of heparin can prevent cells from aggregating; sodium gluconate can provide an energy source for cell metabolism; and other injection solution components can regulate cell signal transduction and maintain acid-base balance and cell osmotic pressure balance.

[0081] The present invention also provides the use of mesenchymal stem cells with respiratory disease therapeutic effects prepared by the above preparation method in the preparation of drugs for treating respiratory diseases.

[0082] The present invention also provides a method for preparing the above-mentioned injection, comprising the following steps: culturing the mesenchymal stem cells obtained by the above-mentioned method to 80% cell confluence and then digesting them, collecting the cells by centrifugation, washing them with cell washing solution, and resuspending them with cell preservation solution to prepare the injection of the mesenchymal stem cells.

[0083] In one embodiment of the present invention, the injection solution is prepared using frozen cells. First, the frozen cells are revived. There is no special limitation on the method for resuscitating the cells. The mesenchymal stem cells can be revived using conventional methods in the art. The present invention uses the cell washing solution (a cell preservation solution that does not contain human albumin and heparin) to wash the cells, such as washing 3 times. The cell washing solution of the present invention comprises: 0.5-1 mmol / L potassium chloride, 2.5-5 mmol / L sodium lactate, 2-5 mmol / L sodium gluconate and 5-10 mmol / L sodium chloride.

[0084] The present invention also provides a stem cell medicine for treating respiratory diseases, comprising mesenchymal stem cells with respiratory disease therapeutic effects prepared by the above preparation method and pharmaceutically acceptable excipients.

[0085] The respiratory diseases described in the present invention include acute lung injury, acute respiratory distress syndrome, pulmonary fibrosis or chronic obstructive pulmonary disease.

[0086] In a preferred embodiment of the present invention, the dosage form of the drug includes any of the following: drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, solutions, pills, pills, buccal tablets, freeze-dried powder injections, gels, suppositories, and aerosols. The present invention does not specifically limit the content of the excipients, and the excipients corresponding to the drug dosage form in the art can be used. The present invention requires that the number of mesenchymal stem cells in the drug reaches (1 to 10) × 10 6 / mL or (1~10)×10 6 / g.

[0087] The present invention also provides a method for detecting the biological efficacy of mesenchymal stem cells in treating respiratory diseases, comprising detecting the ability of mesenchymal stem cells to secrete HGF, KGF and / or TNFR1;

[0088] The sources of the mesenchymal stem cells include any one of the following: mesenchymal stem cells with respiratory disease therapeutic effects prepared by the above preparation method, the above injection solution, and the above stem cell medicine.

[0089] Before using the mesenchymal stem cells or mesenchymal stem cell products of the present invention, especially before using them for the treatment of respiratory diseases, it is necessary to test the biological efficacy of the mesenchymal stem cells in treating respiratory diseases. The judgment criteria for the biological efficacy of the mesenchymal stem cells in treating respiratory diseases of the present invention include at least one of the following: 6 When the HGF secretion of 100-300 ng per mesenchymal stem cell in 48 hours is considered to be of high activity; 6 When the secretion of KGF by mesenchymal stem cells is (1000-2000) pg within 48 hours, the cell activity is considered to be high; 6 When the secretion level of TNFR1 by mesenchymal stem cells is (250-800) pg within 48 hours, the cell activity is considered to be high. The present invention does not specifically limit the detection method of the above three proteins. For example, an ELISA kit is used for detection in the examples.

[0090] It is worth noting that the exemplary data given in the specification of the present invention are only partial examples, and of course they can also be numbers in the form of integers between any two numbers and numbers in the form of non-integer numbers such as decimals or fractions.

[0091] To further illustrate the present invention, the mesenchymal stem cell culture and detection methods for treating respiratory diseases and the therapeutic effects provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0092] In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each example are the same.

[0093] Mesenchymal stem cell medium-1: DMEM / F12 medium containing 10% v / v FBS, 5 μg / L bFGF, and 4 μg / L glutamine;

[0094] Mesenchymal stem cell medium-2: DMEM / F12 culture medium containing 10% v / v FBS, 5 μg / L bFGF, 4 μg / L glutamine, and 0.2 mmol / L SBE-β-CD.

[0095] Mesenchymal stem cell medium-3: DMEM / F12 culture medium containing 10% v / v FBS, 5 μg / L bFGF, 4 μg / L glutamine, and 1.0 μg / ml hydrogen.

[0096] Mesenchymal stem cell medium-4: DMEM / F12 culture medium containing 10% v / v FBS, 5 μg / L bFGF, 4 μg / L glutamine, 1.0 μg / ml hydrogen, and 0.2 mmol / L SBE-β-CD.

[0097] Example 1

[0098] 1. Plate each umbilical cord tissue fragment into a T75 culture flask and add 15 ml of Mesenchymal Stem Cell Medium-1. Incubate the flask at 38°C in a 5% CO2, 20% O2 incubator. Change the culture medium every 3-5 days. When the cell confluence reaches 80%, digest the flask with trypsin to obtain P0 human umbilical cord mesenchymal stem cells.

[0099] 2. The P0 human umbilical cord mesenchymal stem cells obtained in step 1 were centrifuged at 300-400 g for 5 min, the supernatant was discarded, and mesenchymal stem cell culture medium-1 was added. The cells were cultured for two generations to P2 at 37°C in an incubator with a 5% CO2 and 20% O2 environment, and then frozen to obtain a mesenchymal stem cell seed bank.

[0100] 3. The human umbilical cord mesenchymal stem cell seed bank cells obtained in step 2 were further cultured in mesenchymal stem cell medium-1 to passage P5 and frozen to obtain mesenchymal stem cell working bank cells (denoted as W1). The culture conditions were: mesenchymal stem cell medium-1, 37°C, 5% CO2.

[0101] Example 2

[0102] The only difference from Example 1 is that the culture medium used for passage of the mesenchymal stem cell seed bank cells to P5 (step 3) is mesenchymal stem cell medium-2, and all other aspects are the same to obtain mesenchymal stem cell working bank cells (denoted as W2).

[0103] Example 3

[0104] The only difference from Example 1 is that the culture medium used to passage the mesenchymal stem cell seed bank cells to P5 (step 3) is mesenchymal stem cell culture medium-3, and the rest are the same to obtain mesenchymal stem cell working bank cells (denoted as W3).

[0105] Example 4

[0106] The only difference from Example 2 is that the culture medium used to passage the mesenchymal stem cell seed bank cells to P5 (step 3) is mesenchymal stem cell medium-4, and the rest are the same, obtaining mesenchymal stem cell working bank cells (denoted as W4).

[0107] Comparative Example 1

[0108] The only difference from Example 1 is that bFGF was not added to the mesenchymal stem cell culture medium-1. Other procedures were the same as in Example 1, and a mesenchymal stem cell working bank (denoted as W5) was obtained.

[0109] Experimental Example 1

[0110] The mesenchymal stem cell working bank cells (W1-W5) prepared above were tested, and the test items were as follows:

[0111] (1) Safety testing of bacteria, mycoplasma, viruses, etc.

[0112] Sterility testing was performed according to the sterility test method in General Chapter 1101 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0113] Mycoplasma testing was performed according to the first method in General Chapter 3301 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0114] According to the kit instructions, PCR method was used to complete the detection of EBV, HCMV, HIV and other viruses in the working library cells.

[0115] The test results showed that the mesenchymal stem cell working bank cells W1-W5 were all negative for bacteria, mycoplasma and viruses.

[0116] (2) Cell morphology;

[0117] The cell status of the mesenchymal stem cell working bank cells was observed using an inverted microscope. Figure 1 As shown in the figure, it can be seen that W1-W4 cells are in good condition, with individual cells showing spindle-shaped growth and fusion forming a spindle-shaped cell. The morphology of W5 is poor and shows signs of aging.

[0118] (3) Cell proliferation

[0119] The mesenchymal stem cell working bank cells were obtained and seeded into 96-well plates at 5000 cells per well. Cell proliferation was measured using CCK-8 assay at 0h, 24h, 48h, 72h, and 96h after seeding. Figure 2 As shown: the cell proliferation ability of W1-W4 is strong, among which W4 has the strongest cell proliferation ability, followed by W3, W2 and W1, but there is no significant difference among the four groups; W5 cell proliferation ability is poor.

[0120] (4) Flow cytometry phenotyping

[0121] The mesenchymal stem cell working bank cells were obtained and the following operations were performed: 5×106 cells were collected and evenly divided into 10 centrifuge tubes. After centrifugation at 300g for 5 minutes, the supernatant was discarded and the following antibodies were added respectively: PE-IgG1, PE-CD73, PE CD90, PE-CD105, PE-CD45, PE-HLA-DR, PE-CD11b, FITC-IgG1, FITC-CD19 and FITC-CD34 (antibodies from BD Company, USA), and cell surface antigens were detected using a BD Calibur flow cytometer.

[0122] The measurement results are shown in Table 1. The mesenchymal stem cell working bank cells (W1-W5) all highly expressed CD73, CD90 and CD105, and did not express CD19, CD34, CD45, CD11b and HLA-DR.

[0123] Table 1 Flow cytometry phenotypes of mesenchymal stem cell working bank cells

[0124] CD73 CD90 CD105 CD19 CD34 CD45 CD11b HLA-DR W1 99.21 98.99 99.36 0.16 0.19 0.18 0.37 0.18 W2 99.06 99.12 99.26 0.13 0.21 0.26 0.42 0.19 W3 98.92 99.06 98.75 0.09 0.20 0.17 0.34 0.28 W4 99.06 99.17 99.01 0.16 0.28 0.28 0.39 0.26 W5 98.72 98.26 99.17 0.21 0.26 0.27 0.42 0.22

[0125] (5) osteogenic and adipogenic differentiation capacity;

[0126] Take cells W1-W5 from the mesenchymal stem cell working bank and perform the following operations:

[0127] Each group of cells was 1×10 4 / well of a 24-well plate, and when the cell confluence reaches about 80%, replace the adipogenic and osteogenic induction medium, and replace it every 2 to 3 days. On the 21st day of induction culture, fix the cells with 4% paraformaldehyde solution at room temperature for 30 minutes, and then stain with 10% Oil Red-O to observe the adipogenic differentiation under a microscope, and stain with Alizarin Red solution to observe the osteogenic differentiation under a microscope.

[0128] The results are as follows Figure 3 As shown in the figure, mesenchymal stem cells from W1 to W5 all have the ability to differentiate into osteoblasts and adipocytes, among which W4 has the strongest differentiation ability, followed by W2, W3 and W1, and W5 has the weakest differentiation ability.

[0129] (6) Biological efficacy (HGF, KGF, sTNFR1) detection

[0130] Mesenchymal stem cell working bank cells were seeded into 6-well plates and cultured for 48 hours. The supernatant was collected and digested for cell counting. ELISA was used to measure the levels of HGF (detection kit purchased from abcam, catalog number ab275901), KGF (detection kit purchased from abcam, catalog number ab183362), and sTNFR1 (detection kit purchased from R&D, catalog number DRT100) in the supernatant.

[0131] The test results are shown in Table 2. The W1-W5 working bank cells can express and secrete HGF, sTNFR1 and KGF. The factor secretion levels of the four groups W1-W4 are higher than that of the W5 group, among which W4 has the highest secretion level, followed by W2, W3 and W1.

[0132] Table 2 HGF, KGF, sTNFR1 content

[0133]

[0134] Experimental Example 2

[0135] Mesenchymal stem cell working bank cells W1-W5 prepared above were revived and cultured to 80% cell confluency, then trypsinized, centrifuged, and washed three times with cell wash solution (an aqueous solution containing 1 mmol / L potassium chloride, 2.5 mmol / L sodium lactate, 2.5 mmol / L sodium gluconate, and 8 mmol / L sodium chloride). Cells were then resuspended in cell preservation solution (an aqueous solution containing 10 g / L human albumin, 1 mmol / L potassium chloride, 65 IU heparin, 2.5 mmol / L sodium lactate, 2.5 mmol / L sodium gluconate, and 8 mmol / L sodium chloride) to prepare mesenchymal stem cell injections, designated P1-P5. The injection volume was 20 mL, and the cell count was 1E+7.

[0136] 2.1 Inspection of visible foreign matter

[0137] Visible foreign matter was detected according to the Visible Foreign Matter Inspection Method in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0138] 2.2 Insoluble particle inspection

[0139] The insoluble particles were inspected according to the first method of Part III 0903 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0140] 2.3 Filling quantity inspection

[0141] The filling quantity is checked in accordance with General Rule 0102 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0142] 2.4 Osmotic pressure test

[0143] The osmotic pressure molar concentration was determined according to General Chapter 0632 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0144] 2.5 Cell number and cell viability

[0145] A cell counter was used to count cells, and the stem cell preparation to be tested was stained with 0.04% trypan blue (1:1), and the cell number was read and the viability was calculated.

[0146] 2.6 Sterility testing

[0147] Sterility testing was performed according to the sterility test method in General Chapter 1101 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0148] 2.7 Mycoplasma Detection

[0149] Mycoplasma testing was performed according to the first method in General Chapter 3301 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0150] 2.8 Endotoxin detection

[0151] Endotoxin testing was performed according to the gel method in General Chapter 1143 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0152] 2.9 HGF, sTNFR1, and KGF Results

[0153] The obtained mesenchymal stem cell injection (P1-P5) cells were divided into 1×10 4 / cm 2 Inoculate T75 culture flasks and culture for 48 hours, then harvest the supernatant, digest and count the cells, and detect the HGF, sTNFR1 and KGF contents in the samples according to the instructions of the HGF, sTNFR1 and KGF kits.

[0154] Pharmacopeia standards for visible foreign matter, insoluble particles, volume variation, osmotic pressure, cell count, cell viability, sterility testing, mycoplasma testing, and endotoxin testing are shown in Table 3. The test results for mesenchymal stem cell injections (P1-P5) are shown in Table 4, and all tested qualified. The results for HGF, sTNFR1, and KGF testing are shown in Table 5. P1-P4 all expressed high levels of HGF, sTNFR1, and KGF, meeting the required expression levels; however, P5 expressed lower levels of these factors.

[0155] Table 3 Pharmacopoeia standards

[0156]

[0157]

[0158] Table 4 Test results of mesenchymal stem cell injection

[0159]

[0160] Table 5 Results of mesenchymal stem cell injection HGF, sTNFR1, KGF

[0161] <![CDATA[HGF(ng / *10 6 cell)]]> <![CDATA[sTNFR1(pg / *10 6 cell)]]> <![CDATA[KGF(pg / *10 6 cell)]]> P1 171.56 267.88 1099.28 P2 226.62 321.63 1219.31 P3 190.42 282.33 1107.56 P4 240.88 357.69 1298.41 P5 87.69 209.97 821.31

[0162] Experimental Example 3 Effect of Mesenchymal Stem Cell Injection on Acute Lung Injury / Acute Respiratory Distress Syndrome

[0163] 3.1 Model construction

[0164] Establishment of LPS-induced acute lung injury combined with ARDS model in rats:

[0165] One hundred and thirty healthy, male, 240 g Sprague-Dawley rats were randomly divided into a sham-operated group (G1 group, N=10) and a model group (120 rats). After anesthesia, the rats underwent a midline neck skin incision to expose the trachea. 400 μL of LPS solution (containing 4 mg of LPS) was injected into the airway and then sutured. In the sham-operated group, only the trachea was exposed and then sutured to establish an LPS-induced acute lung injury model.

[0166] 3.2 Experimental methods

[0167] 3.2.1 Experimental Animal Grouping and Dosing

[0168] There were 10 rats in the sham operation group (G1 group), and 120 rats with successful model establishment were randomly divided into 6 groups:

[0169] 1) Model control group (G2 group, N=20);

[0170] 2) hUC-MSC treatment group-1 (G3 group, P1 group of Experimental Example 2, N=20 / group);

[0171] 3) hUC-MSC treatment group-2 (G4 group, P2 group of Experimental Example 2, N=20 / group);

[0172] 4) hUC-MSC treatment group-3 (G5 group, P3 group of Experimental Example 2, N=20 / group);

[0173] 5) hUC-MSC treatment group-4 (G6 group, P4 group in Experimental Example 2, N=20 / group).

[0174] 6) hUC-MSC treatment group-5 (G7 group, P5 group of Experimental Example 2, N=20 / group).

[0175] 30 minutes after modeling, the treatment group was treated with 1×10 7 The hUC-MSC suspension was injected into the tail vein at a dose of 2 mL / kg cells / kg body weight. The sham operation group and the model group were given the same volume of normal saline.

[0176] 3.2.2 Detection indicators

[0177] (1) Survival rate curve

[0178] The survival rate of rats was calculated 24 hours after modeling, and a survival rate curve was drawn.

[0179] (2) Blood gas analysis

[0180] 24 hours after modeling, the rats were anesthetized, and the abdominal aorta was isolated to extract about 0.5 mL of arterial blood (anticoagulated with sodium heparin). Blood gas analysis (pO2, sO2, pCO2) was performed using a GEMpremier 3000 blood gas analyzer (Warfen).

[0181] (3) Inflammatory factors in alveolar lavage fluid

[0182] 24 hours after modeling, the rats were dissected, the right lung was ligated, and the left lung alveoli were lavaged with 0.9% sodium chloride injection through tracheal intubation. The lavage fluid was collected and the inflammatory factors rat TNF-α and IL-6 were detected by enzyme-linked immunosorbent assay (rat TNF-α enzyme-linked immunosorbent assay kit, Abcam, USA, batch number: GR3304414-1; rat IL-6 enzyme-linked immunosorbent assay kit, Abcam, USA, batch number: GR3316507-1).

[0183] 3.3 Experimental Results

[0184] 3.3.1 Analysis of rat survival rate

[0185] Twenty-four hours after modeling, clinical observation of deceased animals revealed bloody secretions around the eyes and dirt in the mouth and nose. Gross autopsy revealed watery gastrointestinal contents, flatulence, and heavy, bloody lungs. Analysis suggested that the animals may have died of asphyxiation. Clinical observation of surviving animals revealed obvious abnormalities in body temperature, piloerection, diarrhea, rapid breathing, and lethargy. Gross autopsy revealed no significant abnormalities in any tissue or organ.

[0186] The results are shown in Table 6 and Figure 4 As shown, the number of deaths in the model group, hUC-MSC-treated group-P1, hUC-MSC-treated group-P2, hUC-MSC-treated group-P3, hUC-MSC-treated group-P4, and hUC-MSC-treated group-P5 was 15, 10, 9, 9, 8, and 13 animals, respectively, with survival rates of 25%, 65%, 70%, 65%, 75%, and 50%, respectively. The model group was consistent with severe acute lung injury, and hUC-MSC-treated groups significantly reduced the survival rate of rats. Statistics of rat survival rates showed that the survival rate of rats in the model group was significantly decreased (P < 0.05). Compared with the model group, hUC-MSC-treated groups all had significantly increased survival rates, indicating that hUC-MSCs have a pharmacological effect in reducing the mortality of LPS-induced acute lung injury and multiple organ failure. G6 (hUC-MSC-P4 group) had the most significant effect, followed by G4 (hUC-MSC-P2 group), and G7 had the worst effect.

[0187] Table 6 Effect of hUC-MSC on the survival rate of LPS-induced acute lung injury combined with ARDS model

[0188] Group dose Number of survivors Number of deaths / animal Total / piece Survival rate / % G1 - 10 0 10 100.0 G2 - 5 15 20 25 G3 <![CDATA[1×10 7 / kg]]> 13 7 20 65 G4 <![CDATA[1×10 7 / kg]]> 14 6 20 70 G5 <![CDATA[1×10 7 / kg]]> 13 7 20 65 G6 <![CDATA[1×10 7 / kg]]> 15 5 20 75 G7 <![CDATA[1×10 7 / kg]]> 10 10 20 50

[0189] 3.3.2 Rat blood gas analysis

[0190] The test results are as follows Figure 5-Figure 7 As shown in the results, the arterial oxygen partial pressure of the model rats decreased, the blood oxygen saturation decreased significantly (P<0.01), and the carbon dioxide partial pressure showed an upward trend, indicating that the model rats had impaired lung function and respiratory failure. The hUC-MSC treatment groups (1-5) were able to increase the arterial oxygen partial pressure (P<0.05) and blood oxygen saturation (P<0.01~0.001), and reduce the carbon dioxide partial pressure (P<0.01~0.001), indicating that hUC-MSC has the pharmacological activity of improving the pulmonary ventilation function of the model rats and protecting the lungs; among them, the P4 and P2 groups had the best effects, while the P5 group had weaker improvements on arterial oxygen partial pressure, blood oxygen saturation, carbon dioxide partial pressure and other functions.

[0191] 3.3.3 Detection and analysis of inflammatory factors in rat bronchoalveolar lavage fluid

[0192] The test results are as follows Figure 8-Figure 9 As shown in the data, compared with the sham operation group, IL-6 and TNF-α in the lung lavage fluid of the model group rats 24 hours after modeling were extremely significantly increased (P<0.001), indicating that the inflammatory response of the model group rats was extremely serious. Compared with the model group, IL-6 and TNF-α in the hUC-MSC treatment groups (1-5) were significantly decreased (P<0.001), indicating that hUC-MSC has the pharmacological activity of inhibiting the release of inflammatory factors IL-6 and TNF-α and antagonizing inflammatory response. Among them, the P4 group had the best reduction effect, followed by the P2 group, while the P-5 reduction effect was poor.

[0193] Experimental Example 4 Effect of Mesenchymal Stem Cell Injection on Pulmonary Fibrosis

[0194] 4.1 Model Construction

[0195] Establishment of bleomycin-induced rat pulmonary fibrosis model:

[0196] Ninety male SD rats were enrolled. After approximately one week of acclimatization, the animals were removed, anesthetized with 10% chloral hydrate (40 mg / kg), and fixed on an operating table. After disinfecting the neck with 75% alcohol cotton pads, surgical instruments were used to exfoliate the trachea layer by layer, and the needle was inserted through the interstitial space of the tracheal cartilage rings toward the centripetal end. Bleomycin (MedChemExpress, Lot No. 111561) was injected into the trachea at a dose of 2.5 mg / kg. The animals were then quickly placed upright and rotated briefly to evenly distribute the solution throughout the lungs. The incision was then sutured. Rats in the sham-operated group were injected with saline only.

[0197] 4.2 Experimental methods

[0198] 4.2.1 Experimental Animal Grouping and Dosing

[0199] There were 10 rats in the sham operation group (G1 group), and 90 rats with successful model establishment were randomly divided into 6 groups:

[0200] 1) Model control group (G2 group, N=15);

[0201] 2) hUC-MSC treatment group-1 (G3 group, P1 group of Experimental Example 2, N=15 / group);

[0202] 3) hUC-MSC treatment group-2 (G4 group, P2 group of Experimental Example 2, N=15 / group);

[0203] 4) hUC-MSC treatment group-3 (G5 group, P3 group of Experimental Example 2, N=15 / group);

[0204] 5) hUC-MSC treatment group-4 (G6 group, P4 group in Experimental Example 2, N=15 / group).

[0205] 6) hUC-MSC treatment group-5 (G7 group, P5 group in Experimental Example 2, N=15 / group).

[0206] On the 7th and 21st days after modeling, the treatment group was treated with 1×10 7 The hUC-MSC suspension was injected into the tail vein at a dose of 2 mL / kg cells / kg body weight. The sham operation group and the model group were given the same volume of normal saline.

[0207] 4.2.2 Detection indicators

[0208] (1) Body weight, lung weight and survival rate curves:

[0209] The experimental endpoint was 28 days after modeling, and the rat body weight and survival rate were counted, and a survival rate curve was drawn.

[0210] The rats were sacrificed, the lungs were removed by dissection and weighed, and the organ index was calculated according to the formula: organ index = lung weight / body weight × 100%.

[0211] (2) Hydroxyproline and inflammatory factors in lung tissue:

[0212] When rats were dissected, a certain amount of lung tissue was weighed and quickly placed in a -70°C freezer. During the experiment, the low-temperature freezer tissue was homogenized and the hydroxyproline content in the lung tissue was detected. The collected lung lavage fluid was analyzed for blood cells, and the supernatant was centrifuged and used for ELISA detection of cytokines IL-1β and TGF-β1 (Hydroxyproline kit: Nanjing Jiancheng Bioengineering Institute, production batch number: 20210713; IL-1β detection kit: Shanghai Xinle Biotechnology Co., Ltd., production batch number: 2109GF03LES; TGF-β1 detection kit: Shanghai Xinle Biotechnology Co., Ltd., production batch number: 2109GF18MMK).

[0213] (3) Histopathological examination

[0214] At the end of the trial, lung tissues were pathologically sectioned and Masson stained, and the Ashcroft score for pulmonary fibrosis was performed. The scoring criteria are shown in Table 7.

[0215] Table 7 Scoring criteria

[0216] PF degree Histopathological characteristics Level 0 Normal lung tissue Level 1 Mild fibrosis of the bronchioles and alveolar walls Level 3 Moderate fibrosis of the bronchioles and alveolar walls, but no obvious destruction of lung tissue architecture Level 5 Marked fibrosis with destruction of lung tissue architecture and the formation of fibrous bands or fibrous nodules Level 7 Severe deformation of lung tissue structure or large areas of fibrous lesions Level 8 Fibrotic lesions appear throughout the entire field of view

[0217] 4.3 Experimental Results

[0218] 4.3.1 Analysis of rat body weight, lung weight and survival rate curves

[0219] Weight data such as Figure 10 The results showed that compared with the sham operation group, the body weight of the model group decreased significantly, while the body weight of rats increased after hUC-MSC treatment. There was no significant difference between each treatment group and the model group.

[0220] The lung weight and organ coefficients of rats at the end of the experiment were as follows Figure 11-12 As shown: Compared with the sham-operated group, the lung weight and lung index of the model rats were significantly increased (P<0.001). However, after treatment with hUC-MSC-treated groups (1-5), the lung weight and lung index of the model rats were significantly reduced (P<0.01). In particular, the lung weight and lung index of the hUC-MSC-treated group -4 decreased to the level of the sham-operated group.

[0221] The results of rat mortality at the end of the experiment were as follows Figure 13 As shown, the sham-operated group had a 0% mortality rate, while all other groups experienced mortality. Deaths began occurring in the model and hUC-MSC-treated groups (P1-P5) from day 2-3 after administration, with the model group having the highest mortality rate at 66.67%. Mortality rates in the P1, P2, P3, P4, and P5 groups were 13.33%, 6.67%, 13.33%, 6.67%, and 26.67%, respectively.

[0222] 4.3.2 Detection and analysis of hydroxyproline and inflammatory factors in rat lung tissue

[0223] The levels of hydroxyproline in rat lung tissue and cytokines IL-1β and TGF-β1 in lung lavage fluid at the end of the experiment were as follows: Figure 14-16 As shown in the results: compared with the sham operation group, the levels of hydroxyproline, IL-1β and TGF-β1 in the lung tissue of rats in the model group were significantly increased (P<0.001); after treatment with hUC-MSC treatment groups (P1-P5), the levels of the above three factors decreased, among which the TGF-β1 content of rats in groups P4 and P2 returned to normal levels, and the IL-1β content also decreased significantly (P<0.05).

[0224] 4.3.3 Rat Histopathology

[0225] HE staining and Masson staining pathological scoring Figure 17 As shown: Ashcroft score is the score of lung structural tissue destruction and fibrosis. The sham operation group was 0. Compared with the sham operation group, the fibrosis score of the model group was significantly increased, with an extremely significant difference (P<0.001). The Ashcroft score of the hUC-MSC treatment group (P1-P5) was significantly reduced after treatment (P<0.05), among which P4 and P2 groups had the best reduction in fibrosis.

[0226] Experimental Example 5 Effect of Mesenchymal Stem Cell Injection on Chronic Obstructive Pneumonia

[0227] 5.1 Model construction

[0228] Eight-week-old Balb / c female mice were placed in an aerosol inhalation chamber (60 cm × 60 cm × 80 cm) with cigarettes placed in a smoke generator. After lighting the cigarette, the smoke was automatically pumped into the chamber via a syringe, and burned out within 5 minutes. This was repeated twice daily, morning and evening, for 30 minutes, with an interval of at least 4 hours, for 9 consecutive weeks. On days 27 and 55 of modeling, mice in all groups except the sham-operated control group were anesthetized with intraperitoneal injection of 10% chloral hydrate solution. Following anesthesia, the trachea was exposed, and LPS (30 μg / 6 μL) was rapidly injected into the trachea using a 1 ml syringe. Following injection, the mice were rapidly rotated upright for 10–20 seconds to evenly distribute the LPS solution throughout the lungs.

[0229] 5.2 Experimental methods

[0230] 5.2.1 Experimental Animal Grouping and Dosing

[0231] There were 10 mice in the sham operation group (G1 group), and 90 mice with successful model establishment were randomly divided into 5 groups:

[0232] 1) Model control group (G2 group, N=15);

[0233] 2) hUC-MSC treatment group-1 (G3 group, P1 group of Experimental Example 2, N=15 / group);

[0234] 3) hUC-MSC treatment group-2 (G4 group, P2 group of Experimental Example 2, N=15 / group);

[0235] 4) hUC-MSC treatment group-3 (G5 group, P3 group of Experimental Example 2, N=15 / group);

[0236] 5) hUC-MSC treatment group-4 (G6 group, P4 group in Experimental Example 2, N=15 / group);

[0237] 6) hUC-MSC treatment group-5 (G7 group, P5 group in Experimental Example 2, N=15 / group).

[0238] On days 28 and 56 of modeling, the treatment group was treated with 1×10 6 The hUC-MSC suspension was injected into the tail vein at a dose of 10 cells / body weight, while the sham operation group and the model group were given the same volume of normal saline.

[0239] 5.2.2 Detection indicators

[0240] (1)Weight

[0241] The experimental endpoint was 9 weeks after model establishment. The mice were weighed every 7 days and the weight changes were recorded.

[0242] (2) Lung function

[0243] The lung function of mice was measured by forced pulmonary ventilation (FVC) using a small animal pulmonary function tester.

[0244] (3) Detection of mouse lung immune cells

[0245] Bronchoalveolar lavage fluid (BALF) was collected and smeared, and differential counts of bacteria in the BALF were performed under an optical microscope.

[0246] (4) Bronchial and lung tissue pathology

[0247] The right upper and middle lung tissue was removed, and 10% formalin was perfused into the right main bronchus until the right lower lung expanded. After the pleura was flattened, the tissue was fixed in formalin for 24 hours. Hematoxylin and eosin staining was performed for routine pathological examination to observe the inflammatory infiltration of the bronchial and lung tissues. The area of ​​inflammatory cell infiltration and the mean lining septal index of the mouse lung tissue were observed.

[0248] 5.3 Experimental Results

[0249] 5.3.1 Mouse body weight analysis

[0250] The results are as follows Figure 18 As shown, four weeks after modeling, the weight of all modeling groups was lower than that of the sham-operated group, with a statistically significant difference (P<0.01), indicating that smoking modeling slows weight gain in mice. The weight of mice in each group further decreased after LPS modeling. One week after the second cell treatment, the weight of mice in the hUC-MSC-treated group (P4) increased significantly, with a statistically significant difference compared to the model group (P<0.01).

[0251] 5.3.2 Analysis of mouse lung function

[0252] This experiment uses the volume of gas that animals can exhale forcefully within 200ms (FEV2) to evaluate the lung function of mice. FEV2 / FVC is the ratio of the volume of gas that animals can exhale forcefully within 200ms to their vital capacity. The larger the FEV2 / FVC, the better the respiratory function of the animal.

[0253] The results are as follows Figure 19-20 As shown in the figure, lung function tests were performed one week after the last administration of the drug to the animals. Compared with the sham operation group, the FEV2 and FEV2 / FVC of the model control group were significantly decreased (P<0.01), indicating that smoking + LPS modeling significantly reduced the lung function of mice; while the five hUC-MSC treatment groups could improve the lung function of mice; compared with the model group, the FEV2 and FEV2 / FVC of the mice in the hUC-MSC treatment groups (P1-P5) were significantly increased after treatment (P<0.01), among which the P4 group had the best treatment effect, followed by the P2 group.

[0254] 5.3.3 Detection and Analysis of Mouse Lung Immune Cells

[0255] like Figure 21 As shown in the figure, images of mouse lung lavage fluid were stained and examined microscopically, and neutrophils were counted by microscopically classifying 100 white blood cells and calculating the neutrophil ratio. The microscopic examination results showed that the sham-operated group had fewer cells under the microscopic field of view, but they were basically all neutrophils, so their proportion was relatively large; the model group had neutrophils aggregated into sheets under the microscopic field of view, accounting for a large proportion of the total cells; compared with the model group, the proportion of neutrophils in the lung lavage fluid of mice in the hUC-MSC treatment groups P1-P5 was significantly reduced (P < 0.01 or P < 0.001), indicating that hUC-MSC treatment can significantly improve lung inflammation in mice in the model group, among which P4 had the best reduction effect, followed by P2 group, and P5 group was the worst.

[0256] 5.3.4 Analysis of Inflammatory Cell Infiltration Area and Average Lining Interval Index in Mouse Lung Tissue

[0257] The lungs of mice were fixed and HE staining was performed for microscopic analysis. The lungs of mice in the model control group showed alveolar congestion, inflammatory infiltration, and tracheal wall thickening, which were significantly different from those in the sham operation group (P<0.01). There was a small amount of inflammatory infiltration in the tracheal wall in the P4 group, which was significantly improved compared with the model group (P<0.01), followed by the P2 group, and the P5 group was worse.

[0258] The alveolar lining index of mice was counted and the microscopic examination results were as follows: Figure 22-23 As shown in the figure, the alveolar diameter of the model control group was larger, which was significantly different from that of the sham operation group (P<0.01); the lung pathological staining analysis of mice in each hUC-MSC treatment group showed that the alveolar diameter was smaller, which was significantly different from that of the model group (P<0.05), among which the improvement effect of the P4 group was more obvious (P<0.01), and the improvement effect of the P5 group was the worst.

[0259] Experimental Example 6 Effect of siRNA-treated MSCs on pulmonary fibrosis

[0260] 6.1 Model Construction

[0261] Establishment of bleomycin-induced rat pulmonary fibrosis model:

[0262] Seventy-six male Sprague-Dawley rats were acclimated for approximately one week. After being anesthetized with 10% chloral hydrate (40 mg / kg), the rats were removed and fixed to the operating table. After disinfecting the neck with 75% alcohol cotton pads, surgical instruments were used to exfoliate the trachea layer by layer, and the needle was inserted through the interstitial space between the tracheal cartilage rings toward the centripetal end. Bleomycin (MedChemExpress, Lot No. 111561) was injected into the trachea at a dose of 2.5 mg / kg. The rats were then quickly placed upright and rotated briefly to evenly distribute the solution throughout the lungs. The incision was then sutured. Rats in the sham-operated group were injected with saline only.

[0263] 6.2 Experimental methods

[0264] 6.2.1 Experimental Animal Grouping and Dosing

[0265] There were 12 rats in the sham operation group (G1 group). The 64 rats that successfully established the model and survived were randomly divided into 5 groups:

[0266] 1) Model control group (G2 group, treated with normal saline, N = 16);

[0267] 2) hUC-MSC treatment group-1 (G3 group, P1 group in Experimental Example 2, N=12 / group);

[0268] 3) hUC-MSC treatment group-2 (G4 group, HGF gene silencing group after siRNA gene silencing in group P1 of Experimental Example 2, siHGF-MSCs group, N=12 / group);

[0269] 4) hUC-MSC treatment group-3 (G5 group, P1 group of Experimental Example 2, KGF gene silencing group after siRNA, siKGF-MSCs group, N = 12 / group);

[0270] 5) hUC-MSC treatment group-4 (G6 group, P1 group of Experimental Example 2, TNFR-1 gene silencing group after siRNA, siTNFR1-MSCs group, N=12 / group).

[0271] The siRNA information is shown in Table 8: HGF siRNA and KGF siRNA were synthesized by Suzhou Hongxun, and TNFR1 siRNA was purchased from Thermo with catalog number s14266.

[0272] Table 8 siRNA information

[0273]

[0274] On days 7 and 21 after modeling, the treatment group was treated with 1×10 7 The hUC-MSC suspension was injected into the tail vein at a dose of 2 mL / kg, and the sham operation group and the model group were given the same volume of normal saline.

[0275] 6.2.2 Detection indicators

[0276] (1) Body weight, lung weight and survival rate curves:

[0277] The experimental endpoint was 28 days after modeling, and the rat body weight and survival rate were counted, and a survival rate curve was drawn.

[0278] The rats were sacrificed, the lungs were removed by dissection and weighed, and the organ index was calculated according to the formula: organ index = lung weight / body weight × 100%.

[0279] (2) Hydroxyproline and inflammatory factors in lung tissue:

[0280] When rats were dissected, a certain amount of lung tissue was weighed and quickly placed in a -70°C freezer. During the experiment, the low-temperature freezer tissue was homogenized and the hydroxyproline content in the lung tissue was detected. The collected lung lavage fluid was analyzed for blood cells, and the supernatant was centrifuged and used for ELISA detection of cytokines IL-1β and TGF-β1 (Hydroxyproline kit: Nanjing Jiancheng Bioengineering Institute, production batch number: 20210713; IL-1β detection kit: Shanghai Xinle Biotechnology Co., Ltd., production batch number: 2109GF03LES; TGF-β1 detection kit: Shanghai Xinle Biotechnology Co., Ltd., production batch number: 2109GF18MMK).

[0281] (3) Histopathological examination

[0282] At the end of the trial, lung tissues were pathologically sectioned and Masson stained, and the Ashcroft score for pulmonary fibrosis was performed. The scoring criteria are shown in Table 7.

[0283] 6.3 Experimental Results

[0284] 6.3.1 Secretion of hUC-MSC Factors after siRNA Treatment

[0285] HGF, KGF, and TNFR1 siRNA were used to silence the genes of hUC-MSCs, respectively. The concentrations of HGF, KGF, and TNFR1 in the supernatant of hUC-MSCs were detected by ELISA. Figures 24-25 As shown, the three siRNFs can significantly reduce the secretion levels of HGF, KGF, and TNFR1 by hUC-MSC (P < 0.001), and the inhibition efficiency of HGF, KGF, and TNFR1 expression is more than 80%.

[0286] 6.3.2 Analysis of rat body weight, lung weight and survival rate curves

[0287] Figure 26 Compared with the sham-operated group, the model group had a significant decrease in body weight (P<0.05). Compared with the model group, the hUC-MSCs group had a significant increase in body weight. Compared with the hUC-MSCs group, the siHGF-MSCs, siKGF-MSCs, and siTNFR1-MSCs groups had a significant decrease in body weight. This suggests that the improvement in body weight loss by hUC-MSCs is related to HGF, KGF, or TNFR1.

[0288] Figure 27 The mortality rate was 0% in the sham and hUC-MSCs groups, 37.50% in the model group, 25.00% in the siHGF-MSCs and siKGF-MSCs groups, and 50.00% in the siTNFR1-MSCs group. Except for the sham and hUC-MSCs groups, all other groups experienced mortality within 3-4 days of dosing. This suggests that the improvement of hUC-MSCs in pulmonary fibrosis-related mortality is related to HGF, KGF, or TNFR1.

[0289] 6.3.3 Rat lung weight and organ coefficients

[0290] Figure 28Compared with the sham-operated group, the lung weight and lung coefficient of the model group rats were significantly increased (P<0.001). Compared with the model group, the lung coefficient of the hUC-MSCs group was significantly decreased (P<0.001). Compared with the hUC-MSCs group, the lung coefficient of the siHGF-MSCs group and the siTNFR1-MSCs group was significantly increased (P<0.01). This suggests that the improvement of hUC-MSCs in the lung is related to HGF or TNFR1.

[0291] 6.3.4 Hydroxyproline in Rat Lung Tissue and Cytokines in Lung Lavage Fluid

[0292] Figure 29 The results showed that compared with the sham group, the hydroxyproline content in the model group was significantly increased (P<0.001). Compared with the model group, the content in the hUC-MSCs group was significantly decreased. Compared with the hUC-MSCs group, the content in the siTNFR1-MSCs group was significantly increased. Figure 30 The results showed that compared with the sham operation group, the TNF-α content in the lung lavage fluid of the model group rats was significantly increased (P<0.01). Compared with the model group, the TNF-α content in the lung lavage fluid of the hUC-MSCs group was significantly decreased.

[0293] 6.3.5 Pathological changes of rat lung tissue

[0294] Masson staining pathological scoring Figure 31 The Ashcroft score is a fibrosis score, and the sham surgery group had a score of 0. Compared with the sham surgery group, the model group had a significantly higher score (P < 0.001). Compared with the model control group, the hUC-MSCs group had a significantly lower score (P < 0.001). Compared with the hUC-MSCs group, the siTNFR1-MSCs group had a significantly higher score. This suggests that hUC-MSCs are more closely related to TNFR1 in improving lung function.

[0295] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A culture medium for mesenchymal stem cells having therapeutic effects on respiratory diseases, characterized in that: The basic culture medium is DMEM / F12 culture medium, and further comprises 7.5-10% v / v FBS, 2.5-5 μg / L bFGF and 2-5 μg / L glutamine.

2. The culture medium according to claim 1, characterized in that The culture medium also includes hydrogen and sulfobutyl-β-cyclodextrin. The amount of hydrogen is 0.5-1.0 μg / ml, and the amount of sulfobutyl-β-cyclodextrin is 0.1-0.3 mmol / L.

3. A method for preparing mesenchymal stem cells having therapeutic effects on respiratory diseases, characterized in that: The following steps are involved: placing the tissue containing mesenchymal stem cells in the culture medium of claim 1 for culturing, and digesting the cells after the cell confluence reaches 70-90%, thereby obtaining P0 mesenchymal stem cells; The P0 generation mesenchymal stem cells are collected after centrifugation, and subcultured twice to the P2 generation using the culture medium of claim 1 and frozen to obtain mesenchymal stem cell seed bank cells; After the mesenchymal stem cell seed bank cells are revived, they are continuously passaged using the culture medium described in claim 1 or 2, cultured to the P5 generation and then frozen to obtain mesenchymal stem cells with therapeutic effects on respiratory diseases.

4. The preparation method according to claim 3, characterized in that The types of mesenchymal stem cells include any one of the following: adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells.

5. The preparation method according to claim 3, characterized in that: The culture temperature is 38-39° C., the CO 2 concentration is 4-6%, and the O 2 concentration is 2-20%.

6. The preparation method according to claim 3, characterized in that: The temperature of the subculture and the continuous subculture is 36-37° C., the CO 2 concentration is 4-6%, and the O 2 concentration is 2-20%.

7. An injection of mesenchymal stem cells for treating respiratory diseases, characterized in that: Comprising mesenchymal stem cells and cell preservation solution having a therapeutic effect on respiratory diseases prepared by the preparation method according to any one of claims 3 to 6; The cell preservation solution includes 5-10 g / L human serum albumin, 60-70 IU / ml heparin, 0.5-1 mmol / L potassium chloride, 2.5-5 mmol / L sodium lactate, 2-5 mmol / L sodium gluconate and 5-10 mmol / L sodium chloride.

8. Use of mesenchymal stem cells having therapeutic effects on respiratory diseases prepared by the preparation method according to any one of claims 3 to 6 in the preparation of drugs for treating respiratory diseases.

9. A stem cell drug for treating respiratory diseases, characterized in that: The invention comprises mesenchymal stem cells with respiratory disease therapeutic effects prepared by the preparation method according to any one of claims 3 to 6 and pharmaceutically acceptable excipients.

10. A method for detecting the biological efficacy of mesenchymal stem cells in treating respiratory diseases, characterized in that: This includes detecting the ability of mesenchymal stem cells to secrete HGF, KGF, and / or TNFR1; Every 1×10 6 When the HGF secretion of mesenchymal stem cells is 100-300 ng at 48 hours, the cell activity is considered to be high. Every 1×10 6 When the secretion of KGF by mesenchymal stem cells is 1000-2000 pg within 48 hours, the cell activity is considered to be high. Every 1×10 6 When the secretion of TNFR1 by mesenchymal stem cells is 250-800 pg at 48 hours, the cell activity is considered to be high. The sources of the mesenchymal stem cells include any one of the following: mesenchymal stem cells with therapeutic effects on respiratory diseases prepared by the preparation method according to any one of claims 3 to 6, the injection according to claim 7, and the stem cell medicine according to claim 9.

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