Application of mesenchymal stem cells in the treatment of idiopathic pulmonary fibrosis

Abstract

The present invention relates to the field of biomedicine technology, and in particular, to an application of mesenchymal stem cells in the treatment of idiopathic pulmonary fibrosis. The present invention improves the separation and preparation method of adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells prepared using a new culture medium have higher proliferation activity. TGF-β induction leads to increased expression of SP-C and α-SMA proteins and decreased expression of E-Cadherin, while the addition of a combination of mesenchymal stem cells derived from human adipose tissue and dextromethorphan alleviates this phenomenon, thereby achieving the treatment of idiopathic pulmonary fibrosis, providing a new direction for the treatment of idiopathic pulmonary fibrosis, and having great application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of mesenchymal stem cells in treating idiopathic pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung disease. Current clinical treatments for IPF include anti-fibrotic drugs, glucocorticoids, lung transplantation, etc. These treatments are not very effective and may also cause serious side effects. Currently, IPF patients have a low quality of life, and safe, effective, and convenient therapeutic drugs are urgently needed to improve the prognosis of IPF patients. The multifunctionality of mesenchymal stem cells (MSCs) and their ability to regulate immunity, inhibit inflammation, and promote epithelial tissue repair have led to the rapid application of MSCs in the treatment of IPF.

[0003] In recent years, regenerative medicine technology centered on stem cell therapy, especially MSCs, has brought hope for the treatment of intractable diseases in humans, and also provided new ideas for the transplantation of MSCs to treat IPF. MSCs can be isolated and identified from many tissue sources, including bone marrow, umbilical cord, adipose tissue and placenta. In 1995, Haynesworth et al. first used MSCs as a cell drug preparation for clinical application. In 2003, Ortiz et al. first discovered that MSCs can significantly reduce pulmonary fibrosis in mice.

[0004] Prior art CN115725499A discloses a preparation method and application of human umbilical cord mesenchymal stem cell membrane bionic nanovesicles, wherein the mesenchymal stem cell membrane is extracted and mixed with phospholipid liposomes, and then the bionic nanovesicles containing the mesenchymal stem cell membrane are prepared by thin film method and co-extrusion method. Due to the surface protein of human umbilical cord mesenchymal stem cell membrane, it can target the lesion site of idiopathic pulmonary fibrosis and alleviate the fibrosis degree of idiopathic pulmonary fibrosis.

[0005] Prior art CN111518758 A discloses the use of mesenchymal stem cells in the preparation of drugs for treating lung diseases, wherein the lung diseases are pneumoconiosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, severe novel coronavirus infection, acute respiratory distress syndrome or lung injury; the mesenchymal stem cells are one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0006] Prior art CN110139657A discloses a therapeutic agent for pulmonary fibrosis, which contains mesenchymal stem cells. The pulmonary fibrosis is preferably usual interstitial pneumonia (UIP) or idiopathic pulmonary fibrosis (IPF).

[0007] Prior art CN112823795B discloses an anti-idiopathic pulmonary fibrosis drug combination, comprising pirfenidone and dextromethorphan, wherein the pirfenidone is 200-600 mg and the dextromethorphan is 7.5 mg.

[0008] However, the above existing technologies for the use of MSCs therapy in registered clinical trials for a variety of human diseases have not yet achieved the expected results. The successful application of MSCs to treat IPF patients still needs to overcome many challenges, including the immune compatibility, stability, heterogeneity, differentiation and migration ability of MSCs from different sources, and inconsistency with the host microenvironment. Therefore, it is urgent to develop a new application of mesenchymal stem cells in the treatment of idiopathic pulmonary fibrosis. Summary of the invention

[0009] The present invention first provides, in order to achieve the above purpose, an application of mesenchymal stem cells in treating idiopathic pulmonary fibrosis, wherein the mesenchymal stem cells are one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0010] In some examples, the mesenchymal stem cells are umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells.

[0011] In some examples, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0012] In some examples, the raw material used in the method for preparing adipose-derived mesenchymal stem cells is adipose tissue.

[0013] In some examples, the method for preparing adipose-derived mesenchymal stem cells comprises the following steps:

[0014] (1) Obtaining adipose tissue;

[0015] (2) enzymatically hydrolyzing the adipose tissue of step (1) to obtain an enzymatic hydrolysis product;

[0016] (3) ultrasonically treating the enzymatic hydrolysis product of step (2) to obtain an ultrasonically treated product;

[0017] (4) centrifuging and washing the ultrasonic treatment product of step (3) to obtain a single cell suspension of adipose-derived mesenchymal stem cells;

[0018] (5) Cultivating and passage the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4).

[0019] In some examples, the culture medium used in the culture comprises DMEM, total flavonoids from Epimedium and β-ecdysterone.

[0020] In some examples, the concentration of the total flavonoids from Epimedium is 20 ng / ml, and the concentration of β-ecdysterone is 30 ng / ml.

[0021] In some examples, the mesenchymal stem cells are used in combination with dextromethorphan.

[0022] In some examples, the amount of dextromethorphan is 80-120 μg / mL.

[0023] In some examples, the amount of dextromethorphan is 100 μg / mL.

[0024] In order to achieve the above object, the present invention secondly provides.

[0025] Compared with the prior art, the above technical solution of the present invention has the following beneficial effects:

[0026] The present invention improves the separation and preparation method of adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells prepared using the new culture medium have higher proliferation activity. TGF-β induction leads to increased expression of SP-C and α-SMA proteins and decreased expression of E-Cadherin, while the combination of adding mesenchymal stem cells derived from human adipose tissue and dextromethorphan alleviates this phenomenon, thereby achieving the treatment of idiopathic pulmonary fibrosis, providing a new direction for the treatment of idiopathic pulmonary fibrosis, and having great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the expression level of SP-C in different experimental groups;

[0028] Figure 2 is the expression level of α-SMA in different experimental groups;

[0029] Figure 3 is the expression level of E-Cadherin in different experimental groups. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0031] A549 cell line was purchased from the Cell Bank of the Chinese Academy of Medical Sciences;

[0032] Epimedium total flavonoids were purchased from Sigma;

[0033] β-Ecdysterone was purchased from Biolabs;

[0034] TGF-β1 inhibitor SB431542 was purchased from Sigma, USA;

[0035] Collagenase type I was purchased from Gibco.

[0036] Example 1 Preparation of adipose-derived mesenchymal stem cells

[0037] 1) Obtain adipose tissue obtained from human liposuction under sterile conditions. Rinse the adipose tissue with physiological saline and dispense into 50 ml centrifuge tubes at a rate of 5 ml / tube.

[0038] 2) Add collagenase type I (purchased from Gibco) to the centrifuge tube containing fat, seal it, shake it upside down, and mix it thoroughly. Transfer it to a 37°C constant temperature air bath shaker and digest it for 30 minutes.

[0039] 3) First, ultrasonically treat the tube at 100 W for 60 seconds, then place the centrifuge tube in a 37°C constant temperature shaker and shake at 120 rpm for 20 minutes until the tube is digested into a uniform suspension.

[0040] 4) The obtained suspension was centrifuged at 1500 rpm for 10 minutes, the upper layer of fat cells and the middle liquid were discarded, the cell precipitate was collected, and the fat stem cell clusters at the bottom layer were retained.

[0041] 5) Add 10 ml of physiological saline to the centrifuge tube containing the adipose stem cell mass, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a 40-micron diameter filter, collect single cells, and then centrifuge at 1500 rpm for 5 minutes, discard the upper layer of liquid, and retain the adipose stem cell mass at the bottom; repeat this washing step 2 more times.

[0042] 6) After thorough washing, add an appropriate amount of physiological saline, repeatedly blow and beat the cells to make a single cell suspension of adipose mesenchymal stem cells, add them to a culture bottle containing DMEM + 20ng / ml total flavonoids of epimedium + 30ng / ml β-ecdysterone medium, and culture them in a 5% CO2 concentration 37°C incubator. When the cells are in culture on the 3rd and 6th days, the medium should be changed in time; on the 7th to 10th day, observe that 3-5 cell colonies reach more than 80%, and the cells can be passaged.

[0043] 7) The 3rd and 8th passage adipose-derived mesenchymal stem cells were used for the experiment.

[0044] 8) Cell identification: Adipose-derived mesenchymal stem cells of the 3rd and 8th passages can efficiently express CD49, CD73, CD105 and other adipose-derived mesenchymal stem cell markers, but do not express CD45 and HLA-DR. At the same time, the differentiation potential of adipose-derived mesenchymal stem cells of the 8th passage was detected. The results of Oil Red O staining and silver nitrate staining confirmed that adipose-derived mesenchymal stem cells have obvious adipogenic and osteogenic abilities.

[0045] Example 2

[0046] Except that the culture medium in step 6) is DMEM+20 ng / ml of total flavonoids from Epimedium, the other conditions are the same as those in Example 1.

[0047] Example 3

[0048] Except that the culture medium in step 6) is DMEM+30 ng / ml β-ecdysterone, the other conditions are the same as those in Example 1.

[0049] Example 4

[0050] The adipose-derived mesenchymal stem cells isolated from Examples 1, 2 and 3 were concentrated to 2×10 4 Pieces / cm 2 The cells were inoculated into a T25 culture flask at a density of 100 μg / mL. After 10 days, the number of clones with more than 10 cells was observed and recorded under a microscope. The cells obtained in Example 1 contained more adipose-derived mesenchymal stem cells with proliferation ability, as shown in Table 1.

[0051] Table 1 Proliferation ability of adipose-derived mesenchymal stem cells prepared in different examples

[0052] Group CFU-F number x 1000 Example 1 9.1 Example 2 2.2 Example 3 3.1

[0053] Example 5 Effect of adipose-derived mesenchymal stem cells on the expression of fibrosis-related genes in A549 cells

[0054] Cell groups: normal group, model group, control group, experimental group 1-experimental group 7.

[0055] Normal group (serum-free 1640 medium);

[0056] Model group (TGF-β1: 5 ng / mL);

[0057] Control group (TGF-β1: 5 ng / mL + TGF-β1 inhibitor SB431542 (purchased from Sigma, USA): 5 μmol / L);

[0058] Experimental group 1 (TGF-β1: 5 ng / mL + 2.0 × 10 5adipose-derived mesenchymal stem cells prepared in Example 1 + 100 μg / mL dextromethorphan);

[0059] Experimental group 2 (TGF-β1: 5 ng / mL + 2.0 × 10 5 adipose-derived mesenchymal stem cells prepared in Example 1 + 80 μg / mL dextromethorphan);

[0060] Experimental group 3 (TGF-β1: 5 ng / mL + 2.0 × 10 5 adipose-derived mesenchymal stem cells prepared in Example 1 + 120 μg / mL dextromethorphan);

[0061] Experimental group 4 (TGF-β1: 5 ng / mL + 2.0 × 10 5 adipose-derived mesenchymal stem cells prepared in Example 2 + 100 μg / mL dextromethorphan);

[0062] Experimental group 5 (TGF-β1: 5 ng / mL + 2.0 × 10 5 adipose-derived mesenchymal stem cells prepared in Example 3 + 100 μg / mL dextromethorphan);

[0063] Experimental group 6 (TGF-β1: 5 ng / mL + 2.0 × 10 5 adipose-derived mesenchymal stem cells prepared in Example 1);

[0064] Experimental group 7 (TGF-β1: 5 ng / mL + 100 μg / mL dextromethorphan);

[0065] Alveolar epithelial cell line (A549; 5.0×10 4 After 24 hours of attachment, 1640 without serum was added for synchronization for 24 hours. Reagents were added according to the above groups, and the cells were placed in a CO2 incubator (37°C, 5% CO2) for incubation. After 24 hours of incubation, the expression levels of fibrosis-related proteins SP-C, α-SMA and E-Cadherin were detected by immunofluorescence. The results are shown in Figure 1-Figure 3 . Figure 1-Figure 3 The results showed that TGF-β induction led to increased expression of SP-C and α-SMA proteins and decreased expression of E-Cadherin, while the combination of adding human adipose tissue-derived mesenchymal stem cells and dextromethorphan alleviated this phenomenon, thereby achieving the treatment of idiopathic pulmonary fibrosis.

[0066] It should be understood that the above are only some embodiments of the present invention. It should be pointed out that for ordinary technicians in this field, other modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing adipose-derived mesenchymal stem cells for treating idiopathic pulmonary fibrosis, characterized in that: The preparation method comprises the following steps: (1) Obtaining adipose tissue; (2) enzymatically hydrolyzing the adipose tissue of step (1) to obtain an enzymatic hydrolysis product; (3) ultrasonically treating the enzymatic hydrolysis product of step (2) to obtain an ultrasonically treated product; (4) centrifuging and washing the ultrasonic treatment product of step (3) to obtain a single cell suspension of adipose-derived mesenchymal stem cells; (5) culturing and passaging the adipose-derived mesenchymal stem cell single cell suspension obtained in step (4); The culture medium used in the culture comprises DMEM, total flavonoids from epimedium and β-ecdysterone.

2. The preparation method according to claim 1, characterized in that: The concentration of the total flavonoids of Epimedium is 20 ng / ml, and the concentration of the β-ecdysterone is 30 ng / ml.

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