WO2023217123A1 - Preparation method for and use of lung precursor-like cell - Google Patents

Abstract

A preparation method for a lung precursor-like cell and a use thereof. The preparation method comprises the following steps: taking lung tissue, and subjecting the lung tissue to digestion and separation to obtain primary lung cells; culturing the primary lung cells using a reprogramming medium until the cell confluence is not less than 80% to obtain the lung precursor-like cells. Dedifferentiation of the primary lung cells is achieved, and the lung precursor-like cells can rapidly and massively proliferate in vitro without any exogenous gene, achieving safe and reliable operation and a high batch yield, and achieving the treatment of multiple people multiple times using a single donor. The lung precursor-like cells can be used to repair damaged lung tissue.

Description

Preparation methods and applications of lung precursor-like cells

This application requires the priority of a Chinese patent application with a filing date of May 10, 2022, an application number of 2022105036786, and an invention title of "Biological preparations, cell derivatives, preparation methods and applications". The contents of the above application are incorporated herein by reference.

Technical field

The present invention relates to the field of biotechnology, and in particular to a preparation method and application of lung precursor-like cells.

Background technique

The lungs are important organs of the human body. In the process of performing breathing tasks, they are inevitably exposed to external or internal harmful substances (such as air pollution, tobacco, bacteria, viruses, or toxic substances in the blood), which often lead to large-scale damage to lung tissue. This can lead to acute and chronic lung injury diseases (such as respiratory distress syndrome ARDS, chronic obstructive pulmonary disease COPD, interstitial lung disease ILD, bronchiectasis BE, and idiopathic pulmonary fibrosis IPF, etc.). Currently, there is no effective treatment for these diseases to reverse the progression of the disease. As time goes by, the disease worsens year by year and becomes difficult to cure, eventually leading to death.

Commonly used clinical drugs for lung tissue damage diseases such as pulmonary fibrosis include chemical drugs such as glucocorticoids, immunosuppressants, and anticoagulants. On October 15, 2014, the FDA approved two new oral drugs, Nintedanib (trade name: Ofev) and pirfenidone (trade name: Esbriet), for the treatment of idiopathic pulmonary fibrosis. all It cannot cure pulmonary fibrosis and can only delay the progression of the disease. The treatment effect is not ideal and the adverse reactions are large. With the development of science, cell therapy has gradually appeared in the public eye. Mesenchymal stem cells have been widely used in the treatment of various diseases because of their paracrine, anti-inflammatory, immunomodulatory, antioxidant and other functions. Among them, Includes pulmonary fibrosis and chronic obstructive pulmonary disease. However, mesenchymal stem cells mainly promote lung damage repair and functional recovery by changing the inflammatory microenvironment of the lungs, but cannot induce liver tissue regeneration. At the same time, mesenchymal stem cells have the disadvantages of low transplantation rate and low local survival rate in treating lung injury. , therefore the long-term efficacy of mesenchymal stem cells in treating lung tissue damage such as pulmonary fibrosis remains to be seen.

At present, lung precursor-like cells have been proven to differentiate and regenerate new airway cells and alveolar epithelial cells, reconstruct blood-gas exchange units, repair damaged lung tissue, and are useful in the treatment of chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and other pulmonary diseases. Very good results have been achieved in local diseases. Autologous lung precursor-like cells developed by Professor Zuo Wei of Tongji University are also used in the treatment of pulmonary fibrosis. In 2018, the research team successfully isolated SOX9-positive lung precursor cells from the patient's lung bronchial epithelium and applied them in clinical experiments. The patient's lung function was effectively improved and maintained well 3-12 months after surgery.

However, the current status of lung precursor-like cell therapy for lung tissue damage diseases has the following shortcomings:

1. Treatment of lung precursor-like cells differentiated by ESCs/iPSCs: Currently, there are few reports on the induction of differentiation of ESCs/iPSCs to treat lung diseases. The main reason is that the culture protocol for in vitro induction and differentiation of stem cells into lung cells is still immature and lacks unified and standardized standards. iPSCs are at risk of developing teratomas. Moreover, the lung precursor-like cells differentiated from ESCs/iPSCs have high expression levels of MHC class II molecules, and patients need to receive long-term systemic immunosuppressive treatment. It brings a lot of pain and trouble to patients, and the impure differentiation of iPSCs also carries the risk of tumorigenesis.

2. Autologous lung precursor-like cell therapy: airway cells are brushed from the patient's bronchioles (level 3-4) through bronchoscopy, and the patient is required to have a high tolerance to this method of collecting airway cells. When patients have severe respiratory symptoms, it is difficult to tolerate this method of airway scraping cells. Therefore, the source of autologous lung precursor cells is greatly limited, and the audience group is greatly reduced.

Therefore, it is necessary to provide a novel preparation method and application of lung precursor-like cells to achieve large-scale expansion of lung precursor-like cells in vitro and apply them to the repair of damaged lung tissue.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of lung precursor-like cells, which can achieve a large amount of lung precursor-like cells expansion in vitro and be applied to the repair of damaged lung tissue.

In order to achieve the above object, the preparation method of the lung precursor-like cells of the present invention includes the following steps:

S1: Take lung tissue, digest and separate the lung tissue to obtain primary lung cells;

S2: Use the reprogramming medium to culture the primary lung cells until the cell confluence is no less than 80% to obtain lung precursor-like cells.

The beneficial effects of the preparation method of lung precursor-like cells are:

The lung tissue is digested and separated to obtain primary lung cells, and the reprogramming is used to The primary lung cells are cultured in the culture medium until the cell confluence is not less than 80% to obtain lung precursor-like cells, realize dedifferentiation of the primary lung cells, and enable the lung precursor-like cells to rapidly grow in vitro And a large amount of amplification, without any foreign genes, safe and reliable operation, high batch yield, can achieve multiple people from a single donor, multiple treatments; the lung precursor-like cells can be used to repair damaged lung tissue .

Optionally, in S1, the lung tissue is derived from normal lung tissue that cannot be used for transplantation.

Optionally, in S1, the step of obtaining primary lung cells after digestion and isolation of the lung tissue includes:

S10: Wash and disinfect the lung tissue sequentially with sterile PBS buffer, and then cut the lung tissue into pieces to obtain lung tissue fragments;

S11: Add self-prepared collagenase to the lung tissue fragments, and incubate the lung tissue fragments at 37 degrees Celsius for 30 minutes to obtain a primary lung cell suspension;

S12: Perform screen sorting on the primary lung cell suspension, and then collect the lung cell filtrate;

S13: Centrifuge the lung cell filtrate, discard the supernatant, and obtain the lung cell precipitate;

S14: Add red blood cell lysis balance solution to the lung cell precipitate and resuspend to obtain a lung cell mixture, then centrifuge the lung cell mixture, discard the supernatant, and obtain a lung cell precipitate;

S15: Repeat S14 until no red blood cells are observed in the lung cell pellet. end.

Optionally, in step S10, the size of the lung tissue after cutting is 1-2 mm ³ .

Optionally, in S11, in terms of volume percentage of the self-formulated collagenase, the components of the self-formulated collagenase include: 25%-50% neutral protease II and 50%-75% Type II collagenase. The beneficial effect is that the self-prepared collagenase can fully digest the lung tissue fragments.

Optionally, in step S12, when performing screen sorting on the primary lung cell suspension, the cell filter used has a pore size of 60 to 80 microns.

Optionally, in step S13, when the lung cell filtrate is centrifuged, the centrifugation rate is 1000 rpm and the centrifugation time is 5 minutes.

Optionally, in step S14, when the lung cell mixture is centrifuged, the centrifugation speed is 1000 rpm and the centrifugation time is 5 minutes.

Optionally, in the S2, the reprogramming medium includes basal medium, serum-free supplements, growth factors, TGF-β signaling inhibitors, Wnt signaling pathway activators and ROCK kinase inhibitors.

Optionally, based on the volume of the basal culture medium, the content of the growth factor is 10-50 ng/ml, the content of the ROCK kinase inhibitor is 1-20 micromoles, and the Wnt signaling pathway The content of the activator is 1-10 micromoles, the content of the TGF-β signal inhibitor is 1-10 micromoles, and the volume content of the serum-free additive does not exceed 10%.

Optionally, the basal medium is Ham's F-12 medium.

Optionally, the growth factor is at least one of epidermal growth factor and fibroblast growth factor.

Optionally, the nutritional supplement includes at least one of N2 and B27.

Further optionally, the ROCK kinase inhibitor includes Y-27632.

Further optionally, the Wnt signaling pathway activator includes CHIR-99021.

Further optionally, TGF-β signaling inhibitors include A-8301.

Optionally, in the S2, the reprogramming medium also includes triiodothyronine and hydrocortisone. The beneficial effect is that: the triiodothyronine and the hydrocortisone can ensure the proliferation of the lung precursor-like cells and maintain the markers of the lung precursor-like cells.

Further optionally, the content of triiodothyronine is 2 μg/ml, and the content of hydrocortisone is 0.5 μg/ml.

Optionally, after S2, step S3 is also included: after digesting the lung precursor-like cells, using the reprogramming medium for subculture to obtain the subcultured lung precursor-like cells, and the subcultured cells The number of generations is no less than 20. The beneficial effect is that after the lung precursor-like cells are digested and processed, the reprogramming medium is used for subculture to obtain the lung precursor-like cells, and the lung precursor-like cells can be used to maintain epithelial precursor morphology. Stable passage under low conditions achieved large-scale expansion of the lung precursor-like cells in vitro.

Optionally, in the S3, the step of using the reprogramming medium for subculture includes:

S31: Aspirate the supernatant of the reprogramming culture medium, wash the lung precursor-like cells twice with sterile PBS buffer, and add trypsin digestion solution to the lung precursor-like cells for 5-8 seconds. minutes of digestion to obtain a mixed solution;

S32: Centrifuge the mixed solution, discard the supernatant, and obtain lung precursor-like cell precipitates;

S33: Precipitate the lung precursor-like cells for cell counting, and then inoculate the lung precursor-like cells in the reprogramming medium to obtain the first generation of lung precursor-like cells;

S34: Use the reprogramming medium to subculture the first-generation lung precursor-like cells.

Optionally, in S32, when the mixed solution is centrifuged, the centrifugation rate is 200g and the centrifugation time is 5 minutes.

Optionally, the lung precursor-like cells positively express at least one characteristic marker.

Optionally, the expression rate of the characteristic marker is no less than 50% and no more than 99%. The beneficial effect is that the cells whose expression rate of the characteristic marker reaches the target are the lung precursor-like cells defined in the patent of the present invention.

Further optionally, the expression rate of the characteristic marker is higher than 70%.

Optionally, at least one of the characteristic markers includes at least one of CD24, CD73, CD326, CK19, and Sox9.

Optionally, the lung precursor-like cells negatively express at least one MHC class II molecule, and at least one of the MHC class II molecules includes at least one of HLA-DR/DP/DQ. That The beneficial effect is that the lung precursor-like cells do not express MHC-II antigens. After the patient transplants the lung precursor-like cells, the patient's body cannot recognize the exogenous cells through MHC-II molecules and will not treat them. Generates immune attack, so rejection is less likely.

Optionally, the lung precursor-like cells are differentiated and cultured using a differentiation medium to obtain at least one of airway secretory cells, type I alveolar cells and type II alveolar cells.

Optionally, the differentiation medium includes basal medium, serum-free supplements, and growth factors.

Further optionally, the basic culture medium is DMEM/F-12 culture medium.

Further optionally, the growth factor includes at least one of fibroblast growth factor and hepatocyte growth factor.

Further optionally, the serum-free additive includes at least one of transferrin and bovine serum albumin.

Further optionally, based on the volume of the basal culture medium, the content of the fibroblast growth factor is 10-100 ng/ml, and the content of transferrin is 2-10 μg/ml, so The volume content of bovine serum albumin does not exceed 10%.

The present invention also provides an application of lung precursor-like cells, using the lung precursor-like cells prepared by the above-mentioned preparation method of lung precursor-like cells to intervene in in vivo animal models. The beneficial effect is that the lung precursor-like cells can be used to repair damaged lung tissue.

Optionally, the in vivo animal model includes any one of a rat chronic obstructive pulmonary disease model and a mouse idiopathic pulmonary fibrosis model.

Description of the drawings

Figure 1 is a schematic diagram of an inverted microscope imaging photograph of P3 generation lung precursor-like cells in Example 2-1 provided by the present invention;

Figure 2 is a schematic diagram of an inverted microscope imaging photograph of P23 generation lung precursor-like cells in Example 2-1 provided by the present invention;

Figure 3 is a schematic diagram of an inverted microscope imaging photograph of P5 generation lung precursor-like cells in Example 2-1 provided by the present invention;

Figure 4 is a schematic diagram of an inverted microscope imaging photograph of P5 lung precursor-like cells subcultured using a control medium in Example 2-2 provided by the present invention;

Figure 5 is a growth curve of lung precursor-like cells on different culture media in Examples 2-3 provided by the present invention;

Figure 6 is a flow chart of surface markers of P3 generation lung precursor-like cells in Example 3 provided by the present invention;

Figure 7 is a flow chart of intracellular markers of P3 generation lung precursor-like cells in Example 3 provided by the present invention;

Figure 8 is a gene expression diagram before and after differentiation of P6 generation lung precursor-like cells in Example 4 provided by the present invention;

Figure 9 shows the modeling group, airway drug administration group, and tail vein drug administration in Example 5-1 provided by the present invention. Change curve of gas consumption of rats in the group and normal group;

Figure 10 is a diagram of the dry and wet weight ratio of rat lung tissue in the modeling group, airway administration group, tail vein administration group and normal group in Example 5-2 provided by the present invention;

Figure 11 is a graph showing Masson staining results of rat lung tissue in the modeling group, airway administration group, tail vein administration group and normal group in Example 5-3 provided by the present invention;

Figure 12 is a graph showing changes in respiratory frequency of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-1 provided by the present invention;

Figure 13 is a graph showing the arterial blood gas analysis results of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-2 provided by the present invention.

Figure 14 is a graph showing HE staining results of lung tissue of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-3 provided by the present invention;

Figure 15 is a graph showing Masson staining results of lung tissue of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-3 provided by the present invention;

Figure 16 is a graph showing the detection results of pulmonary tissue fibrosis molecule protein concentration of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-4 provided by the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the following will be combined with the present invention. The accompanying drawings clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. Unless otherwise defined, technical or scientific terms used herein shall have their ordinary meaning understood by one of ordinary skill in the art to which this invention belongs. The use of "comprising" and similar words herein means that the elements or things appearing before the word include the elements or things listed after the word and their equivalents, without excluding other elements or things.

In view of the problems existing in the prior art, embodiments of the present invention provide a preparation method and application of lung precursor-like cells, which can achieve large-scale expansion of lung precursor-like cells in vitro and be applied to damaged lung tissue. of repair.

The preparation method of the lung precursor-like cells of the present invention includes the following steps:

S1: Take lung tissue, digest and separate the lung tissue to obtain primary lung cells;

S2: Use the reprogramming medium to culture the primary lung cells until the cell confluence is no less than 80% to obtain lung precursor-like cells.

The advantages of the preparation method of lung precursor-like cells are:

Primary lung cells are obtained after digestion and separation of the lung tissue, and the primary lung cells are cultured using the reprogramming medium until the cell confluence is not less than 80% to obtain lung precursor-like cells to achieve the desired goal. The primary lung cells are dedifferentiated and the lung precursor-like cells are rapidly and massively expanded in vitro without any exogenous genes. The operation is safe and reliable, with high batch yield, and can realize multiple people from a single donor. Multiple treatments; the lung precursor-like cells can Used to repair damaged lung tissue.

In some embodiments, in S1, the lung tissue is derived from normal lung tissue that cannot be used for transplantation.

In some embodiments, in S1, the step of obtaining primary lung cells after digestion and isolation of the lung tissue includes:

S10: Wash and disinfect the lung tissue sequentially with sterile PBS buffer, and then cut the lung tissue into pieces to obtain lung tissue fragments;

S11: Add self-prepared collagenase to the lung tissue fragments, and incubate the lung tissue fragments at 37 degrees Celsius for 30 minutes to obtain a primary lung cell suspension;

S12: Perform screen sorting on the primary lung cell suspension, and then collect the lung cell filtrate;

S13: Centrifuge the lung cell filtrate, discard the supernatant, and obtain the lung cell precipitate;

S14: Add red blood cell lysis balance solution to the lung cell precipitate and resuspend to obtain a lung cell mixture, then centrifuge the lung cell mixture, discard the supernatant, and obtain a lung cell precipitate;

S15: Repeat S14 until no red blood cells are observed in the lung cell pellet.

In some embodiments, in S10, the lung tissue has a size of 1-2 mm ³ after being minced.

In some embodiments, in S11, based on the volume percentage of the self-formulated collagenase, the components of the self-formulated collagenase include: 25%-50% neutral protease II and 50%-75 % type II collagenase. The advantage is that the self-prepared collagenase can fully digest the lung tissue fragments.

In some embodiments, in step S12, when performing screen sorting on the primary lung cell suspension, the cell filter used has a pore size of 60 to 80 microns.

In some embodiments, in S13, when the lung cell filtrate is centrifuged, the centrifugation rate is 1000 rpm and the centrifugation time is 5 minutes.

In some embodiments, in step S14, when the lung cell mixture is centrifuged, the centrifugation speed is 1000 rpm and the centrifugation time is 5 minutes.

In some embodiments, in the S2, the reprogramming medium includes basal medium, serum-free supplements, growth factors, TGF-β signaling inhibitors, Wnt signaling pathway activators, and ROCK kinase inhibitors.

In some embodiments, the content of the growth factor is 10-50 ng/ml based on the volume of the basal culture medium, the content of the ROCK kinase inhibitor is 1-20 micromoles, and the Wnt signal The content of the pathway activator is 1-10 micromoles, the content of the TGF-β signal inhibitor is 1-10 micromoles, and the volume content of the serum-free additives does not exceed 10%.

In some embodiments, the basal medium is Ham's F-12 medium.

In some embodiments, the growth factor is at least one of epidermal growth factor and fibroblast growth factor.

In some embodiments, the nutritional supplement includes at least one of N2 and B27.

In some embodiments, the ROCK kinase inhibitor includes Y-27632.

In some specific embodiments, the Wnt signaling pathway activator includes CHIR-99021.

In some embodiments, the TGF-β signaling inhibitor includes A-8301.

In some embodiments, in the S2, the reprogramming medium further includes triiodothyronine and hydrocortisone. The advantage is that the triiodothyronine and the hydrocortisone can ensure the proliferation of the lung precursor-like cells and maintain the markers of the lung precursor-like cells.

In some embodiments, the content of triiodothyronine is 2 μg/ml, and the content of hydrocortisone is 0.5 μg/ml.

In some embodiments, after S2, step S3 is also included: after digesting the lung precursor-like cells, using the reprogramming medium for subculture to obtain subcultured lung precursor-like cells, and the subcultured lung precursor-like cells are The cell passage number should not be less than 20 generations. The beneficial effect is that after the lung precursor-like cells are digested and processed, the reprogramming medium is used for subculture to obtain the lung precursor-like cells, and the lung precursor-like cells can be used to maintain epithelial precursor morphology. Stable passage under low conditions achieved large-scale expansion of the lung precursor-like cells in vitro.

In some embodiments, in the S3, the step of using the reprogramming medium for subculture includes:

S31: Aspirate the supernatant of the reprogramming culture medium, wash it twice with sterile PBS buffer, add trypsin digestion solution to the lung precursor-like cells and perform digestion for 5-8 minutes to obtain a mixture. solution;

S32: Centrifuge the mixed solution, discard the supernatant, and obtain lung precursor-like cell precipitates;

S33: Precipitate the lung precursor-like cells for cell counting, and then inoculate the lung precursor-like cells in the reprogramming medium to obtain the first generation of lung precursor-like cells;

S34: Use the reprogramming medium to subculture the first-generation lung precursor-like cells.

In some embodiments, in S32, when the mixed solution is centrifuged, the centrifugation rate is 200g and the centrifugation time is 5 minutes.

In some embodiments, the lung precursor-like cells positively express at least one characteristic marker.

In some embodiments, the expression rate of the characteristic marker is no less than 50% and no more than 99%. The advantage is that the cells whose expression rate of the characteristic marker reaches the target are the lung precursor-like cells defined in the patent of the present invention.

In some specific embodiments, the expression rate of the characteristic marker is higher than 70%.

In some embodiments, at least one of the characteristic markers includes at least one of CD24, CD73, CD326, CK19, and Sox9.

In some embodiments, the lung precursor-like cells negatively express at least one MHC class II molecule, and at least one of the MHC class II molecules includes at least one of HLA-DR/DP/DQ. The advantage is that the lung precursor-like cells do not express MHC-II antigens. After the patient transplants the lung precursor-like cells, the patient's body cannot recognize the exogenous cells through MHC-II molecules and will not treat them. Generates immune attack, so rejection is less likely.

In some embodiments, at least one of airway secretory cells, type I alveolar cells and type II alveolar cells can be obtained by using a differentiation medium to differentiate and culture the lung precursor-like cells.

In some embodiments, the differentiation medium includes basal medium, serum-free supplements, and growth factors.

In some specific embodiments, the basal culture medium is DMEM/F-12 culture medium.

In some embodiments, the growth factor includes at least one of fibroblast growth factor and hepatocyte growth factor.

In some specific embodiments, the serum-free additive includes at least one of transferrin and bovine serum albumin.

In some specific embodiments, based on the volume of the basal culture medium, the content of the fibroblast growth factor is 10-100 ng/ml, and the content of transferrin is 2-10 μg/ml, The volume content of bovine serum albumin does not exceed 10%.

The present invention also provides an application of lung precursor-like cells, using the lung precursor-like cells prepared by the above-mentioned preparation method of lung precursor-like cells to intervene in in vivo animal models. The advantage is that the lung precursor-like cells can be used to repair damaged lung tissue.

In some embodiments, the in vivo animal model includes any one of a rat chronic obstructive pulmonary disease model and a mouse idiopathic pulmonary fibrosis model.

The following is a detailed description through specific examples:

Example 1

This example provides the acquisition of primary lung cells

1. Statement on the nature of the starting organization and the legality of the source:

Normal lung tissue that cannot be used for transplantation is used as the starting material.

Specifically, the normal lung tissue that cannot be used for transplantation is shown to be normal lung tissue by pathological examination.

Specifically, the normal lung tissue that cannot be used for transplantation is a surgical sample derived from a patient who is no more than 70 years old. The patient has no infectious viral infection after medical examination, and the patient has not used steroid hormones within 6 months before surgery. drug. The patient was fully informed about the purpose of obtaining surgical samples before surgery and signed an informed consent form.

2. Specific steps:

Preparation of self-prepared collagenase: Use sterile PBS buffer to prepare self-prepared collagenase containing 25% neutral protease II and 75% type II collagenase based on the volume percentage of the self-prepared collagenase.

Among them, neutral protease II was purchased from Sigma, product number d4693; type II collagenase was purchased from Aibixin Biotech, product number abs47048001; sterile PBS buffer was purchased from Yuanpei, product number B310KJ.

Obtain normal lung tissue, use sterile PBS buffer to clean the acquired lung tissue, and soak the cleaned lung tissue in 10 mL of PBS buffer containing 1% gentamicin for 5 minutes for disinfection. Cut the sterilized lung tissue into pieces so that the size of the chopped lung tissue is 1-2mm ³ . Transfer the chopped lung tissue to a 15mL centrifuge tube, and add 5mL of self-prepared solution to the centrifuge tube. Collagenase and place the centrifuge tube Obtain the cell suspension by incubating for 30 min at 37 °C in an incubator containing 5% _CO2 . Among them, the volume of self-prepared collagenase and the weight ratio of lung tissue are 10:1, that is, 10 mL of collagenase is added to 1 g of tissue.

Add an equal volume of sterile PBS buffer to the cell suspension for dilution, and then use a 70-micron cell strainer to screen the cell suspension to collect the cell filtrate and remove mucus and undigested tissue from the cell suspension. Then, the cell filtrate was put into a centrifuge, and the cell filtrate was centrifuged at 1000 rpm for 5 minutes. The supernatant of the centrifuged cell filtrate was discarded to obtain a cell pellet.

Add 2 mL of red blood cell lysis balance solution to the obtained cell pellet to obtain a cell mixture, resuspend the cell mixture, put the resuspended cell mixture into a centrifuge again, and centrifuge the cell mixture at 1000 rpm for 5 seconds. minutes, discard the supernatant of the centrifuged cell mixture to obtain the centrifuged cell pellet, repeatedly add red blood cell lysis balance solution to the centrifuged cell pellet, and perform the process of resuspension and centrifugation again until Until no red blood cells are observed in the cell pellet obtained by centrifugation again, the red blood cells are lysed and removed to obtain primary lung cells, which are recorded as Pr-generation lung cells. Specifically, the rate of each centrifugation process was 1000 rpm, and the centrifugation time was 5 minutes.

Among them, the red blood cell lysing balance solution was purchased from Soleba, and the product number is R1010.

Example 2-1

The Pr-generation lung cells obtained in Example 1 were seeded on a 6-well culture plate at a seeding density of 1E+04 cells/cm2, and 2 ml of reprogramming medium was added to each well of the culture plate for cell culture. , replace the new culture medium every two days until each If the cell confluence in the well is not less than 80% and the cells are in good growth status, the expansion culture is completed. Among them, the culture plate was purchased from Corning, the product number is 3516.

Aspirate the supernatant of the reprogramming medium on the culture plate, wash the culture plate twice with sterile PBS buffer, and drop 1 mL of trypsin digestion solution into each well of the culture plate for 5-8 minutes of digestion. Process to obtain a mixed solution; centrifuge the mixed solution at a centrifugation rate of 200g for 5 minutes to obtain a cell pellet, count the cells in the cell pellet, and inoculate the reprogramming culture at a density of 1E+04 cells/ ^cm2 In the base, the first generation lung precursor-like cells were obtained, which were designated as P1 generation lung precursor-like cells. Among them, trypsin digestion solution was purchased from Yuanpei, and the product number is S310JV.

Inoculate the P1 generation lung precursor-like cells into the new reprogramming medium at a ratio of 1E+04 cells/ ^cm2 and subculture to the third generation according to the above operations. The cell confluence is not less than 80% and the growth status is good. , the third generation lung precursor-like cells were recorded as P3 generation lung precursor-like cells.

P3 generation lung precursor-like cells were inoculated into the new reprogramming medium at a density of 1E+04 cells/ ^cm2 and subcultured to the 23rd generation according to the above operations. The 23rd generation lung precursor-like cells were recorded as the P23 generation. Lung precursor-like cells, the cell morphology has not changed significantly and the growth status is good.

The reprogramming medium used consists of the following: Ham's F-12 basal medium, and 1% N2 nutritional supplement and 2% B27 nutritional supplement based on the volume of Ham's F-12 basal medium. ; Epithelial cell growth factor EGF at a content of 20ng/mL, fibroblast growth factor bFGF at a content of 50ng/mL, triiodothyronine (Taoshu, T1653) at a content of 2ug/mL, 0.5μg/mL Hydrogen can It contains 10uM of ROCK kinase inhibitor Y-27632, 3uM of Wnt signaling pathway activator CHIR-99021, and 1uM of TGF-β signaling inhibitor A8301.

Among them, the epithelial cell growth factor EGF was purchased from abcam, the product number is ab259398; the ROCK kinase inhibitor Y-27632 was purchased from TargetMol, the product number is T1870; the Wnt signaling pathway activator CHIR-99021 was purchased from TargetMol, the product number is T2310; TGF-β signal Inhibitor A-8301 was purchased from TargetMol, product number: T3031; fibroblast growth factor bFGF was purchased from biorbyt, product number: orb80024; Ham's F-12 basal medium was purchased from Thermo Fisher, product number: 31765035; triiodothyronine was purchased from From Taoshu Biotech, the product number is T1653; Hydrocortisone was purchased from Taoshu Biotech, the product number is T1614; N2 nutritional supplement (1X) was purchased from Thermo Fisher, the product number is 17502048; B27 nutritional supplement (1X) was purchased from Thermo Fisher , the item number is 12587010.

In this example, after the Pr generation lung cells obtained in Example 1 were subcultured using reprogramming medium, the expansion fold of each generation of lung precursor-like cells was calculated. The specific data are shown in Table 1.

Table 1

It can be seen from the data in Table 1 that the expansion rate of P23 generation lung precursor-like cells has not changed significantly compared with that of P3 generation lung precursor-like cells, which shows that when reprogramming medium is used, and lung precursor-like cells in vitro Proliferation is stable.

In this example, P3 generation lung precursor-like cells and P23 generation lung precursor-like cells were imaged through an inverted microscope. The cell pictures are shown in Figures 1 and 2. Figure 1 shows the P3 generation lung precursor-like cells in Example 2-1 provided by the present invention. Inverted microscope imaging of somatic cells; Figure 2 is an inverted microscope imaging of P23 generation lung precursor-like cells in Example 2-1 provided by the present invention.

Referring to Figures 1 and 2, it can be seen that the cell morphology of the P23 generation lung precursor-like cells has not changed significantly compared with the P3 generation lung precursor-like cells, indicating that the lung precursor-like cells proliferate stably in vitro when using reprogramming medium. .

Example 2-2

The Pr generation lung cells obtained in Example 1 were inoculated on the control medium for amplification, culture and subculture to the P5 generation, which was the same as the Pr generation lung cells using the reprogramming medium in Example 2-1. A control group was formed, and the culture steps in this example were consistent with those in Example 2-1.

Compared with the reprogramming medium, the control medium lacked triiodothyronine and hydrocortisone, and the remaining components and contents were consistent with the reprogramming medium.

The P5 lung precursor-like cells subcultured in this example and the P5 lung precursor-like cells subcultured using the reprogramming medium in Example 2-1 were imaged and photographed under an inverted microscope. The cell pictures are shown in Figures 3 and 4 , Figure 3 is an inverted microscope image of P5 lung precursor-like cells in Example 2-1 provided by the present invention; Figure 4 is a P5 lung precursor-like cell subcultured using a control medium in Example 2-2 provided by the present invention. Inverted microscope image of somatic cells.

Referring to Figures 3 and 4, it can be seen that when the Pr generation lung cells obtained in Example 1 were subcultured to the P5 generation in the control medium, the lung precursor-like cells deteriorated, their diameters became larger, and their cytoplasm became transparent and spread out, making it impossible to continue. Therefore, the cell culture capacity of the reprogramming medium is much greater than that of the control medium, and the triiodothyronine and hydrocortisone in the reprogramming medium can ensure the proliferation of the lung precursor-like cells.

Example 2-3

The Pr-generation lung cells obtained in Example 1 were expanded, cultured and sub-cultured using DMEM+FBS medium, and the Pr-generation lung cells were expanded, cultured and sub-cultured using the reprogramming medium in Example 2-1 to form a control group. , the culture steps in this example are consistent with those in Example 2-1.

The composition of the DMEM+FBS medium used is as follows: based on the volume percentage of the DMEM+FBS medium, it includes 90% DMEM medium and 10% FBS medium.

Among them, DMEM culture medium was purchased from Yuanpei with the product number L110KJ; FBS culture medium was purchased from Corning with the product number 35081-CV.

The growth curve of lung precursor-like cells cultured using reprogramming medium in Example 2-1 and In this example, the growth curve of lung precursor-like cells cultured using DMEM+FBS medium is plotted on a graph, see Figure 5 .

Figure 5 is the growth curve of lung precursor-like cells on different culture media in Examples 2-3 provided by the present invention. Referring to Figure 5, it can be seen that the lung precursor-like cells cultured in the reprogramming medium can be expanded to at least the 23rd passage in vitro, while the lung precursor-like cells cultured in the DMEM+FBS medium can be expanded to the 4th passage at most. generation, indicating that the proliferation ability of lung precursor-like cells cultured in reprogramming medium is much greater than that of lung precursor-like cells cultured in DMEM+FBS medium, and the cell culture ability of reprogramming medium is much greater than that of DMEM+FBS medium.

Example 3

Use flow cytometry to perform identification analysis on the P3 generation lung precursor-like cells obtained in Example 2-1.

Surface marker staining was performed on the P3 generation lung precursor-like cells in Example 2-1:

Sample the P3 generation lung precursor-like cells in Example 2-1, aspirate the reprogramming medium, rinse with 5 mL of sterile PBS buffer, and then drop 2 mL of trypsin digestion solution into the culture dish for digestion to obtain For the cell mixture, place the cell mixture in a 15mL centrifuge tube, put the centrifuge tube into a centrifuge, and centrifuge at a speed of 200g for 5 minutes. Discard the supernatant of the centrifuged cell mixture to obtain the cells. Precipitate.

Add 700 μL of staining buffer to the cell pellet to resuspend the cell pellet. Transfer the resuspended cell pellet to six 1.5 mL centrifuge tubes with a specification of 100 μ L/tube. Add 5 μL of the flow cytometry antibody to be tested into the six 1.5 mL centrifuge tubes mentioned above, and mix by pipetting. Place the centrifuge tubes in a refrigerator at 2-8°C for 30 minutes. Add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube. Then put the centrifuge tubes into a centrifuge and mix at 300 g. Centrifuge at high speed for 5 minutes. After centrifugation, discard the supernatant, add 400 μL of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture will be used for flow cytometric detection of surface markers. .

The names of antibodies used in surface marker flow cytometric detection are: CD326, CD90, CD73, CD24, CD44, HLV-DR/DP/DQ.

Among them, CD326 was purchased from BD Biosciences, the catalog number is 565399; CD90 was purchased from BD Biosciences, the catalog number is 555595; CD73 was purchased from BD Biosciences, the catalog number is 561254; CD24 was purchased from abcam, the catalog number is ab290730; CD44 was purchased from abcam, the catalog number is ab254530; HLV-DR/DP/DQ was purchased from abcam, the catalog number is ab7856; the staining buffer was purchased from BD Biosciences, the catalog number is 554656, and the trypsin digestion solution was purchased from Yuanpei, the catalog number is S310JV.

Perform intracellular marker staining on the P3 generation lung precursor-like cells in Example 2-1:

Sample the P3 generation lung precursor-like cells in Example 2-1, aspirate the reprogramming medium, rinse with 5 mL of sterile PBS buffer, and then drop 2 mL of trypsin digestion solution into the culture dish for digestion to obtain For the cell mixture, place the cell mixture in a 15mL centrifuge tube, put the centrifuge tube into a centrifuge, and centrifuge at a speed of 200g for 5 minutes. Discard the supernatant of the centrifuged cell mixture to obtain the cells. sink sediment.

Add 1 mL of fixed membrane-penetrating solution to the cell pellet, place the centrifuge tube in a refrigerator at 2-8°C for 50 minutes, add 2 mL of sterile PBS buffer to the centrifuge tube, and rotate the centrifuge tube at 300g for 5 minutes. After centrifugation, add 500 μl of staining buffer to the centrifuge tube to resuspend. Transfer the resuspended cell pellet to four 1.5 mL centrifuge tubes, with a specification of 100 μL/tube. Add 5 μL of the flow cytometry antibody to be tested to each of the above four 1.5 mL centrifuge tubes, mix by pipetting, and place the centrifuge tubes in an incubator containing 5% _CO2 at 37°C for 30 minutes. After the incubation, add sterile PBS buffer to each centrifuge tube at a dose of 800 μl/tube, then place the centrifuge tube into a centrifuge and centrifuge at a speed of 300g for 5 minutes. After centrifugation, discard the supernatant, add 400 μL of staining buffer to each centrifuge tube to resuspend the cell pellet, and then transfer it to a flow tube. The resuspended cell mixture is subjected to flow cytometry for intracellular markers. detection.

The names of antibodies used in flow cytometric detection of intracellular markers are: krt5, P63, CK19, and Sox9.

Among them, krt5 was purchased from abcam, the product number is ab270900; P63 was purchased from abcam, the product number is ab246727; CK19 was purchased from abcam, the product number is ab205445; Sox9 was purchased from abcam, the product number is ab208427; the fixed transmembrane solution was purchased from BD Biosciences, the product number is 554714 , the trypsin digestion solution was purchased from Yuanpei, the catalog number is S310JV, and the staining buffer was purchased from BD Biosciences, the catalog number is 554656.

Flow cytometric detection of surface markers and intracellular markers of P3 generation lung precursor-like cells The flow cytometric detection diagrams are shown in Figures 6 and 7. Figure 6 is a flow chart of surface markers of P3 generation lung precursor-like cells in Example 3 provided by the present invention; Figure 7 is a flow chart of intracellular markers of P3 generation lung precursor-like cells in Example 3 provided by the present invention. Detection chart.

Referring to Figure 6, it can be seen that the P3 generation lung precursor-like cells cultured using the reprogramming medium in Example 2 positively expressed the surface markers CD24, CD73, and CD326, and negatively expressed the surface marker HLA-DRPQ; referring to Figure 7, it can be seen that , the P3 generation lung precursor-like cells cultured using the reprogramming medium in Example 2 positively expressed the intracellular markers SOX9 and CK19. In Example 2, the P3 generation lung precursor-like cells cultured using the reprogramming medium positively expressed the lung precursor-related markers CD24, CD73, CD326, CK19, and SOX9, and the expression rate of CD326/CD24/CD73/CK19/SOX9 More than 70%, P3 generation lung precursor-like cells show characteristics of lung precursor-like cells; in Example 2, the P3 generation lung precursor-like cells cultured using reprogramming medium negatively express HLA-DRPQ, indicating that the cells do not express MHC-II antigen. After the patient transplants the cells, the body cannot recognize the cells through the MHC-II antigen of the immune system. The patient's immune system will not launch an immune attack on them, so the patient's body may have a rejection reaction. Small.

Example 4

The P6 generation lung precursor-like cells subcultured in Example 2-1 were sampled, differentiated and cultured, and the markers of the lung precursor-like cells before and after differentiation were detected by real-time fluorescence quantitative PCR.

The types of gene expression markers before and after differentiation of lung precursor-like cells are shown in Table 2.

Table 2

When the confluence of the P6 generation lung precursor-like cells cultured in the reprogramming medium in Example 2-1 reaches 80%, sample some of the P6 generation lung precursor-like cells and transfer them into a 15 mL centrifuge tube. Put it into a centrifuge and centrifuge the cell mixture in the centrifuge tube at a speed of 200g for 5 minutes. Discard the supernatant after centrifugation to obtain the cell precipitate. Then collect the cells and detect them with real-time fluorescence quantitative PCR.

When the confluence of the P6 generation lung precursor-like cells cultured in the reprogramming medium in Example 2-1 reaches 80%, sample some of the P6 generation lung precursor-like cells, discard the supernatant of the reprogramming medium, and use Wash twice with 10 mL sterile PBS buffer, then add the P6 generation lung precursor-like cells to the differentiation medium, place them in an incubator containing 5% _CO2 at 37°C for differentiation for 7 days, and then collect the cells and perform real-time fluorescence quantitative PCR. detection.

Use a fluorescence quantitative PCR instrument (Manufacturer: Applied Biosystems, Catalog No.: 7300Plus) to perform a fluorescence test on lung precursor-like cells after differentiation and culture. The specific test method is a routine technical method for those skilled in the art, and will not be described in detail here.

The composition of the differentiation medium used is as follows: DMEM/F-12 basal medium, and fibroblast growth factor bFGF at a content of 50 ng/mL based on the volume of DMEM/F-12 basal medium and a content of 5 μg/mL. transferrin, hepatocyte growth factor HGF at 20ng/mL and bovine serum albumin at 5%.

Among them, DMEM/F-12 basal medium was purchased from Yuanpei, with the product number of L310KJ; fibroblast growth factor bFGF was purchased from biorbyt, with the product number of orb80024; hepatocyte growth factor HGF was purchased from abcam, with the product number of ab632; transferrin was purchased from It was purchased from Solebao, the product number is T8010; bovine serum albumin was purchased from Solebao, the product number is A8020.

The gene expression of the P6 generation lung precursor-like cells before and after differentiation is shown in Figure 8. Figure 8 is a gene expression diagram of the P6 generation lung precursor-like cells before and after differentiation in Example 4 provided by the present invention. The ordinate of Figure 8 is the expression level of gene expression markers in lung precursors. Referring to Figure 8, it can be seen that the HOPX expression level of lung precursor-like cells cultured in differentiation medium is significantly increased by 28 times compared with lung precursor-like cells before differentiation, and HOPX is a marker of alveolar type I cells, proving that lung precursor-like cells The cells can differentiate and regenerate to form alveolar type I cells.

Example 5

This example provides a modeling method for a rat chronic obstructive pulmonary disease (COPD) model, and uses the lung precursor-like cells in Example 2 to intervene in the rat chronic obstructive pulmonary disease (COPD) model. Examine Example 2 Role of pulmonary precursor-like cells in chronic obstructive pulmonary disease.

In this example, 16 7-week-old SPF male SD rats purchased from Beijing Vitong Lever Company were used for modeling. The average weight of the rats reached 180g.

Preparation of inducer: Weigh 10 mg of LPS and pour it into a beaker, add 1 mL of NS into the beaker, vortex thoroughly to dissolve, and prepare a 10 mg/mL LPS mother solution. Weigh 6 mg of powdered 24U PPE into another beaker, add 1.8 mL of NS and 0.2 mL of the LPS mother liquor configured above into the beaker, and vortex thoroughly to dissolve, to prepare a solution containing 1 mg/mL. LPS and 24U/mL PPE as inducers. Store the inducer temporarily in the dark at 2 to 8°C and prepare it for immediate use.

Among them, LPS was purchased from Sigma, the product number is L2630; NS was purchased from Chenxin Pharmaceutical Co., Ltd., the product number is 2102020728; PPE was purchased from Sigma, the product number is E1250.

Modeling of the rat chronic obstructive pulmonary disease (COPD) model: use isoflurane to anesthetize the rat, load the induction agent into the drug delivery device, and insert the drug delivery device orally into the trachea of the anesthetized rat. Quickly push 100 μL of inducer into the trachea of the rat. After the induction of the inducer is completed, put the rat back into the cage and raise it. The inducer was injected once a day for 3 consecutive days to complete the rat modeling.

Intervention experiment of lung precursor-like cells on rat model of chronic obstructive pulmonary disease (COPD):

The P3 generation lung precursor-like cells in Example 2-1 were sampled, and NS was added to form a cell preparation of lung precursor-like cells. NS was purchased from Chenxin Pharmaceutical Co., Ltd., the product number is 2102020728.

The grouping of rats is as follows: Twelve rats were modeled. After the modeling was completed, the rats were returned to the cage and raised for one day without any treatment. Then the rats were randomly divided into three groups according to their breathing frequency and air consumption. There were four rats in each group, namely the modeling group, the airway administration group and the tail vein administration group; the remaining four rats were not subjected to any treatment and served as the normal group.

The situation of rats in the modeling group, airway administration group and tail vein administration group using cell preparations of lung precursor-like cells is as follows:

Modeling group: No processing is done.

Airway drug delivery group: The cell preparation of lung precursor-like cells is loaded into the drug delivery device and administered The drug device is inserted into the rat's trachea through the mouth, and the cell preparation of lung precursor-like cells is quickly pushed into the rat's trachea, and a single dose is administered at a standard of 0.1 mL/rat.

Tail vein administration group: The cell preparation of lung precursor-like cells was injected into the rats through the tail vein, and a single injection was performed at a standard of 1 mL/animal.

The specific dosing data for rats are shown in Table 3.

table 3

All rats were then returned to their cages for 21 days.

Example 5-1

Specific steps for measuring air consumption in rats: The rat is anesthetized with isoflurane and tracheally intubated. Connect a 2mL syringe (containing 20 μL water column) to the tracheal tube. Record the drop volume of the water column in the syringe within 10 seconds. This volume is the maximum Rat gas consumption.

Among them, the syringe was purchased from KDL, and the batch number was 20191123.

The air consumption of all rats was measured on the 4th day before the intervention experiment, on the day of the intervention experiment when no medication was administered, on the 7th day after the intervention experiment, and on the 21st day after the intervention experiment.

Figure 9 is a graph showing changes in air consumption of rats in the modeling group, airway administration group, tail vein administration group and normal group in Example 5-1 provided by the present invention. Referring to Figure 9, it can be seen that the air consumption of the rats in the modeling group, airway administration group and tail vein administration group was seriously reduced compared with the rats in the normal group after modeling. The rats after modeling had difficulty breathing and their lungs were Damage; after using lung precursor-like cells to rats, the air consumption of rats in the airway administration group and tail vein administration group was significantly greater than the air consumption of rats in the modeling group, indicating that lung precursor-like cells can Improve the lung function of rats after modeling.

Example 5-2

The dry-to-wet weight ratio of lung tissue is a direct indicator of pulmonary edema and a sensitive indicator of the severity of lung injury. The greater the dry-to-wet weight ratio (W/D), the higher the degree of lung injury.

All rats were administered the drug for 21 days. Serta 50 was injected from the tail vein of the rats. The rats were deeply anesthetized and then bled from the abdominal aorta. Finally, the rats were sacrificed by cervical dislocation. Among them, Serta 50 was purchased from Vick, with the item number 785T.

The postmortem right lung tissues of the rats in the modeling group, airway administration group, tail vein administration group and normal group were taken out and weighed, and the data were recorded. The postmortem right lung tissues of the rats in the intravenous administration group and the normal group were placed in an oven and dried at 70°C for 72 hours. They were weighed again and calculated for the modeling group, airway administration group, and tail vein administration group. The dry and wet weight ratio of the right lung tissue of rats in the normal group after death.

Figure 10 is a diagram of the dry and wet weight ratio of rat lung tissue in the modeling group, airway administration group, tail vein administration group and normal group in Example 5-2 provided by the present invention. Referring to Figure 10, it can be seen that the dry and wet weight ratio of the lung tissue of the rats in the modeling group, airway administration group and tail vein administration group after modeling was higher than that of the rats in the normal group; before using the lungs on the rats, After using somatic cells, the dry and wet weight ratios of the lung tissue of rats in the airway administration group and tail vein administration group were significantly smaller than those in the modeling group, indicating that lung precursor-like cells can repair The lung injury in rats after modeling was reduced.

Example 5-3

The postmortem left lung tissues of the rats in the modeling group, airway administration group, tail vein administration group and normal group in Example 5-2 were respectively taken out for Masson staining detection.

Figure 11 is a graph showing Masson staining results of rat lung tissue in the modeling group, airway administration group, tail vein administration group and normal group in Example 5-3 provided by the present invention; the scale bar in the figure is 1000 microns. Referring to Figure 11, it can be seen that obvious inflammatory cell infiltration can be seen in the left lung tissue of rats in the modeling group, airway administration group and tail vein administration group after modeling, and the integrity of the tissue structure is lower than that in the normal group. Rats; after using lung precursor-like cells in rats, compared with the rats in the modeling group, airway administration group and tail vein administration group, the rats in the airway administration group and tail vein administration group had better The degree of fibrosis in the left lung tissue was significantly lower than that in the modeling group, and the degree of infiltration of inflammatory cells in the left lung tissue of rats in the airway administration group and tail vein administration group was significantly lower than that in the modeling group, indicating that the prepulmonary Somatic cells can improve inflammation in rat lung tissue after modeling and reduce the infiltration of inflammatory cells in lung tissue.

Combining Examples 5-1 to 5-3, it is concluded that the reprogrammed culture in Example 2-1 The lung precursor-like cells cultured in the medium can repair damaged lung tissue, reduce the infiltration of inflammatory cells, and effectively improve lung function when treating chronic obstructive pulmonary disease in rats.

Example 6

This example provides a modeling method for a mouse idiopathic pulmonary fibrosis (IPF) model, and uses the lung precursor-like cells in Example 2 to intervene in the mouse idiopathic pulmonary fibrosis (IPF) model. The effect of the lung precursor-like cells in Example 2 on idiopathic pulmonary fibrosis was examined.

In this example, 12 7-week-old male C57BL/6 mice purchased from Charles Lihua Pharmaceutical Technology (Shanghai) Co., Ltd. were used for modeling. The average weight of the mice reached 25g.

Modeling of the mouse idiopathic pulmonary fibrosis (IPF) model: Nine mice in the modeling group were anesthetized using isoflurane, tracheally intubated with a 20G arterial indwelling needle, and then breathed with small animals. The mice were mechanically ventilated for 2 hours to induce and establish a mouse pulmonary fibrosis model. Among them, the parameters of the small animal ventilator are set as follows: FiO2: 0.2, VT: 20mL/kg, and respiratory rate (RR) 70 times/minute.

Intervention experiments of lung precursor-like cells on mouse idiopathic pulmonary fibrosis (IPF) model:

The P3 generation lung precursor-like cells in Example 2-1 were sampled, and NS was added to form a lung precursor-like cell preparation. NS was purchased from Chenxin Pharmaceutical Co., Ltd., the product number is 2102020728.

The mice were grouped as follows: nine mice were randomly modeled. After the modeling was completed, the mice were randomly divided into three groups, with three mice in each group, namely the model control group, the airway administration group and the tail vein administration group. group, and the cell preparations of lung precursor-like cells were immediately used on the mice in the airway administration group and the tail vein administration group; the remaining three mice were not subjected to any treatment and served as the normal control group.

The situation of mice in the model control group, airway administration group and tail vein administration group using cell preparations of lung precursor-like cells is as follows:

Model control group: no treatment.

Airway drug administration group: Load the cells of lung precursor-like cells into the drug delivery device, insert the drug delivery device orally into the trachea of mice, and quickly push the cell preparation of lung precursor-like cells into the trachea of mice. A single dose was administered at the standard of 1×10 ⁶ /50 μL.

Tail vein administration group: Lung precursor-like cells were injected into mice through the tail vein, and administered as a single injection at the standard of 1×10 ⁶ /100 μL.

The specific administration to mice is shown in Table 4.

Table 4

Then the mice in the modeling group and the normal control group were raised in cages for 7 days.

Example 6-1

Specific steps for measuring the respiratory frequency of mice: Anesthetize the mice with isoflurane and then intubate the trachea. Connect a 1mL syringe (containing 20 μL water column) to the tracheal tube. Record the number of times the water column moves up and down in the syringe within 10 seconds. The number is Respiratory rate of mice.

Among them, the syringe was purchased from KDL, and the batch number was 20191123.

The respiratory frequency of all mice was measured on the day of the intervention experiment before administration and on the 7th day after the intervention experiment.

Figure 12 is a graph showing changes in respiratory frequency of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-1 provided by the present invention; referring to Figure 12, it can be seen that the model control group, airway administration group The respiratory frequency of the mice in the tract administration group and the tail vein administration group increased significantly compared with the mice in the normal control group after the modeling. The mice after the modeling showed obvious shortness of breath, the respiratory rate increased significantly, and the lungs Damage; after administering lung precursor-like cells to mice, the respiratory rates of mice in the airway administration group and tail vein administration group were significantly lower than those in the model control group, indicating that lung precursor-like cells Can improve the lung function of modeled mice.

Example 6-2

On the 7th day after the intervention experiment, 1 mL of arterial blood was taken from the mice in the model control group, airway administration group, tail vein administration group and normal control group, and the venous blood of the mice was dripped into the heparin sodium anticoagulant tube. , then put the heparin sodium anticoagulation tube into the blood gas analyzer, and use the blood gas analyzer to detect the blood gas indicators of the mouse arterial blood.

Among them, the heparin sodium anticoagulant tube was purchased from BD with the item number 367874; the blood gas analyzer was purchased from From Silman Technology, model number is G100.

Figure 13 is a graph showing the arterial blood gas analysis results of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-2 provided by the present invention. Referring to Figure 13, it can be seen that compared with the normal control group, the partial pressure of carbon dioxide in the arterial blood of mice in the model control group, airway administration group, and tail vein administration group was significantly increased, the partial pressure of oxygen was significantly reduced, and the partial pressure of carbon dioxide was significantly lower. The increase led to a decrease in the blood pH value, proving that the pulmonary ventilation function of the mice after modeling was blocked; after using lung precursor-like cells in the mice, the mice in the airway administration group and the tail vein administration group were better than the model. The partial pressure of carbon dioxide in the arterial blood of mice in the control group was significantly reduced, and the partial pressure of oxygen was significantly increased. The decrease in partial pressure of carbon dioxide led to an increase in blood pH, indicating that lung precursor-like cells can improve the lung ventilation function of mice to a certain extent.

Example 6-3

The left lungs of mice in the model control group, airway administration group, tail vein administration group and normal control group were perfused with 2 ml of 10% formalin solution through the trachea, the trachea was ligated, and the left lungs were soaked in 10% formalin solution. The left lung was fixed with malin solution, and the left lung was sent to a third party for HE and Masson staining.

Among them, formalin solution was purchased from Shanghai Ruiyu Biotechnology Co., Ltd., with the product number #Bry-0018.

(1) HE staining is used. Figure 14 shows the HE staining results of the lung tissue of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-3 provided by the present invention; Figure The middle scale bar is 1000 microns. Referring to Figure 14, it can be seen that the mice in the model control group, airway administration group and tail vein administration group had obvious inflammation in the lung tissue after modeling. The infiltration of sex cells proved that the lung tissue of mice after modeling was damaged and caused lung tissue inflammation; after administration of lung precursor-like cells, inflammation of the lung tissue of mice in the airway administration group and tail vein administration group was The degree of infiltration of sex cells was significantly lower than the degree of inflammatory cell infiltration in the lung tissue of mice in the model control group, indicating that lung precursor-like cells can improve inflammation in the lung tissue of modeled mice and reduce the infiltration of inflammatory cells in the lung tissue. .

(2) Masson staining is used. Figure 15 shows the Masson staining results of the lung tissue of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-3 provided by the present invention; Figure The middle scale bar is 1000 microns. Referring to Figure 15, it can be seen that the area of blue-stained area of the lung tissue of mice in the model control group, airway administration group and tail vein administration group after modeling is significantly larger than that of mice in the normal control group. The area of the blue-stained area proves the fibrosis of the lung tissue of mice after modeling; after administration of lung precursor-like cells, the area of blue-stained area of the lung tissue of mice in the airway administration group and tail vein administration group is obvious Lower than that of model control mice, indicating that lung precursor-like cells can significantly reduce the degree of fibrosis in the lung tissue of modeled mice.

Example 6-4

The right bronchi of mice in the model control group, airway administration group, tail vein administration group and normal control group were bidirectionally ligated, and the right lungs of the mice were cut off. Cut out ^1cm2 right lung tissue, quickly freeze it in liquid nitrogen for 2 minutes, and use Western blotting to detect the right lung tissue of mice in the model control group, airway administration group, tail vein administration group and normal control group. Fibrosis factors COL1A1, Fibronectin, α-SMA. Figure 16 is a graph showing the detection results of pulmonary tissue fibrosis molecule protein concentration of mice in the model control group, airway administration group, tail vein administration group and normal control group in Example 6-4 provided by the present invention. Referring to Figure 16, it can be seen that the protein expression of COL1A1 in the model control group, airway administration group, and tail vein administration group was significantly higher after modeling. The protein expression of COL1A1 was significantly higher than that of mice in the normal control group; after administration of lung precursor-like cells, the protein expression of COL1A1 in mice in the airway administration group and tail vein administration group was significantly lower than that in the model The protein expression of COL1A1 in mice in the control group shows that lung precursor-like cells can significantly reduce the degree of fibrosis in the lung tissue of modeled mice.

Combined with Examples 6-1 to 6-4, it is concluded that the lung precursor-like cells cultured in the reprogramming medium in Example 2-1 can reduce the degree of lung tissue fibrosis and reduce inflammation in the treatment of mouse pulmonary fibrosis. The infiltration of sex cells can effectively improve respiratory function.

Although the embodiments of the present invention have been described in detail above, it will be obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are within the scope and spirit of the invention as described in the claims. Furthermore, the invention described herein is capable of other embodiments and of being practiced or carried out in various ways.

Claims (12)

1. A method for preparing lung precursor-like cells, characterized by comprising the following steps:

   S1: Take lung tissue, digest and separate the lung tissue to obtain primary lung cells;

   S2: Use the reprogramming medium to culture the primary lung cells until the cell confluence is no less than 80% to obtain lung precursor-like cells.
2. The method for preparing lung precursor-like cells according to claim 1, wherein in S1, the step of obtaining primary lung cells after digestion and separation of the lung tissue includes:

   S10: Wash and disinfect the lung tissue sequentially with sterile PBS buffer, and then cut the lung tissue into pieces to obtain lung tissue fragments;

   S11: Add self-prepared collagenase to the lung tissue fragments, and incubate the lung tissue fragments at 37 degrees Celsius for 30 minutes to obtain a primary lung cell suspension;

   S12: Perform screen sorting on the primary lung cell suspension, and then collect the lung cell filtrate;

   S13: Centrifuge the lung cell filtrate, discard the supernatant, and obtain the lung cell precipitate;

   S14: Add red blood cell lysis balance solution to the lung cell pellet and resuspend to obtain a lung cell mixture. Then, centrifuge the lung cell mixture and discard the supernatant. Pulmonary cell pellets were obtained;

   S15: Repeat S14 until no red blood cells are observed in the lung cell pellet.
3. The method for preparing lung precursor-like cells according to claim 2, wherein in S11, the components of the self-prepared collagenase include: 25%-50% Neutral Protease II and 50%-75% Type II Collagenase.
4. The preparation method of lung precursor-like cells according to claim 1, characterized in that, in the S2, the reprogramming medium includes a basal medium, serum-free additives, growth factors, and TGF-β signal inhibition agent, Wnt signaling pathway activator and ROCK kinase inhibitor.
5. The method for preparing lung precursor-like cells according to claim 4, wherein the content of the growth factor is 10-50 ng/ml based on the volume of the basal culture medium, and the ROCK kinase The content of the inhibitor is 1-20 micromoles, the content of the Wnt signaling pathway activator is 1-10 micromoles, the content of the TGF-β signaling inhibitor is 1-10 micromoles, and the serum-free additive The volume content does not exceed 10%.
6. The method for preparing lung precursor-like cells according to claim 1, wherein in the S2, the reprogramming medium further includes triiodothyronine and hydrocortisone.
7. The preparation method of lung precursor-like cells according to claim 1, characterized in that, The lung precursor-like cells positively express at least one characteristic marker.
8. The method for preparing lung precursor-like cells according to claim 7, wherein the expression rate of the characteristic marker is no less than 50% and no more than 99%.
9. The method for preparing lung precursor-like cells according to claim 7, wherein at least one of the characteristic markers includes at least one of CD24, CD73, CD326, CK19, and Sox9.
10. The method for preparing lung precursor-like cells according to claim 1, wherein the lung precursor-like cells negatively express at least one MHC class II molecule, and at least one of the MHC class II molecules includes HLA-DR. At least one of /DP/DQ.
11. An application of lung precursor-like cells, characterized in that the lung precursor-like cells prepared by the method for preparing lung precursor-like cells according to claim 1 are used to intervene in an in vivo animal model.
12. The application of lung precursor-like cells according to claim 11, wherein the in vivo animal model includes any one of a rat chronic obstructive pulmonary disease model and a mouse idiopathic pulmonary fibrosis model.