CN111514164A - Adipose-derived mesenchymal stem cells for treating lung diseases and preparation method thereof - Google Patents
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Abstract
The invention provides adipose-derived mesenchymal stem cells for treating lung diseases and a preparation method thereof. The adipose-derived mesenchymal stem cells provided by the invention can be used for preparing medicines for preventing or treating lung injury and fibrosis. The medicine is applied to the treatment of lung diseases, particularly lung injuries and fibrosis thereof, has high safety, easily obtained materials and good intervention and treatment effects, can realize quantitative production by mature cell culture technical means, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to adipose-derived mesenchymal stem cells for treating lung diseases and a preparation method thereof.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells with the potential for long-term self-renewal and multipotent differentiation. Mesenchymal stem cells generally refer to undifferentiated primitive APSC pluripotent cells that survive embryonic development in a variety of adult mesenchymal tissues, such as: bone marrow stroma, fat, skin dermis, placenta, skeletal muscle, umbilical cord blood, umbilical cord, and the like. Studies have demonstrated that mesenchymal stem cells can ameliorate or reduce lung injury.
Pulmonary fibrosis is a chronic progressive, diffuse interstitial pulmonary disease caused by a variety of etiologies, and is mainly characterized by proliferation and aggregation of a large number of fibroblasts (fibroplasts), deposition of extracellular matrix (ECM), and gradual replacement of the lung parenchyma, thereby losing respiratory function, with accompanying inflammatory reactions and damage resulting in destruction of lung tissue and impaired lung function. The pulmonary fibrosis can be classified into idiopathic pulmonary fibrosis, pneumoconiosis, sarcoidosis, hypersensitivity pneumonitis, and pulmonary fibrosis caused by drugs and radioactivity, etc. according to the causes of the pulmonary fibrosis. The idiopathic pulmonary fibrosis is the most common, the incidence rate is gradually increased and is increased with age, and the median survival time of patients after diagnosis is only 3.8 years.
The mesenchymal stem cells are mainly used for treating lung diseases such as pneumoconiosis at present. Panyong and the like discover that exogenous bone marrow mesenchymal stem cells transplanted to an early silicosis model mouse can reduce lung inflammation and slow down the progress of pulmonary fibrosis [1 ]. Morales [2] and the like implant Bone Marrow Derived Mononuclear Cells (BMDMCs) into 5 silicosis patients for treatment through a bronchoscope, and scan 360 days before treatment and after BMDMCs treatment, and perform whole body and plane scanning 2h and 24h after perfusion, and the results show that no adverse reaction exists after treatment, the lung function of the patients is improved to a certain extent, and the fact that the treatment of acute and chronic silicosis through the administration of BMDMCs through bronchus is feasible and safe. The most classical animal model for pulmonary fibrosis disease research is the bleomycin murine model, and multiple research data indicate that MSCs have good efficacy in the treatment of pulmonary fibrosis. Lee SH et al [3] use intravenous BMDMSCs to treat rats 4 days after Bleomycin (BLM) inhalation, and research shows that MSCs can effectively reduce lung injury and fibrosis induced by BLM by down-regulating nitric oxide metabolites, proinflammatory factors and angiogenic factors. However, the mesenchymal stem cells are derived from bone marrow stroma, and the materials are mostly difficult to obtain.
[1] Panyon, yankun and Liu Yongtai. Bone marrow mesenchymal stem cell transplantation is used for treating pneumonitis and pulmonary fibrosis of silicosis mice [ J ]. Chinese tissue engineering research, 2013, 000(006): P.1106-1106.
[2]Morales MM，Souza SA，Loivos LP，et al.Pilot safety study ofintrabronchial instillation of bone marrow-derived mononuclear cells inpatients with silicosis[J].BMC Pulm Med，2015，15(1):1-9.
[3]Lee SH,Jang AS,Kim YE,et al.Modulation of cytokine and nitricoxide by mesenchymal stem cell transfer in lung injury/fibrosis[J].Respiratory research.2010,11(1).
Disclosure of Invention
In order to solve the problems, the invention provides the adipose-derived mesenchymal stem cell for treating the lung diseases, which is applied to the treatment of lung diseases, particularly pneumoconiosis injury and fibrosis, and has the characteristics of simple preparation, high safety, good treatment effect and the like.
In one aspect, the invention provides the use of mesenchymal stem cells in the preparation of a medicament for the treatment of a pulmonary disease.
The mesenchymal stem cells include, but are not limited to, one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells and bone marrow mesenchymal stem cells.
The lung diseases include, but are not limited to, pneumoconiosis, Idiopathic Pulmonary Fibrosis (IPF), Chronic Obstructive Pulmonary Disease (COPD), severe novel coronavirus pneumonia, Acute Respiratory Distress Syndrome (ARDS), various acute and chronic lung injuries.
The adipose-derived mesenchymal stem cells comprise primary cells separated from human adipose tissues and passage cells thereof.
The passage cell of the adipose derived mesenchymal stem cell can be 1-50 generations.
The adipose-derived mesenchymal stem cells are used for treating lung diseases by means of injection.
The injection mode of the adipose-derived mesenchymal stem cells is one-time injection or staged injection.
The injection amount of the adipose-derived mesenchymal stem cells in one-time injection is 1 × 106-6×106Preferably 2 × 10/kg body weight, preferably 26One per kg body weight.
The fat mesenchymal stem cells are injected for 1 to 6 times in equal amount in stages, and the total injection amount is 1 × 106-36×106One injection per kg body weight, preferably 4 times, and total injection amount of 8 × 106One per kg body weight.
The injection methods include but are not limited to: intravenous injection and tracheal administration.
The preparation method of the adipose tissue-derived mesenchymal stem cells comprises the following steps:
(1) aseptically collecting adipose tissue, performing separation culture by collagenase digestion method within 12h, washing adipose tissue, removing blood cells, adding preheated 0.075% collagenase I or collagenase IV working solution with the same amount as the adipose tissue suspension, vigorously shaking the culture bottle for 5-10 s, placing in a vibration gas bath kettle, digesting at 37 deg.C and 70rpm for 50-70min, taking out the culture bottle every 15 min, vigorously shaking the culture bottle for 5-10 s, and continuously placing in the vibration gas bath kettle for digestion;
(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, and centrifuging the centrifuge tubes for 10min at the room temperature of 300g-500g to obtain precipitates, namely the matrix blood vessel component;
(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 300-500 g for 10 min;
(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; suspending the stromal vascular fraction with 10mL of culture medium, then collecting the stromal vascular fraction into 1 new 50mL centrifuge tube, centrifuging again at room temperature, 200g-400g, 10 min;
(5) a bottle is planted: centrifuging, collecting 15mL supernatant, inspecting, performing sterile detection, resuspending tissue with 20mL culture medium, uniformly distributing 20mL tissue suspension into culture flask, placing the flask at 37 deg.C and 5% CO2An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; floating non-adherent medium in culture flaskPouring the blood vessel component and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 700g-900 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution into each culture bottle again, then adding a proper amount of complete culture solution to a constant volume, blowing and uniformly mixing, and respectively adding 2mL of matrix blood vessel components into each culture bottle; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;
(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; standing at 37 deg.C for 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P1 generation adipose-derived stem cells;
(7) passage: lightly blowing and beating the resuspension cells, inoculating to a new culture container after constant volume, wherein the density of the passage cells is 5000-6000 cells/cm2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells;
(8) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
In another aspect, the present invention provides an adipose-derived mesenchymal stem cell.
The adipose-derived mesenchymal stem cells are isolated from human adipose tissues.
The preparation method of the adipose tissue-derived mesenchymal stem cells comprises the following steps:
(1) aseptically collecting adipose tissue, performing separation culture by collagenase digestion method within 12h, washing adipose tissue, removing blood cells, adding preheated 0.075% collagenase I or collagenase IV working solution with the same amount as the adipose tissue suspension, vigorously shaking the culture bottle for 5-10 s, placing in a vibration gas bath kettle, digesting at 37 deg.C and 70rpm for 50-70min, taking out the culture bottle every 15 min, vigorously shaking the culture bottle for 5-10 s, and continuously placing in the vibration gas bath kettle for digestion;
(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, and centrifuging the centrifuge tubes for 10min at the room temperature of 300g-500g to obtain precipitates, namely the matrix blood vessel component;
(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 300-500 g for 10 min;
(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; suspending the stromal vascular fraction with 10mL of culture medium, then collecting the stromal vascular fraction into 1 new 50mL centrifuge tube, centrifuging again at room temperature, 200g-400g, 10 min;
(5) a bottle is planted: centrifuging, collecting 15mL supernatant, inspecting, performing sterile detection, resuspending tissue with 20mL culture medium, uniformly distributing 20mL tissue suspension into culture flask, placing the flask at 37 deg.C and 5% CO2An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; pouring the matrix blood vessel components floating in the culture bottle and having no adherence and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 700g-900 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution into each culture bottle again, then adding a proper amount of complete culture solution to a constant volume, blowing and uniformly mixing, and respectively adding 2mL of matrix blood vessel components into each culture bottle; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;
(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; standing at 37 deg.C for 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain P1 generationAdipose-derived stem cells;
(7) passage: lightly blowing and beating the resuspension cells, inoculating to a new culture container after constant volume, wherein the density of the passage cells is 5000-6000 cells/cm2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells;
(8) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
In yet another aspect, the invention also provides a medicament for treating a pulmonary disease.
The medicament comprises human mesenchymal stem cells.
The mesenchymal stem cell sources include but are not limited to umbilical cord, fat, placenta and bone marrow.
Preferably, the human mesenchymal stem cells in the medicament are adipose mesenchymal stem cells prepared by the preparation method.
Preferably, the medicament is an injection.
The medicine also comprises other pharmaceutically acceptable carriers, such as: trehalose, anhydrous or aqueous sodium chloride, glucose, sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride and magnesium chloride.
In some embodiments, the medicament is an injection solution, and the injection solution is 1 × 105-25×105Adipose-derived mesenchymal stem cells per mL, human serum albumin of 0.01-0.05g/mL and sodium chloride injection of 0.9%.
The adipose-derived mesenchymal stem cells provided by the invention are high in safety when being applied to treating lung diseases, particularly lung injuries and fibrosis thereof, have good intervention and treatment effects on corresponding diseases, can realize quantitative production through mature cell culture technical means, and have good application prospects.
Drawings
Fig. 1 shows the results of adipose-derived mesenchymal stem cell adipogenesis induction.
Fig. 2 shows the results of osteogenesis induction of adipose-derived mesenchymal stem cells.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 preparation method of adipose-derived mesenchymal stem cells
(1) Collecting human adipose tissues in a sterile operation, performing separation culture by a collagenase digestion method within 12h after collection, washing the adipose tissues, removing blood cells, adding a working solution of collagenase I (purchased from Life corporation of America) with the concentration of 0.075 percent and newly prepared in an equal amount with an adipose tissue suspension, preheating (preheating condition is preheating by a gas bath shaker at 37 ℃ in advance for half an hour), wherein the preheating concentration is 0.075 percent, the working solution is violently shaken for 5 to 10 seconds, placed in a vibrating gas bath kettle, digested for 60min at 37 ℃, and continuously placed in the vibrating gas bath kettle after the culture bottle is taken out and violently shaken for 5 to 10 seconds every 15 min;
(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, centrifuging the centrifuged tissue at the room temperature of 400g for 10min, and obtaining a precipitate which is a matrix blood vessel component;
(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 400g for 10 min;
(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; resuspending the stromal vascular fraction in 10mL of medium (10% fetal bovine serum in LG-DMEM), then pooling the stromal vascular fraction in 1 new 50mL centrifuge tube, and centrifuging again at room temperature for 300g for 10 min;
(5) a bottle is planted: centrifuging and taking15mL of the supernatant was submitted for sterility testing, the tissue was resuspended thoroughly with 20mL of medium, 20mL of the tissue suspension was aliquoted into flasks, the flasks were placed at 37 ℃ and 5% CO2An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; pouring the matrix blood vessel components floating in the culture bottle and having no adherence and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 800 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution (general serum-free basal culture medium containing 2% serum substitute or DMEM/F12 basal culture medium) into each culture bottle again, metering the volume with a proper amount of complete culture solution, blowing and uniformly mixing, and adding 2mL of matrix blood vessel components into each culture bottle respectively; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;
(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; standing at 37 deg.C for 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P1 generation adipose-derived stem cells;
(7) passage: lightly blowing and beating the resuspension cells, inoculating to a new culture container after constant volume, wherein the density of the passage cells is 5000-6000 cells/cm2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells.
(8) And sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
Adipose-derived mesenchymal stem cells used in the subsequent examples were prepared using the adipose-derived mesenchymal stem cell stem cells of each generation prepared in example 1.
Example 2 differentiation induced by adipose-derived mesenchymal Stem cells
(1) Adipose tissue-derived mesenchymal stem cell adipocyte induction: taking the 3 rd generation adipose-derived mesenchymal stem cells, culturing for 3d in DMEM culture solution, and adding adipogenic cell induction solution after the cell climbing sheet is pasted for 50%. The components of the composition are 1 mu mol/L dexamethasone, 10 mu mol/L insulin, 200 mu mol/L indomethacin and 0.5mmol/L IBMX which are added into a DMEM basic culture solution. Adipogenesis was induced for 10 days and cell morphology was observed under the microscope. And then carrying out oil red O dyeing: fixing adipose-derived mesenchymal stem cells for 10 days after adipogenesis induction by using 100g/L paraformaldehyde at 4 ℃ for 1h, washing by using 70% isopropanol, adding 2% oil red at room temperature for dyeing for 5-10min, washing off redundant dye, washing by using PBS (phosphate buffer solution), and observing whether lipid drops are formed in the cells under a microscope. As a result, as shown in FIG. 1, lipid droplets were observed under the mirror.
(2) Osteogenic induction of adipose tissue-derived mesenchymal stem cells: taking 3 rd generation adipose-derived mesenchymal stem cells, culturing for 3 days in a DMEM culture solution, and after 50% of cell climbing tablets are pasted, inducing by an osteogenic induction solution, wherein the ingredients are 10nmol/L vitamin D3, 37.5mg/L vitamin C and 2.16g/L beta-glycerophosphate and adding into a DMEM basic culture solution. Osteogenic induction 14d, alizarin red staining, and observing cell morphology under a microscope and detecting. As a result, as shown in fig. 2, a red colored spot (calcium nodule) was observed under a mirror, and it was found that adipose-derived mesenchymal stem cells had an osteogenic ability.
(3) Inducing adipose tissue-derived mesenchymal stem cells into chondroblast, namely taking the 3 rd generation adherent adipose tissue-derived mesenchymal stem cells, and performing induction on the adherent adipose tissue-derived mesenchymal stem cells by using 2 × 105After inoculation of individual cells, the cells were centrifuged at 1000r/min for 6min and placed at 37 ℃ with 5% CO2Culturing in a saturated humidity incubator in a cartilage induction basic culture (TGF- β 310 ng/mL + FGF-22 ng/mL + retinoic acid 1nmol/L + vitamin C phosphate 50 μ g/mL + proline 40 μ g/mL + pyruvic acid 100 μ g/mL + DEX 100nmol/L + ITS + premix 50mg/mL), removing the culture medium after 3 weeks of induction, washing with PBS 2 times, fixing with 4% paraformaldehyde for 30min, embedding the paraffin-embedded section, washing with distilled water, staining with toluidine blue, and observing the acid mucopolysaccharide with blue staining under a low power microscope.
Example 3 lymphocyte inhibition test results
The method comprises the steps of adopting adipose-derived mesenchymal stem cells obtained through culture as research objects, detecting the inhibition effect of the adipose-derived mesenchymal stem cells on peripheral blood mononuclear cells by a CCK8 method, evaluating the influence of the adipose-derived mesenchymal stem cells on the peripheral blood mononuclear cells by flow cytometry, and analyzing the effect of the adipose-derived mesenchymal stem cells on immune regulation. The results show that the adipose-derived mesenchymal stem cells have an immunoregulation effect on lymphocytes, and can promote the production of Treg cells and inhibit the secretion of inflammatory factors such as IL-17 and the like.
The CCK8 kit used in this example was purchased from Sigma under the cat number 96992.
(1) CCK8 test inhibition effect of adipose-derived mesenchymal stem cells on peripheral blood mononuclear cells
Peripheral blood density gradient centrifugation mononuclear cells, adipose-derived mesenchymal stem cells of generation P2 and generation P5 were co-cultured with the isolated peripheral blood mononuclear cells.
Results 3 days after the inhibition of peripheral blood mononuclear cells by the adipose-derived mesenchymal stem cells of generation P2: the expansion of peripheral blood mononuclear cells is inhibited as the proportion of adipose mesenchymal stem cells increases, and when adipose mesenchymal stem cells: peripheral blood mononuclear cells greater than 1: at 50 hours, compared with a normal control group, the difference has significance, and P is less than 0.001; the proliferation of peripheral blood mononuclear cells is enhanced along with the increase of adipose mesenchymal stem cells.
Results 3 days after the inhibition of peripheral blood mononuclear cells by the adipose-derived mesenchymal stem cells of generation P5: similar to the results of P2 generation, expansion of peripheral blood mononuclear cells was inhibited as the proportion of adipose mesenchymal stem cells increased, and when adipose mesenchymal stem cells: peripheral blood mononuclear cells greater than 1: at 50 hours, compared with a normal control group, the difference has significance, and P is less than 0.001; the proliferation of peripheral blood mononuclear cells is enhanced along with the increase of adipose mesenchymal stem cells.
(2) And (3) flow-detecting the influence of the adipose-derived mesenchymal stem cells on IL17 cells in peripheral blood mononuclear cells.
Effect of P2 generation adipose-derived mesenchymal stem cells on Th17 cell population: the results show that the CD4 is treated along with the increase of the amount of the adipose mesenchymal stem cells+IL17+The inhibition of the cells is enhanced.
P5 generation adipose mesenchymal stem cellsThe effect of the cells on the Th17 cell population was similar to that of the P2 generation on CD4+IL17+The cytostatic effect of (a) is enhanced with the increase in the amount of adipose mesenchymal stem cells.
(3) Flow detection of Foxp3 in adipose mesenchymal stem cell pairs PBMC (peripheral blood mononuclear cell)+CD4+CD25+Influence of cells
P2 generation adipose-derived mesenchymal stem cell pair Foxp3+CD4+CD25+Influence of cell population: the P2 generation adipose-derived mesenchymal stem cells can remarkably induce Foxp3+CD4+CD25+The survival of the cells, the induction effect is enhanced along with the increase of the cell proportion of the adipose mesenchymal stem cells.
Effect of P5 generation adipose-derived mesenchymal stem cells on the population of Foxp3+ CD4+ CD25+ cells: similar to the results of the adipose-derived stem cells of the P2 generation, the adipose-derived mesenchymal stem cells of the P5 generation can remarkably induce Foxp3+CD4+CD25+The survival of the cells, the induction effect is enhanced along with the increase of the cell proportion of the adipose mesenchymal stem cells. .
Example 4 measurement of the ability of inflammatory factors to stimulate the secretion of indoleamine 2, 3-dioxygenase (IDO) from adipose mesenchymal stem cells
TNF-alpha and INF-gamma stimulate P1 and P5 adipose-derived mesenchymal stem cells respectively, and indoleamine 2, 3-dioxygenase ELISA kits (Shanghai scrupulously and respectfully sensitive Biotech Co., Ltd., product number KB2296) are used for measuring the expression of indoleamine 2, 3-dioxygenase in the adipose-derived mesenchymal stem cells and evaluating the immunoregulation effect of the adipose-derived mesenchymal stem cells. Adipose-derived mesenchymal stem cells of P1 and P5 generation which were not stimulated by TNF-alpha and INF-gamma were set as a control group.
The results show that compared with the control group, the concentration of indoleamine 2, 3-dioxygenase in the stimulated group is obviously improved, and P is less than 0.05; indicating that the cells can play a good immune regulation function in the inflammatory environment. The cells have good paracrine function, and the P1 has the same function with the P5 generation cells.
Example 5 therapeutic Effect of adipose-derived mesenchymal Stem cells on rats with pulmonary fibrosis
This example reflects the therapeutic effect of adipose-derived mesenchymal stem cells on rats with pulmonary fibrosis through several aspects:
the method for constructing the pulmonary fibrosis rat model comprises the following steps: rat intratracheal injection of bleomycin solution 0.05mL (5 mg/kg).
This example was carried out using the adipose mesenchymal stem cells obtained in example 1.
Groups were set up with the following conditions, with 20 rats per group:
control group: normal rats;
model group: a model rat;
the treatment group comprises injecting adipose-derived mesenchymal stem cells via tail vein at 4 th, 8 th, 15 th and 22 th days after first rat model building with cell density of 3 × 106one/mL, the injection amount is 0.5 mL/mouse.
The method comprises the following steps: analysis of lung tissue Wet to Dry ratio (W/D), morphology of bronchoalveolar lavage cells, changes in lung tissue morphology and collagen deposition in lung tissue, and determination of IL-1 β, TGF- β and IL-6 and TNF- α concentrations in bronchoalveolar lavage fluid in lung tissue lysates.
(1) Rats in each group were sacrificed on day 29 post-molding and lung specimens were taken after bronchoalveolar lavage.
(2) After the animals were sacrificed, the right lung was subjected to protein or histological analysis after bronchoalveolar lavage. The left lung lobes were excised for wet/dry ratio analysis. Cells in bronchoalveolar lavage fluid were counted using a hemocytometer. Alveolar lavage fluid was centrifuged at 500 Xg for 10 minutes and the supernatant was stored at-70 ℃. The right lung was taken from each animal, fixed with 4% paraformaldehyde, and the specimens were dehydrated and embedded in paraffin.
(3) Lung tissues of each group of rats are taken and prepared into 5 mu m sections by a conventional method, Hematoxylin-Eosin (HE) and Masson staining is respectively carried out, and morphological change of the lung tissues and collagen fiber staining conditions are observed under an optical microscope.
(4) The ELISA method is used for measuring the IL-1 beta, TGF-beta and IL-6 and TNF-alpha concentration in bronchoalveolar lavage fluid in rat lung tissue lysate.
And (4) analyzing results:
researches show that the lung neutrophilic granulocyte inflammation and collagen deposition can be effectively eliminated after the adipose-derived mesenchymal stem cells are infused, and the lung proinflammatory cytokine induced by bleomycin can be reduced.
After treatment, compared with the model group rats, the adipose-derived mesenchymal stem cells can obviously reduce the increasing conditions of the levels of cytokines such as bleomycin-induced rat wet/dry ratio, lymphocyte infiltration degree, collagen deposition, IL-1 beta, TGF-beta and the like (p is less than 0.05).
Claims (10)
1. The application of the mesenchymal stem cells in preparing the medicine for treating the lung diseases is characterized in that the lung diseases are idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, severe novel coronavirus pneumonia, acute respiratory distress syndrome, pneumoconiosis or lung injury; the mesenchymal stem cells are one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells and bone marrow mesenchymal stem cells.
2. The use of claim 1, wherein the mesenchymal stem cell is an adipose mesenchymal stem cell.
3. The use of claim 2, wherein the adipose-derived mesenchymal stem cells comprise primary cells isolated from human adipose tissue and passaged cells thereof.
4. The use of claim 2, wherein the total injection amount of adipose derived mesenchymal stem cells is 1 × 106-36×106One per kg body weight.
5. The use according to claim 2, wherein the adipose-derived mesenchymal stem cells are prepared by the following steps:
(1) aseptically collecting adipose tissue, performing separation culture by collagenase digestion method within 12h, washing adipose tissue, removing blood cells, adding preheated 0.075% collagenase I or collagenase IV working solution with the same amount as the adipose tissue suspension, vigorously shaking the culture bottle for 5-10 s, placing in a vibration gas bath kettle, digesting at 37 deg.C and 70rpm for 50-70min, taking out the culture bottle every 15 min, vigorously shaking the culture bottle for 5-10 s, and continuously placing in the vibration gas bath kettle for digestion;
(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, and centrifuging the centrifuge tubes for 10min at the room temperature of 300g-500g to obtain precipitates, namely the matrix blood vessel component;
(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 300-500 g for 10 min;
(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; suspending the stromal vascular fraction with 10mL of culture medium, then collecting the stromal vascular fraction into 1 new 50mL centrifuge tube, centrifuging again at room temperature, 200g-400g, 10 min;
(5) a bottle is planted: centrifuging, collecting 15mL supernatant, inspecting, performing sterile detection, resuspending tissue with 20mL culture medium, uniformly distributing 20mL tissue suspension into culture flask, placing the flask at 37 deg.C and 5% CO2An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; pouring the matrix blood vessel components floating in the culture bottle and having no adherence and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 700g-900 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution into each culture bottle again, then adding a proper amount of complete culture solution to a constant volume, blowing and uniformly mixing, and respectively adding 2mL of matrix blood vessel components into each culture bottle; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;
(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; placing at 37 deg.C，5％CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P1 generation adipose-derived stem cells;
(7) passage: lightly blowing and beating the resuspension cells, inoculating to a new culture container after constant volume, wherein the density of the passage cells is 5000-6000 cells/cm2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells;
(8) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
6. The method according to claim 5, wherein the preheating condition in the step (1) is preheating by a gas bath rocking bed at 37 ℃ in advance for half an hour.
7. The adipose-derived mesenchymal stem cell is characterized in that the preparation method of the adipose-derived mesenchymal stem cell comprises the following steps:
(1) collecting adipose tissue under aseptic operation, performing separation culture by collagenase digestion method within 12h, washing adipose tissue, removing blood cells, adding preheated 0.075% collagenase I working solution with the same amount as the adipose tissue suspension, vigorously shaking the culture bottle for 5-10 s, placing in a vibration gas bath kettle, digesting at 37 deg.C and 70rpm for 50-70min, taking out the culture bottle every 15 min, vigorously shaking the culture bottle for 5-10 s, and continuing to digest in the vibration gas bath kettle;
(2) separation of stromal vascular fraction: filtering the digested tissue by using a blood transfusion device filter screen, then averagely subpackaging the digested tissue into 2 centrifuge tubes with the volume of 50mL, and centrifuging the centrifuge tubes for 10min at the room temperature of 300g-500g to obtain precipitates, namely the matrix blood vessel component;
(3) purifying and precipitating: after centrifugation, the stromal vascular fraction is deposited at the bottom of the centrifuge tube, and the upper layer of oil and the lower layer of collagenase solution are carefully removed from top to bottom by a pipette; resuspending matrix blood vessel component with appropriate amount of normal saline, blowing, centrifuging at room temperature of 300-500 g for 10 min;
(4) after centrifugation, carefully removing the supernatant, and taking care not to directly pour the supernatant; suspending the stromal vascular fraction with 10mL of culture medium, then collecting the stromal vascular fraction into 1 new 50mL centrifuge tube, centrifuging again at room temperature, 200g-400g, 10 min;
(5) a bottle is planted: centrifuging, collecting 15mL supernatant, inspecting, performing sterile detection, resuspending tissue with 20mL culture medium, uniformly distributing 20mL tissue suspension into culture flask, placing the flask at 37 deg.C and 5% CO2An incubator; performing primary culture for 24 hours, performing full-scale liquid change, and observing the cell state under a mirror; pouring the matrix blood vessel components floating in the culture bottle and having no adherence and the culture solution into a new 50mL centrifuge tube, and centrifuging for 5min at 700g-900 g; pouring out the centrifugal supernatant, adding 8mL of new complete culture solution into each culture bottle again, then adding a proper amount of complete culture solution to a constant volume, blowing and uniformly mixing, and respectively adding 2mL of matrix blood vessel components into each culture bottle; on the fifth day, the liquid is changed fully; collecting cells on the seventh day, carrying out passage or cryopreservation, and culturing for about 7 days, wherein when the area percentage of the cell clone group of the primary culture reaches 70-80%, digesting and harvesting;
(6) after the cell suspension is obtained, the cell suspension is inoculated to a new culture container after constant volume, and the density of passage cells is 5000-2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in each culture bottle, and adding culture medium to 10mL in each bottle; standing at 37 deg.C for 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P1 generation adipose-derived stem cells;
(7) passage: lightly blowing and beating the resuspension cells, inoculating to a new culture container after constant volume, wherein the density of the passage cells is 5000-6000 cells/cm2I.e. 3.75 × 105-4.5×105Culture flask according to 4.5 × 105Subculturing in one/culture flask, adding culture medium to 10 mL/flask, standing at 37 deg.C and 5% CO2Culturing in an incubator until the cell fusion degree reaches 85-90 percent to obtain the P2 generation adipose-derived stem cells;
(8) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
8. A medicament for treating pneumoconiosis, comprising the adipose-derived mesenchymal stem cell according to claim 7.
9. The medicament of claim 8, further comprising other pharmaceutically acceptable carriers, such as: trehalose, anhydrous or aqueous sodium chloride, glucose, sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride and magnesium chloride.
10. The drug of claim 8, wherein the drug is an injection solution consisting of 1 × 105-25×105Adipose-derived mesenchymal stem cells per mL, human serum albumin of 0.01-0.05g/mL and sodium chloride injection of 0.9%.
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