CN111518758A - Umbilical cord mesenchymal stem cells for treating lung diseases and preparation method thereof - Google Patents
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Abstract
The invention provides umbilical cord mesenchymal stem cells for treating lung diseases and a preparation method thereof. The umbilical cord mesenchymal stem cells provided by the invention can be used for preparing medicines for preventing or treating pneumoconiosis injury and fibrosis. The medicine is applied to lung disease treatment, particularly pneumoconiosis lung injury and fibrosis thereof, has high safety, good intervention and treatment effects on corresponding diseases, 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 umbilical cord mesenchymal stem cells for treating lung diseases and a preparation method thereof.
Background
The stem cells have the characteristics of self-renewal capacity, multidirectional differentiation potential and the like, and mesenchymal stem cells (mesenchymal stem cells) are one of important members of a stem cell family, originate from early mesoderm and ectoderm, are firstly found in bone marrow, are gradually found in the characteristics of multidirectional differentiation, hematopoietic assistance and immune regulation, and can be induced and differentiated into various tissue cells in vivo or in vitro.
Kotton in 2001 suggested that adult tissue-derived mesenchymal stem cells could home to the lungs, which brought a new opportunity for stem cells to treat respiratory diseases. Burt et al report: the stem cells can be fused in the lung by contacting with somatic cells, show corresponding epithelial-like tissue morphological characteristics and functions, and fulfill the aim of repairing damaged lung tissues. For the mechanism of the stem cells for relieving inflammation and resisting fibrosis, one hypothesis is that the stem cells can replace alveolar epithelial cells and play a role in promoting proliferation, regeneration and repair of damaged alveolar cells; another hypothesis is that stem cells can alter the microenvironment at the damaged site, promoting the secretion of cytokines.
Once pneumoconiosis happens, the pneumoconiosis develops progressively, and currently, the worldwide treatment of the pneumoconiosis is lack of specific drugs, clinically applied drugs mainly inhibit pulmonary fibrosis, and drug therapy can only relieve the ventilation function of patients to a certain extent and cannot effectively cure the diseases. Another direct method is lung lavage, which can physically remove residual dust in alveolar cavities, swallow pneumoconiosis macrophages, inflammation-causing factors, fibrosis-causing factors and other substances, dredge air passages, reverse air passage spasm, increase lung ventilation and blood oxygen ratio and the like, and is considered as the only effective treatment method for the pneumoconiosis at present. The curative effect of the large-capacity whole lung lavage is mainly related to the course of the pneumoconiosis, in the early stage (0-I stage) of the pneumoconiosis, a large amount of dust is still in the alveoli, and the whole lung lavage can wash out the dust in the alveoli, so the curative effect is good; in the late stage (stage iii), a large amount of dust has been transported into the pulmonary interstitium and thus cannot be washed out, the therapeutic effect is far less effective than in the early stage, and the late stage often fails to perform a large volume whole lung lavage due to complications. In addition, studies have demonstrated that there may be loss of alveolar surfactant (PS) during lung lavage.
The application of mesenchymal stem cells in the treatment of various severe lung diseases is a hotspot of research in recent years, and research has proved that the mesenchymal stem cells can improve or reduce lung injury. The self-renewal and multidirectional differentiation potential of the mesenchymal stem cells and the low immunogenicity and immunosuppressive effects of the mesenchymal stem cells provide a reliable theoretical basis for the safety and effectiveness of clinical application of the mesenchymal stem cells; in addition, after being infused into the body, the mesenchymal stem cells can not only migrate to the damaged tissue part, but also inhibit the release of proinflammatory factors and promote the repair of the damaged tissue, thereby providing a more effective treatment means for improving the symptoms of patients with pneumoconiosis and delaying the pulmonary fibrosis of the patients.
Chinese patent CN 106038597A discloses an application of human amniotic mesenchymal stem cells in preparation of a preparation for treating acute lung injury, wherein the mesenchymal stem cells separated from human amniotic tissues are adopted, PQ is injected into a peritoneal cavity to prepare an acute lung injury animal model, and an hAD-MSCs preparation is transplanted through a sublingual vein, so that the acute lung injury treatment effect is good. However, this method is only directed to the treatment of paraquat-induced acute lung injury.
Chinese patent CN 110680833A discloses a set of medicines for treating pneumoconiosis by combining lung lavage, which consists of mesenchymal stem cells and lung lavage liquid. The medicine set can improve serum immunological index, lung ventilation function, serum fibrosis index and CT density histogram of pneumoconiosis patient and improve pulmonary fibrosis. However, the method is complicated in operation and complex in components, and needs to be further optimized.
Disclosure of Invention
The invention aims to provide new application of mesenchymal stem cells, including new application in pneumoconiosis injury and fibrosis thereof.
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 umbilical cord mesenchymal stem cells comprise primary cells separated from an umbilical cord of a fetus and passage cells thereof.
The passage cell of the umbilical cord mesenchymal stem cell can be 1-50 generations.
The preparation method of the umbilical cord mesenchymal stem cells comprises the steps of taking a healthy fetal umbilical cord produced by caesarean section of gestation at term, collecting the umbilical cord, and then performing isolated culture by using a tissue block wall-adhering method within 12h, wherein the specific culture steps are as follows:
(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO2An incubator;
(2) on day 7, total fluid change, half fluid change on day 12, colony count on day 14, colony count over 50 cells, when the cells reach 70% -80% confluence, passage cells are digested with 0.1% EDTA containing 0.125% trypsin, and passage cells are digested with 2.5 × 103-5×103/cm2Density inoculation, marked as first generation cells, i.e. P1;
(3) taking P1 generation cells to be 3-6 × 103/cm2Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO2When the cells reached 70% -80% confluence in the incubator, the passaged cells were digested with 0.1% EDTA with 0.125% trypsin, and scored as second generation cells, P2.
(4) Sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8;
the complete culture medium in the step (1) is a Lonza UltraCULTURE general serum-free basal medium or a DMEM/F12 culture medium containing 2% of serum substitute; CO in the step (1) and the step (3)2The set conditions of the incubator are 37 ℃ and 5% CO2And saturated humidity.
The umbilical cord mesenchymal stem cells treat lung diseases by means of injection.
The umbilical cord mesenchymal stem cell injection mode is one-time injection or staged injection.
Preferably, theThe injection amount of the umbilical cord mesenchymal stem cells in one-time injection is 1 × 106-6×106Per kg body weight, more preferably 2 × 106One per kg body weight.
Preferably, the umbilical cord mesenchymal stem cells are injected for 1 to 6 times in equal or unequal phases, 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.
Preferably, the cell density of the human umbilical cord mesenchymal stem cells for injection is 2.5 × 105-7.5×105one/mL, preferably 5 × 105one/mL.
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; 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.
In another aspect, the present invention provides an umbilical cord mesenchymal stem cell.
The umbilical cord mesenchymal stem cells are obtained from the umbilical cord of a fetus produced by healthy full-term gestation cesarean section, and are isolated and cultured by a tissue block adherence method within 12 hours, wherein the culture method comprises the following specific steps:
(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO2An incubator;
(2) on day 7, total fluid change, half fluid change on day 12, and on day 14, counting colonies, counting more than 50 cells as colonies, and when the cells reach 70% -80% confluence, digesting the passage cells with 0.1% EDTA containing 0.125% trypsin, and digesting with 2.5-5 × 103/cm2Density inoculation, marked as first generation cells, i.e. P1;
(3) taking P1 generation cells to be 3-6 × 103/cm2Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO2In the incubator, when the cells reach 70% -80% fusion, the subculture cells are digested by 0.1% EDTA containing 0.125% trypsin, and are marked as second generation cells, namely P2;
(4) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
The complete culture medium in the step (1) is a Lonza UltraCULTURE general serum-free basal medium or a DMEM/F12 culture medium containing 2% of serum substitute; CO in the step (1) and the step (3)2The set conditions of the incubator are 37 ℃ and 5% CO2And saturated humidity.
In still another aspect, the present invention also provides an adipose-derived mesenchymal stem cell.
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 the P1 generation adipose-derived stem cells;
(7) passage: blowing and beating the re-suspended cells gently, inoculating the re-suspended cells into a new culture container after constant volume, and carrying out passageThe cell density is 5000-2I.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 umbilical cord 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 medicine is an injection solution, and the components of the injection solution comprise mesenchymal stem cells, human serum albumin and 0.9% sodium chloride injection, wherein the content of the mesenchymal stem cells is 1 × 105-25×105The content of human serum albumin is 0.01-0.05 g/mL.
The method provided by the invention is applied to treating lung diseases, particularly pneumoconiosis injury and fibrosis thereof, has high safety and good intervention and treatment effects on corresponding diseases, can realize quantitative production by mature cell culture technical means, and has good application prospect.
Drawings
FIG. 1 is a graph showing the results of the cell colony formation rate of umbilical cord mesenchymal stem cells at the generations P1, P5 and P8. Where A is the total number of formations and B is the formation rate.
FIG. 2 shows the results of flow cytometry detection using umbilical cord mesenchymal stem cells from generations P1, P2, P5 and P8 as research objects.
FIG. 3 is a graph analyzing the percentage of different cell cycles of umbilical cord mesenchymal stem cells of generations P1, P2, P5 and P8.
FIG. 4 shows the expression result of indoleamine 2, 3-dioxygenase after TNF-alpha and INF-gamma stimulation of umbilical cord 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 umbilical cord mesenchymal stem cells
The method is characterized in that the umbilical cord of a fetus, which is obtained from healthy full-term gestation cesarean section, is isolated and cultured by a tissue block adherence method within 12 hours, and the method comprises the following specific steps:
(1) cutting blood vessel along the lumen of umbilical vein, peeling off the intima of umbilical vein, and cutting the rest tissue into 3-5mm pieces3The small pieces were transferred to a T75 cell culture flask, and 10mL of complete medium (DMEM/F12 medium with 2% serum replacement) was added to make uniform distribution, and placed at 37 ℃ with 5% CO2Saturated humidity CO2An incubator.
(2) Total fluid change on day 7, half fluid change on day 12, and colony count on day 14 of culture, more than 50 cells are counted as colonies when the cells reach 70% -80% confluence, the passaged cells are digested with 0.1% EDTA containing 0.125% trypsin, and the passaged cells are digested with 2.5-5 × 103/cm2Density inoculation, scored as first generation cells (P1).
(3) Taking P1 generation cells and adding 5.7 × 103/cm2Performing density inoculation, performing subculture, adding complete culture medium 20mL to T175 flask, placing at 37 deg.C and 5% CO2Saturated humidity CO2In the incubator, the culture medium is filled with a culture medium,when the cells reached 70% -80% confluence, the passaged cells were digested with 0.1% EDTA with 0.125% trypsin, and scored as second generation cells (P2).
(4) Sequentially preparing and culturing P3-P8 generation secondary cells. Comparing the morphological characteristics of the obtained generation cells, analyzing the surface markers of the cells by using a flow cytometry technology, and identifying the differentiation potential of the obtained generation cells to osteoblasts and adipocytes under different induction conditions by using a specific staining method.
The detection and identification method of umbilical cord mesenchymal stem cells in the embodiment is as follows:
preliminary characterization of morphology and growth characteristics: human umbilical cord mesenchymal stem cells (hUC-MSCs) of generations P1, P3, P5 and P8 are used as research objects, the cell morphology is observed and photographed, and compared with the classical cell picture from other approaches (literature/books/graphs). The results show that: all generation cells show the characteristic of adherent growth; the P1/P3/P5 cells are roughly divided into three types according to the area, 0-2000 μm2The cell proportion is the largest, P80-2000 mu m2The cell proportion is reduced to more than or equal to 6000 mu m2The cell proportion is increased, and the statistical data are consistent with the observation result under a visual inspection mirror. Through research, all generation cells of the project meet the ISCT standard.
Cell proliferation assay: the umbilical cord mesenchymal stem cells of generation P1-P5 and generation P8 are used as research objects, a CCK8 kit (purchased from Sigma and having a product number of 96992) is used for detecting the proliferation conditions at different time points, and a growth curve is drawn at the same time. The results show that the proliferation trend of the umbilical cord mesenchymal stem cells in the P8 generation and the proliferation curve of the umbilical cord mesenchymal stem cells in the P1-P5 generation are different significantly, while the proliferation curve of the umbilical cord mesenchymal stem cells in the P1-P5 generation is not different, and the two-to-two comparison of the single-factor anova is not different significantly (P > 0.05).
Cell population doubling experiments: the umbilical cord mesenchymal stem cells of P1-P5 generation and P8 generation are used as research objects, the doubling time of cell populations is detected, and the differences of the cell populations are analyzed. The doubling time is calculated by analyzing different cell numbers, and the result shows that the average doubling time of the P1 generation is 25.10 h; the doubling times of P2 and P3 are 22.72h and 23.33h respectively, and the difference between the doubling times is not large; the doubling times for P4 and P5 were also similar, 27.87h and 27.45h, respectively; the P8 doubling time was relatively long, 36.86 h.
Cellular CFU formation capacity: the umbilical cord mesenchymal stem cells of P1 generation, P5 generation and P8 generation are used as research objects to detect the formation rate of cell colony. The cells of P1, P5 and P8 generation were all able to form CFU cell colonies by crystal violet staining, and the total number of colonies formed in the 6-well plate of the CFU experiment of P1 generation was: 24, the formation rate is: 20% (24/120 ═ 0.1083); in the P5 CFU experiment 6-well plate, the total is: 13, the formation rate is: 10.83% (13/120 ═ 0.1083); the total number of CFU of generation P8 is 7, and the formation rate is: 5.83% (7/120 ═ 0.0583) (see fig. 1).
Cell surface marker detection: the P1-P5 generation umbilical cord mesenchymal stem cells are used as research objects, and a flow cytometer is used for detecting cell surface markers. The results show that: the expression levels of CD90, CD73, CD105, HLA-ABC, CD29 and CD166 increase along with generations, and the expression levels of P2-P5 generations are all more than or equal to 95.0 percent; the expression levels of CD14, CD34, HLA-DR, CD271, CD45, CD19 and CD314 are reduced, and the expression levels of P2-P5 generations are less than or equal to 2 percent. The cells of generation P2-P5 all met the international standard for ISCT (high expression of CD105, CD73 and CD90, low expression of CD34, CD45, CD19 and HLA-DR).
Cell differentiation capacity assay: the umbilical cord mesenchymal stem cells of P2 generation and P5 generation are used as research objects, and the differentiation capacity of different generation umbilical cord mesenchymal stem cells for in vitro induction osteogenesis, adipogenesis and chondrogenesis is analyzed and compared. The results show that the cells of the P2 and P5 generations successfully induce adipogenic differentiation and osteogenic differentiation in vitro, the fat-forming differentiation capacity of the P2 and the fat-forming differentiation capacity of the P5 generations in vitro do not have obvious difference, the osteogenic differentiation capacity of the P2 and the P5 generations in vitro do not have obvious difference, and the cartilage differentiation capacity of the P2 and the P5 generations in vitro induction does not have obvious difference. The lipogenic differentiation capacity of the same generation of cells induced in vitro is relatively weaker than the osteogenic and chondrogenic capacities.
Example 2 umbilical cord mesenchymal stem cell safety assessment
Safety evaluation was performed on the human umbilical cord mesenchymal stem cells provided in example 1.
Cell cycle analysis experiment: the umbilical cord mesenchymal stem cells of the generations P1, P2, P5 and P8 are used as research objects, and the percentage of each cell cycle of the umbilical cord mesenchymal stem cells of the generations P1, P2, P5 and P8 is detected and analyzed by a flow cytometer. As can be seen from FIGS. 2 and 3, most of the cell cycles of the umbilical cord mesenchymal stem cells of P1-P8 are gathered in the G0-G1 stages, although a little difference exists between the cell cycles, the G1 stage can be maintained at more than 84%, the number of the cells undergoing division is small, and the DNA ploidy is not abnormal.
A telomerase activity analysis experiment includes that umbilical cord mesenchymal stem cells of P0, P1, P2 and P5 generations are used as research objects, a PCR amplification combined ELISA method is used for detecting the relative activity of the telomerase of cells, and results show that the relative activity of the telomerase is reduced after P2 generations, and the telomerase activity is estimated to be related to cell growth and senescence but not affect other functions of the cells.
And (3) soft agar clone formation experiment, namely detecting the clone formation capability and clone size of cells in soft agar by using P1, P2 and P5 umbilical cord mesenchymal stem cells as research objects. The results show that the P1/2/5 generation cells and the positive control Hela cells have clonogenic cells, and the fibroblasts have negative results. Observations of the colony of the P5 generation showed that cell clustering was due to self-proliferation. The cells of the P1, P2 and P5 generations all have the capability of forming cloning clusters, and the clustering capability of each generation has no significant difference.
Karyotype analysis, namely, using umbilical cord mesenchymal stem cells producing P1 and P5 as research objects, observing whether the cell is normal after passage under a mirror, and displaying that no abnormality is found in the cells of P1 and P5 generations.
Example 3 lymphocyte inhibition test results
In this example, human umbilical cord mesenchymal stem cells obtained by culturing in example 1 were used as a study object, the CCK8 method was used to detect the inhibitory effect of umbilical cord mesenchymal stem cells on peripheral blood mononuclear cells (PMBC), and the effect of umbilical cord mesenchymal stem cells on peripheral blood mononuclear cells was evaluated by flow cytometry, and the immunomodulatory effect thereof was analyzed. The result shows that the umbilical cord mesenchymal stem cells have an immunoregulation effect on lymphocytes, can promote the production of Treg cells and inhibit the secretion of inflammatory factors such as IL-17 and the like.
(1) CCK8 test inhibition effect of umbilical cord mesenchymal stem cells on peripheral blood mononuclear cells
The results after 3 days of inhibition of peripheral blood mononuclear cells by the umbilical cord mesenchymal stem cells generation P2 show that: the expansion of peripheral blood mononuclear cells is inhibited as the proportion of umbilical cord mesenchymal stem cells increases, and the umbilical cord 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; and the inhibition of proliferation of peripheral blood mononuclear cells is enhanced along with the increase of umbilical cord mesenchymal stem cells, (umbilical cord mesenchymal stem cells: peripheral blood mononuclear cells ═ 1:10) vs (umbilical cord mesenchymal stem cells: peripheral blood mononuclear cells ═ 1: 50), and P is less than 0.05.
Results 3 days after inhibition of peripheral blood mononuclear cells by P5 umbilical cord mesenchymal stem cells: the effect of the umbilical cord mesenchymal stem cells of the P2 generation is similar to that of the umbilical cord mesenchymal stem cells of the P5 generation, the expansion of the peripheral blood mononuclear cells is inhibited along with the increase of the proportion of the umbilical cord mesenchymal stem cells of the P5 generation, and the ratio of the peripheral blood mononuclear cells to the umbilical cord mesenchymal stem cells is more than 1: at 50 hours, compared with a normal control group, the composition has a significant difference, and P is less than 0.001; and the inhibition of proliferation of peripheral blood mononuclear cells is enhanced along with the increase of umbilical cord mesenchymal stem cells, (umbilical cord mesenchymal stem cells: peripheral blood mononuclear cells ═ 1:10) vs (umbilical cord mesenchymal stem cells: peripheral blood mononuclear cells ═ 1: 50), and P is less than 0.05.
(2) Flow detection of influence of umbilical cord mesenchymal stem cells on IL17 cells in peripheral blood mononuclear cells
Effect of P2 passage umbilical cord mesenchymal stem cells on Th17 cell population: the results show that umbilical cord mesenchymal stem cells are paired with CD4+IL17+The cytostatic effect of (a) was enhanced with increasing concentration, and there was a significant difference, namely (umbilical cord mesenchymal stem cell: peripheral blood mononuclear cell ═ 1:10) VS (umbilical cord mesenchymal stem cell: peripheral blood mononuclear cell ═ 1: 50), P<0.01。
Effect of P5 passage umbilical cord mesenchymal stem cells on Th17 cell population: the result shows that P5 generation umbilical cord mesenchymal stem cell pairs CD4+IL17+The cell inhibition effect of the compound is similar to that of the umbilical cord mesenchymal stem cells generation P2, and the cell inhibition effect is enhanced along with the increase of the amount of the umbilical cord mesenchymal stem cells.
(3) Flow detection of umbilical cord mesenchymal stem cells to peripheryFoxp3 in blood mononuclear cells+CD4+CD25+Influence of cells
P2 generation umbilical cord mesenchymal stem cell pair Foxp3+CD4+CD25+Influence of cell population: the P2 generation umbilical cord mesenchymal stem cells can remarkably induce Foxp3+CD4+CD25+The survival of the cells is also increased along with the increase of the proportion of the umbilical cord mesenchymal stem cells.
Effect of P5 generation umbilical cord mesenchymal stem cells on Foxp3+ CD4+ CD25+ cell population: the experiment that the umbilical cord mesenchymal stem cells of generation P5 can obviously induce Foxp3+CD4+CD25+The survival of the cells is also increased along with the increase of the proportion of the umbilical cord mesenchymal stem cells.
Example 4 measurement of the ability of inflammatory factors to stimulate the secretion of indoleamine 2, 3-dioxygenase by human umbilical cord mesenchymal Stem cells
This example was carried out using the human umbilical cord mesenchymal stem cells obtained in example 1.
The expression of indoleamine 2, 3-dioxygenase after TNF-alpha and INF-gamma stimulation of umbilical cord mesenchymal stem cells is measured by an ELISA method by using P1 and P5 umbilical cord mesenchymal stem cells as research objects, and the immunoregulation effect of the umbilical cord mesenchymal stem cells is evaluated. The results show (fig. 4) that compared with the control group, the indoleamine 2, 3-dioxygenase concentration of the P1 and the P5 generation cells in the stimulated group are 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 complete culture Medium for umbilical cord mesenchymal Stem cells induced angiogenesis experiment of human umbilical vein endothelial cells
This example was carried out using the human mesenchymal stem cells obtained in example 1.
The umbilical cord mesenchymal stem cells of P2 and P5 generations are taken as research objects, and the full culture medium of the umbilical cord mesenchymal stem cells is applied to stimulate the angiogenesis capacity of Human Umbilical Vein Endothelial Cells (HUVECs). The results showed that the number of rings generated at 4h or 8h, P2 and P5, were both significantly higher than the control group at the same time, while there was no significant difference between the periods P2 and P5; the 4h node numbers P2 and P5 generation cells are higher than those of the control group, and the time slices P2 and P5 have no significant difference.
Example 6 intervention and therapeutic Effect of human umbilical cord mesenchymal Stem cells on pneumoconiosis rats
This example was carried out using the human mesenchymal stem cells obtained in example 1. The construction method of the pneumoconiosis rat model comprises the following steps: non-exposed type trachea cannula is filled with silicon dioxide (SiO)2) Suspension method.
Control group: normal rats;
model group: a model rat;
the intervention group comprises umbilical cord mesenchymal stem cells injected via tail vein on 1 st day, 4 th day, 8 th day and 15 th day after the first model building of pneumoconiosis rat, and the cell density is 3 × 106one/mL, the injection amount is 0.5 mL/mouse.
The treatment group comprises umbilical cord mesenchymal stem cells injected via tail vein at 29 days, 36 days, 43 days and 50 days after the first model building of pneumoconiosis rat, and the cell density is 3 × 106one/mL, the injection amount is 0.5 mL/mouse.
(1) CT scan of the lungs of each group of rats: CT scanning examination is respectively carried out on the rats of each group at the 60 th day and the 75 th day after the model building, and after the anesthetized rats are injected into the abdominal cavity, the normal posture adhesive tapes of the rats are fixed on an examination bed. Scanning was performed using somatommemotion 16 slice spiral CT, siemens germany.
(2) Animal treatment: animals were treated on day 75 after the first dust. Anesthetized rats, abdominal aorta are bled and sacrificed. Separating serum, collecting lung, heart, liver and kidney of rat, fixing part of formaldehyde, and freezing part of liquid nitrogen.
(3) Lung coefficient detection: after each group of rats died, the whole lung was taken, and the general morphology of the lung was observed after the blood stain was washed with physiological saline. The lung organ coefficient was calculated by "lung organ coefficient (mg/g) ═ wet mass of lung (mg)/mass of lung (g)".
(4) Histopathological observation of lung: 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.
(5) Lung tissue Hydroxyproline (HYP) level assay: according to the steps of the specification of a hydroxyproline detection kit (purchased from Nanjing institute of bioengineering), the hydroxyproline level of lung tissues is detected by an enzyme-linked immunosorbent assay (ELISA) instrument at the wavelength of 450nm by adopting an alkaline hydrolysis method. The calculation formula is as follows: hydroxyproline level (mg/g wet mass) — (measured tube optical density-control tube optical density)/(standard tube optical density-blank tube optical density) × standard tube level (5mg/L) × total hydrolysate volume (10 mL)/tissue wet mass (mg).
(6) Detecting the TGF-beta 1 of rat lung tissue and the IL-6 level of serum by an ELISA method: according to the experimental steps of the double-antibody sandwich method of the ELISA detection kit specification, the TGF-beta and serum IL-6 levels in lung tissues are detected by an enzyme-linked immunosorbent assay (ELISA) instrument under the wavelength of 450 nm.
Analysis of results
(1) The general observation of the lung shows that the appearance of the lung of the rat in the control group is normal, which indicates that the lung is smooth, the lung tissue is pink, and the color is uniform; the lung tissue surfaces of the model group are scattered in grey spots, the texture is tough, frosted feeling is generated when the model group is cut, and the lung tissues of the intervention group and the treatment group are seen in grey spots, so that the lung tissues are obviously reduced compared with the model group.
(2) Pathological examination of the lungs of rats in each group
HE staining of lung tissue showed that the control group showed normal lung tissue structure and 20 model groups all had significant pathological changes. The control group has complete alveolar structure, no obvious inflammatory cell infiltration, edema and fibroplasia, and no thickening of the vascular wall. Compared with the control group, the lung tissue pathological change of the model group is obvious: it is seen that various inflammatory cell infiltrates, a large amount of effusion in the alveolar cavity, the alveolar wall is thickened, interstitial congestion of the lung occurs, cellular nodules (granuloma) appear, fibrosis appears in the center of part of nodules, or cellular nodules with different sizes and fibrous nodules coexist, the nodules are gradually enlarged and fused, even become flaky, and the normal alveolar structure is obviously reduced. Visible part of alveolar, bronchiole or interstitial bifold silica particles. The pulmonary alveolar structure of rats in the intervention group and the treatment group is slightly damaged, the normal pulmonary alveolar structure is increased, the pulmonary alveolar epithelium is slightly thickened, and sporadic cellular nodules are generated. Masson staining results show that a small amount of normal blue-stained collagen fiber scaffolds are observed in the lung tissues of rats in the control group; the lung tissues of rats in the model group are greatly stained with blue collagen fibers; only a small amount of blue collagen fibers were seen in the lung tissue of rats in the pretreatment group and the treatment group.
(3) Lung CT scan of rats in each group: the control group showed normal imaging, and showed clear and transparent lung field, and formed texture image of larger bronchus or larger blood vessel, and had natural and smooth running, no thickening, uniform density in lung parenchyma, and no local increase or decrease area. The model group is mainly expressed as diffuse cloud fog sheet-shaped high-density images of the lung, rough lung texture can be seen, and frosted fine-particle high-density images can be seen in lung interstitium. The diffuse cloud and fog flaky shadow range of the lung can be reduced, the density is reduced, the lung texture is thickened and reduced, and the lung field definition is increased in the intervention group and the treatment group.
(4) Rats (12 per group) lung organ coefficients, lung hydroxyproline and TGF- β 1 and serum IL-6 levels: the organ coefficient, the lung tissue hydroxyproline acid level and TGF-beta of the model group rat and the serum IL-6 level are all obviously higher than those of the control group (the lung coefficient of each group is respectively 2.45 +/-0.31, 8.54 +/-0.47, 5.87 +/-0.82 and 5.64 +/-0.34), the 4 indexes of the rats of the intervention group and the treatment group are all lower than those of the model group, the difference has statistical significance, and the difference between the intervention group and the treatment group has no statistical significance.
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 pneumoconiosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, severe novel coronavirus pneumonia, acute respiratory distress syndrome or lung injury; the mesenchymal stem cells are one or more of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells and bone marrow mesenchymal stem cells.
2. The use of claim 1, wherein the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
3. The use of claim 2, wherein the umbilical cord mesenchymal stem cells comprise primary cells isolated from human umbilical cord tissue and passaged cells thereof.
4. The use of claim 2, wherein the total injection amount of the umbilical cord mesenchymal stem cells is 1 × 106-36×106One per kg body weight.
5. The application of claim 2, wherein the umbilical cord mesenchymal stem cells are prepared by collecting healthy umbilical cord of fetus produced by caesarean section of full-term pregnancy, and performing isolated culture within 12h by tissue block adherence method, and the culture comprises the following specific steps:
(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO2An incubator;
(2) on day 7, total fluid change, half fluid change on day 12, colony count on day 14, colony count over 50 cells, when the cells reach 70% -80% confluence, passage cells are digested with 0.1% EDTA containing 0.125% trypsin, and passage cells are digested with 2.5 × 103-5×103Per cm2Density inoculation, marked as first generation cells, i.e. P1;
(3) taking P1 generation cells to be 3-6 × 103/cm2Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO2In the incubator, when the cells reach 70% -80% fusion, the subculture cells are digested by 0.1% EDTA containing 0.125% trypsin, and are marked as second generation cells, namely P2;
(4) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
6. The use according to claim 5, wherein the complete medium in step (1) is Lonza UltraCULTURE Universal serum-free basal medium containing 2% serum replacement or DMEM/F12 basal medium; the step (A) of1) And CO in step (3)2The set conditions of the incubator are 37 ℃ and 5% CO2And saturated humidity.
7. The umbilical cord mesenchymal stem cells are obtained from the umbilical cord of a healthy fetus produced by caesarean section of full-term pregnancy, and are separated and cultured by a tissue block adherence method within 12 hours, wherein the culture method comprises the following specific steps:
(1) cutting blood vessel along the umbilical vein lumen longitudinally, peeling off umbilical vein intima, cutting the rest tissue into small pieces, transferring into cell culture flask, adding 10mL complete culture medium to make it uniformly distributed, placing in CO2An incubator;
(2) on day 7, total fluid change, half fluid change on day 12, colony count on day 14, colony count over 50 cells, when the cells reach 70% -80% confluence, passage cells are digested with 0.1% EDTA containing 0.125% trypsin, and passage cells are digested with 2.5 × 103-5×103/cm2Density inoculation, marked as first generation cells, i.e. P1;
(3) taking P1 generation cells to be 3-6 × 103/cm2Performing density inoculation, performing subculture, adding 20mL of complete culture medium into a culture flask, and placing in CO2In the incubator, when the cells reach 70% -80% fusion, the subculture cells are digested by 0.1% EDTA containing 0.125% trypsin, and are marked as second generation cells, namely P2;
(4) and sequentially preparing and culturing third to eighth generation secondary cells, namely P3-P8.
8. A medicament for treating pneumoconiosis, comprising the umbilical cord mesenchymal stem cells 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 9, wherein the drug is an injection solution consisting of 1 × 105-25×105Mesenchymal stem cells per mL, human serum albumin of 0.01-0.05g/mL, and sodium chloride injection of 0.9%.
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