CN111346110A - Application of mesenchymal stem cell supernatant in preparation of preparation for treating lung cell injury - Google Patents
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The scheme discloses the application of mesenchymal stem cell supernatant in the technical field of stem cell application in preparing a preparation for treating lung cell injury; in particular to application of a human amniotic mesenchymal stem cell culture supernatant preparation in treating acute lung cell injury caused by paraquat. Compared with human amniotic mesenchymal stem cells, the biological product of the invention can reduce the problems of ethic and biological safety brought by cell therapy and convert the biological product into clinical treatment medicaments more safely and effectively.
Description
Technical Field
The invention belongs to the technical field of stem cell application, and particularly relates to application of mesenchymal stem cell supernatant in preparation of a preparation for treating lung cell injury; in particular to application of a human amniotic mesenchymal stem cell culture supernatant preparation in treating acute lung cell injury caused by paraquat.
Background
Paraquat (PQ) is a fast-acting herbicide and has been widely used due to its high herbicidal effect, but PQ is extremely toxic to human bodies, and is absorbed and distributed to various tissues and organs of the whole body along with blood circulation, most of PQ is absorbed by lung tissues to cause extensive alveolar cell injury, so that Acute Lung Injury (ALI) and subsequent fibrosis are caused, wherein the strong inflammatory response of the lung tissues is the central link leading to ALI and pulmonary fibrosis, and the mortality rate of oral poisoning reaches over 90%. Although China stops the sale and use of PQ water aqua at home, the patients who are diagnosed with PQ poisoning are still rare at present, and no specific antidote is available at present. The clinical routine treatment measures mainly include: 1. inhibiting the absorption of toxic substances, such as cleaning contaminated skin, promoting vomiting, gastric lavage and catharsis; 2. accelerating PQ excretion in vivo, such as blood perfusion, hemodialysis, etc.; 3. the medicine treatment, such as oxygen free radical elimination, pulmonary fibrosis reduction, symptomatic support and the like. However, the treatment measures of inhibiting inflammatory factors, improving oxygenation and promoting absorption of fluid in the lung and the like cannot effectively relieve the progress of the disease course of lung injury caused by paraquat poisoning, and the death rate is high.
With the rapid development of stem cell related technologies, the characteristics of stem cells such as low immunogenicity, differentiation to multiple germ layers and paracrine create new ideas and methods for treating lung injury diseases. Studies have found that transplanted Bone Marrow Mesenchymal Stem Cells (BMMSCs) have a protective effect on PQ-induced lung injury, and can reduce pulmonary edema, lipid peroxidation and inhibit the release of inflammatory mediators. The human umbilical cord mesenchymal stem cells have certain therapeutic effect on paraquat poisoning acute and chronic lung injury, and the effect can be realized through a paracrine mechanism.
Human amnion-derived mesenchymal stem cells (hAD-MSCs) have stronger amplification capacity than bone marrow and umbilical cord-derived mesenchymal stem cells. Under specific in vitro induction culture conditions, hAD-MSCs can differentiate into all cells from three germ layers, including nerve cells, cardiac muscle cells, osteogenic and chondrogenic cells, pancreatic cells, and the like. As stem cells, the stem cells have the potential of migrating to damaged tissues, and in addition, the hAD-MSCs have immunoregulation characteristics and can inhibit inflammatory response of the damaged tissues. And the hAD-MSCs have rich sources, short separation period, strong amplification capacity, stable properties and obvious curative effect. These properties have made it more clinically useful in the treatment of ALI due to PQ poisoning.
The applicant finds the effect of hAD-MSCs in preparing the preparation for treating acute lung injury (application number: CN201610359976.7) through previous researches, and the invention further deeply researches the protective effect of hAD-MSCs supernatant on lung cells in vitro on the basis of the original researches and explores the possible paracrine mechanism. The cell culture supernatant is used for replacing cell products, so that the effect of treating lung cell injury can be achieved, and the ethical and biological safety problems caused by cell treatment can be reduced.
Disclosure of Invention
The invention aims to provide application of mesenchymal stem cell culture supernatant in a preparation for treating paraquat induced acute lung cell injury.
The invention discovers that the mesenchymal stem cell supernatant has a good effect on treating the lung cell injury, and the mesenchymal stem cell supernatant is from a serum-free culture medium of the mesenchymal stem cell, does not contain cells and belongs to a non-cell supernatant preparation.
The mesenchymal stem cell supernatant from bone marrow, placenta, amniotic fluid, umbilical vein subendothelial layer, peripheral blood, liver, fat, muscle, skin or umbilical cord tissue is adopted, and the effect of treating lung cell injury is better.
Particularly, the human amniotic mesenchymal stem cell culture supernatant preparation has a better treatment effect on the lung cell injury, and particularly has an obvious treatment effect on the paraquat-induced acute lung cell injury, and the human amniotic mesenchymal stem cell culture supernatant preparation can be applied to the paraquat-induced acute lung cell injury. The mesenchymal stem cells are derived from a amniotic membrane layer on the surface of the human placenta tissue of the post-partum discarded matter, namely human amniotic mesenchymal stem cells, and the culture supernatant is obtained by culturing in a serum-free culture medium for 24 hours after subculture and is free of cells and cell debris residues after filtering by a 0.22 mu m small filter.
The lung cells cultured in vitro are infected with PQ with different concentration gradients, and are subjected to CCK-8 detection after immediately adding hAD-MSCs-CM (human amniotic mesenchymal stem cell supernatant) for intervention for 24 hours; detecting the apoptosis rate by adopting flow cytometry; observing the shapes of the cells and the organelles by using an electron microscope; observing the fluorescent probe under a fluorescent microscope, and detecting the level of Reactive Oxygen Species (ROS); detecting the expression level of related proteins in cells by using a Western blot method; the content of cytokines in the culture supernatant was measured by ELISA.
Compared with human amniotic mesenchymal stem cells, the biological product of the invention can reduce the problems of ethic and biological safety brought by cell therapy and convert the biological product into clinical treatment medicaments more safely and effectively.
Drawings
FIG. 1 is a graph showing the growth of hAD-MSCs;
FIG. 2 is a graph showing that PQ decreases the proliferative activity of A549 cells and hAD-MSCs-CM relieves the decrease in cell proliferation caused by PQ;
FIG. 3 is a graph of hAD-MSCs-CM reducing the level of oxidative stress and inflammatory mediators by PQ;
FIG. 4 is a graph of apoptosis caused by hAD-MSCs-CM decreasing PQ;
FIG. 5 is a diagram of Western blot for detecting the intracellular CHOP and GRP78 protein expression.
Detailed Description
Separation, culture, amplification and identification of hAD-MSCs: the amniotic membrane was separated from the placenta under sterile conditions with forceps, washed once with D-PBS containing double antibody (final concentration: penicillin: 100IU/ml, streptomycin: 0.1mg/ml, ready for use), and then repeatedly washed with D-PBS containing no double antibody to remove residual blood stain and mucus. Cutting amnion, adding 0.05% trypsin-0.02% EDTA-2Na solution, digesting for 30min at 37 ℃ by a constant temperature horizontal shaking table (200rpm/min), filtering by a 200-mesh stainless steel filter screen, discarding the filtrate, washing the rest amnion tissue fragments by a D-PBS solution, adding 0.75mg/ml II type collagenase-0.075 mg/ml DNase I digestive solution, digesting for 2-3 h at 37 ℃ and 200rpm/min until the tissue is completely digested, filtering by a 200-mesh stainless steel filter screen, collecting the cell filtrate, centrifuging at 2400rpm/min for 16min, discarding the supernatant, and suspending the precipitate by LG-DMEM culture medium containing 2mM L-glutamine, 10% fetal calf serum and 10ng/ml FGF, namely suspension of hAD-MSCs and 2% Taiwan FGFDetecting cell viability by trypan blue staining, inoculating to 25cm2In the culture flask, the cell density is 5 × 105Inoculating in 5% CO at 37 deg.C/ml2Culturing under the saturated humidity condition, replacing a new culture medium after 24h, flushing with D-PBS (phosphate buffer solution) when adherent cells are fused to be about 80-90%, adding 0.25% trypsin-0.02% EDTA-2Na digestive juice, incubating at 37 ℃ for 1min, observing the digestion condition under a microscope, stopping digestion by using a culture medium containing 10% FBS when the cells begin to shrink, collecting the cells, carrying out parallel passage amplification culture, and observing the cell growth condition by an inverted microscope.
The purity of hAD-MSCs is determined by taking 3 rd generation hAD-MSCs, centrifuging at 1000rpm/min for 5min, discarding supernatant, washing with D-PBS once, suspending, adjusting cell density to 2.0 × 106Taking 200 mul of cell suspension, adding 10 mul of fluorescein labeled CD44, CD73, CD90, CD105, CD34, CD45, CD14, CD19, CD80, CD86 and HLA-DR antibody according to a combination scheme, shaking and mixing uniformly, incubating at room temperature in a dark place for 30min, adding 2ml of D-PBS containing 0.1% BSA into each tube, mixing uniformly, centrifuging at 1000rpm/min for 5min, discarding supernatant, shaking and suspending cells, adding 200 mul of 1% paraformaldehyde into each tube, mixing uniformly, storing at 2-8 ℃ in a dark place, detecting and analyzing by using a flow cytometer within 24h, wherein the number of collected cells of each sample is more than or equal to 2 × 104Cell Quest software analysis. Isotype control antibodies were mouse IgG labeled with the corresponding fluorescein.
Collecting culture supernatant of hAD-MSCs: and taking 3-6 generation hAD-MSCs, when the hAD-MSCs are approximately spread to 80-90% of the bottom of the dish, abandoning the culture medium, washing the culture medium for 2-3 times by using D-PBS, and replacing the serum-free L-DMEM culture medium without any additive factor component. After further culturing for 24 hours, the culture supernatant was aspirated, centrifuged at 3000rpm, and the supernatant was aspirated without the cell and debris fraction, filtered through a 0.22 μm filter, and dispensed into an EP tube and stored at-80 ℃ to obtain hAD-MSCs-CM (hAD-MSCs culture supernatant).
Acute lung cell injury model preparation, taking a549 cells (a continuous tumor cell line from human lung cancer with type II alveolar epithelial cell characteristics) as an example: after A549 cells are cultured, PQ solutions (0, 250, 500, 750 and 1000 mu M) with different concentrations are added for 24h (6 multiple wells in each group), 10 mu L of CCK-8 reagent is added into each well, the cells are incubated in an incubator for 2h, and the absorbance value at the wavelength of 450nm is measured; cell proliferation activity was calculated according to the formula: cell proliferation activity (%) — (detection well OD value-zero well OD value)/(control group OD value-zero well OD value). In the same way, the concentration of hAD-MSCs-CM intervention was divided into 25%, 50%, 75% (volume fraction containing medium components). The culture medium, PQ solution and hAD-MSCs-CM were added in this order, and the assay was performed after 24h of culture.
A549 cells are cultured by a 96-well plate, PQ is infected (in the embodiment, the PQ is dichloroparaquat), and CCK-8 detection is carried out after the intervention of hAD-MSCs-CM for 24 h. A549 cells are cultured by using a 6-well plate, and the apoptosis rate is detected by adopting flow cytometry after the intervention for 24 hours. Using 25cm2Culturing A549 cells in a cell culture bottle, intervening for 24h, observing cell and organelle forms by an electron microscope, detecting the level of Reactive Oxygen Species (ROS), detecting the expression level of related proteins in the cells by a Western blot method, and detecting the content of cytokines in culture supernatant by an ELISA method.
Qualitative observation of intracellular Reactive Oxygen Species (ROS): DCFH-DA (2, 7-dichlorofluoroescin diacetate) has no fluorescence, is hydrolyzed into DCFH by esterase after entering cells, is oxidized into a strong green fluorescent substance DCF which cannot permeate cell membranes in the presence of active oxygen, has the maximum peak at the excitation wavelength of 502nm and the maximum peak at the emission wavelength of 530nm, and has the intensity which is in direct proportion to the level of active oxygen in the cells. After each group of cells are cultured for 24 hours, the cells are washed 1 time by PBS, A549 cells are added into the culture medium and 40 mu M of DCFH-DA dye (added according to the volume of 3: 1), the cells are incubated for 30min, and the fluorescence coloration of each group of cells is observed by a fluorescence microscope under a green light source.
Cell morphology observation after 24 hours of cell culture, cell morphology was observed using an inverted phase contrast microscope Each group of cells was washed 2 times with PBS, digested, centrifuged (2200 × g, 5min), the supernatant was discarded, fixed with 1% osmic acid, dehydrated with different concentrations of ethanol and acetone in a gradient, soaked, embedded, trimmed, sectioned and stained, and placed under a transmission electron microscope to observe changes in tissue structure.
Detecting oxidation indexes and inflammatory factor levels of cells and supernatant, namely operating each sample according to a kit specification, respectively detecting the activity of SOD in A549 cells by using a xanthine oxidase method, detecting the content of MDA in the A549 cells by using a thiobarbituric acid method, respectively detecting the content of IL-1 β, IL-6 and IL-10 in the culture supernatant of the A549 cells by using an ELISA method, arranging blank control holes and standard product holes in experiments, selecting a wavelength of 450nm to detect absorbance, making a standard curve, and calculating the concentration value of a sample to be detected.
Annexin V-FITC/PI detection of apoptosis levels: annexin V-FITC can be specifically combined with phosphatidylserine (phosphatidylserine) which is turned to the outside of a membrane in the process of apoptosis, and emits green fluorescence under the excitation of blue light, so that apoptotic cells and normal cells are distinguished. Propidium iodide (propidium iodide) is a nucleic acid dye that does not penetrate intact cell membranes, can penetrate broken cell membranes (including late apoptotic and dead cells), and red stains cell nuclei. The two are matched to distinguish early and late apoptosis. After the cells are cultured for 24 hours, digesting and collecting the cells; washing with cold PBS for 2 times, adding Binding Buffer for resuspension, subpackaging cell suspension, adding Annexin V-FITC and PI respectively, mixing, reacting in dark for 5min, and detecting with 1h internal flow cytometer.
Western blot detection of expression of related proteins includes collecting A549 cells of each group, adding proper amount of lysate, centrifuging at 4 deg.c (39588 × g for 10min), taking supernatant, determining protein concentration by BCA method, adding loading Buffer and denatured protein at 100 deg.c for 5min, adding 20 ug of total protein sample into each lane, SDS-PAGE electrophoresis, transferring to PVDF microporous membrane at 200mA for 70min, sealing with 3% BSA TBST sealing solution at normal temperature for 2 hr, adding diluted primary antibody, incubating overnight at 4 deg.c, washing with TBST for 3 times (15 min each), oscillating with secondary antibody at normal temperature for 2 hr, washing with TBST for 3 times, dropping A, B mixture onto membrane, imaging in Bio-Rad gel imaging system, and grey scanning analysis with Western-blot J software.
The statistical method comprises the following steps: data related to experiment, measured data are mean ± standard deviation
Representing that SPSS17.0 statistical software is adopted for statistical analysis, and single-factor analysis of variance is adopted for comparison among groups (amplitude groups); comparing two groups (between 2groups) with difference, and adopting independent sample t test; p < 0.05 is statistically significant.
As a result:
the hAD-MSCs have the characteristics of adherent growth, cells are slowly attached during primary culture, the primary cells can be fused by more than 80% after about 3 days of culture, and the cell morphology has diversity and is polygonal, fusiform and the like (part A in figure 1). As the cells were subcultured for purification, the morphology of hAD-MSCs gradually appeared as spindle or fiber-like, growing as radial or vortex-like (FIG. 1, panel B).
After the A549 cells are infected with PQ with the concentrations of 250 muM, 500 muM, 750 muM and 1000 muM for 24 hours, the cell proliferation activity is measured by a CCK-8 method. Cell proliferation activity decreased (fig. 2A). The IC50 PQ concentration was calculated to be 816. mu.M, approaching 750. mu.M, so we chose 750. mu.M concentration of PQ as the molding concentration, followed by intervention with hAD-MSCs-CM.
Adding hAD-MSCs-CM with different concentrations for intervention, culturing for 24h, and detecting cell proliferation activity to find that the cell proliferation activity of the PQ + CM group is higher than that of the PQ group; compared with hAD-MSCs-CM of different concentrations, the A549 cell proliferation activity of 50% volume fraction dry prognosis is the highest (FIG. 2B).
And detecting the content of the ROS in the cells by adopting a DCFH-DA probe. The green fluorescence in the visual fields of the control group and the CM group is less, and the ROS content is low; the PQ group and PQ + CM group fluoresced more green and contained more ROS, but the PQ + CM group fluoresced less and contained less ROS than the two groups (fig. 3A).
The cells of the control group and the CM group are uniform in size, distributed like a paving stone, good in wall adhesion and tight in connection when observed by an inverted microscope; after 24h exposure to PQ, the cells were not tightly connected and the number of dead cells increased, but the PQ + CM group had a higher cell density and more adherent cells than the PQ group (FIG. 3B). Observing the control group and the CM group through a transmission electron microscope, wherein the cell forms are regular, the sizes are consistent, the cell membranes are complete, microvilli can be seen on the cell surfaces, the cytoplasm is uniform, various organelles are intact, the nuclear membrane is complete, and the chromatin is uniform; after PQ is infected, microvilli and protrusions on the surface of an A549 cell are reduced, the cell membrane is accompanied with the expression of buds and apoptotic bodies, endoplasmic reticulum is swollen, autophagosome is increased, the organelles such as mitochondria and the like are reduced, and the distribution of chromatin is abnormal; organelle damage such as endoplasmic reticulum was reduced in the PQ + CM group compared to the PQ group (fig. 3C).
The MDA kit is adopted to detect the MDA content in each group of cells, the content of the PQ group is increased compared with that of the control group, and the MDA content of the PQ + CM group is reduced compared with that of the PQ group (figure 3D). The SOD content in the cells of each group was determined, and the SOD content in the PQ group was decreased compared to the control group, while the SOD content in the PQ + CM group was significantly increased compared to the PQ group (FIG. 3E). Compared with the control group, the CM group has no obvious difference in MDA and SOD content.
IL-1 β and IL-6 levels in the supernatant were significantly higher in the PQ group than in the Control group, while the PQ + CM group was significantly lower than the PQ group (FIGS. 3F and 3G). the IL-10 level in the supernatant, the Control group as a base value, was significantly higher in the remaining 3 groups than the Control group and was highest in the CM group, whereas the PQ + CM group was significantly higher than the PQ group (FIG. 3H).
After each group is infected with virus (PQ with 750 muM concentration) and intervened (hAD-MSCs-CM with 50% concentration) for 24h, the apoptosis condition of each group of cells is detected by using an annexinV-FITC/PI apoptosis kit, and the detection shows that the apoptosis rate of the PQ group of cells is higher than that of other groups. The early apoptosis rates of the control group, PQ + CM group and CM group were (1.96. + -. 0.50)%, (14.16. + -. 1.15)%, (7.52. + -. 2.35)% and (1.71. + -. 0.88)%; and the rate of early apoptosis was reduced in the PQ + CM group compared with that in the PQ group cells (FIG. 4A, FIG. 4B).
Detecting the protein expression conditions of cell apoptosis and related pathways of endoplasmic reticulum by western blot technology, and prompting the Bax/Bcl-2 level and the PQ group to be obviously higher than the Control group by results; whereas the PQ + CM group was significantly decreased compared to the PQ group, the CM group was not significantly different from the control group (FIGS. 4C and 4D).
After each group was infected with virus (PQ at a concentration of 750. mu.M) and intervened (hAD-MSCs-CM at a concentration of 50%) for 24h, western blot detection revealed that the expression levels of CHOP and GRP78 proteins in the PQ group were increased as compared with the Control group (FIG. 5A); after 50% concentration of hAD-MSCs-CM intervention, CHOP protein expression was significantly reduced (P < 0.05) (FIG. 5B), while GRP78 protein expression was not significantly changed (P > 0.05) (FIG. 5C).
Of course, the above is only a specific application example of the present invention, and other embodiments of the present invention, such as replacing a lung tumor cell line or a lung normal cell line, or replacing a toxic substance that can be used for lung cell injury, etc., are within the scope of the present invention as claimed by using equivalent substitution or equivalent transformation.
Claims (5)
1. The application of the mesenchymal stem cell supernatant in preparing a preparation for treating lung cell injury is characterized in that: the mesenchymal stem cell supernatant is from a serum-free culture medium of the mesenchymal stem cells, does not contain cells, and belongs to a non-cell supernatant preparation.
2. Use of mesenchymal stem cell supernatant according to claim 1 in the preparation of a formulation for the treatment of lung cell injury, wherein: the mesenchymal stem cell supernatant is from a serum-free culture medium of bone marrow, placenta, amniotic fluid, umbilical vein subendothelial layer, peripheral blood, liver, fat, muscle, skin or umbilical cord tissue, does not contain cells, and belongs to a non-cell supernatant preparation.
3. Use of mesenchymal stem cell supernatant according to claim 1 or 2 in the preparation of a formulation for the treatment of lung cell injury, wherein: the mesenchymal stem cells are derived from a amniotic membrane layer on the surface of the human placenta tissue of the postpartum abandoned object, namely, the human amniotic mesenchymal stem cells, and the supernatant of the mesenchymal stem cells is derived from a serum-free culture medium for culturing the human amniotic mesenchymal stem cells, does not contain cells, and belongs to a non-cell supernatant preparation.
4. Use of mesenchymal stem cell supernatant according to claim 3 in the preparation of a formulation for the treatment of lung cell injury, wherein: the lung cell injury model is prepared by paraquat with different concentration gradients.
5. Use of mesenchymal stem cell supernatant according to claim 4 in the preparation of a formulation for the treatment of lung cell injury, wherein: when a lung cell injury model is prepared, hAD-MSCs-CM is added to intervene for 24 hours immediately after PQ is infected to lung cells cultured in vitro.
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