CN113209134A - Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by mustard gas - Google Patents
Application of exosome derived from mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by mustard gas Download PDFInfo
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Abstract
The invention provides application of exosome derived from mesenchymal stem cells in preparation of a medicine for treating acute lung injury caused by mustard gas. In particular to a method for separating the supernatant of mesenchymal stem cells to obtain exosome and applying the exosome to acute lung injury caused by mustard gas. The exosome obtained by the method can obviously improve the survival rate of experimental animals and the activity of lung cells under the exposure of mustard gas, and has the effect of resisting the lung injury of the mustard gas by improving the change of the lung tight junction protein caused by the mustard gas. Therefore, the invention provides a new treatment way for acute lung injury caused by mustard gas.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of exosomes derived from mesenchymal stem cells in preparation of a medicine for treating acute lung injury caused by mustard gas and a pharmaceutical composition containing the exosomes.
Background
Mustard (SM) is a blister poison, and causes blistering of skin and involvement of multiple organs such as eyes, lungs, liver, spleen, etc. after poisoning, but at present, the poisoning mechanism is unknown, and no specific treatment medicine exists, wherein Exposure-induced lung injury is the main cause of death, and is manifested by acute lung injury mainly caused by short-Term inflammatory reaction and Long-Term pulmonary fibrosis (Wolfe GA, Petteys SM, Phelps JF, Wasmund JB, Plackett TP. Sulfur Mustard Exposure: Review of Acute, Subacute, and Long-Term Effects and theory management. journal of special opportunities: a consumer's vision jjnnal for SOF services 2019; 81-6).
Bone marrow mesenchymal stem cells (BMSCs) are stem cells with multi-differentiation potential, have rich sources and simple and convenient collection operation, and a plurality of studies prove that the BMSCs can improve acute lung injury by reducing the expression of inflammatory factors and regulating immune response. Previously, the treatment effect of BMSC on acute lung injury caused by mustard gas has been studied by subjects, and it was found that BMSC can promote repair of acute lung injury caused by mustard gas by improving lung inflammatory response and barrier function. (FengY, Xu Q, Yang Y, Shi W, Meng W, Zhang H, et al. the thermal effects of bone ground-derived sensory structural cells in the acid solution in jet induced by sulfur atom mustard. Stem cell research & thermal 2019; 10: 90). On one hand, BMSCs still have the potential and long-term safety problem of tumor formation, and certain risks still exist in application (Lee HY, Hong IS. double-edited swing of sensory stem cells: Cancer-promoting therapeutic potential. Cancer science 2017; 108: 1939-46); on the other hand, the cell culture conditions are harsh and difficult to form preparations, which limits the clinical application thereof.
Exosomes (Exosomes) are lipid bilayer structures secreted by various cells and having diameters of 40-150 nm, contain various bioactive substances (proteins, DNA, RNA and the like), can be taken into cells through endocytosis to perform information transmission, regulate signal transduction, and play various biological functions such as anti-inflammation and antioxidation. It has low immunogenicity and can be well absorbed by cells, and is an excellent information carrier (van Niel G, D' Angelo G, Raposo G. cutting light on the Cell biology of extracellular vehicles. Nat Rev Mol Cell Biol 2018; 19: 213-28).
At present, no relevant research report on the association between exosomes and acute lung injury caused by mustard gas is found.
Disclosure of Invention
The present invention is made to solve the above technical problems, and an object of the present invention is to provide a novel use of exosome derived from mesenchymal stem cells of bone marrow, a preparation method of the exosome, and a pharmaceutical composition comprising the exosome.
The research of the invention discovers that the BMSC-derived exosome can treat acute lung injury caused by mustard gas. Exosomes from BMSCs are obtained through an ultracentrifugation method, an experimental animal model under mustard gas and a mouse lung epithelial cell (MLE-12) model are established, and then exosomes are added for treatment, so that the survival rate and in-vitro cell activity of the experimental animal under mustard gas exposure are detected, and the distribution and expression of cell tight junction protein are determined. The results show that the survival rate of mice under SM exposure is obviously reduced, the lung pathological manifestations are hemorrhage, edema and inflammation, the lung epithelial barrier function is obviously damaged, tight connection is not continuous, the molecular expression quantity of the tight connection protein is reduced, and exosome from BMSC obviously relieves lung injury caused by mustard gas, improves the molecular expression and positioning of the tight connection protein, and restores the lung barrier function.
In a first aspect of the invention, the application of exosome derived from mesenchymal stem cells in preparing a medicament for treating acute lung injury caused by mustard gas is provided.
Preferably, the drug is a drug that enhances the activity of lung epithelial cells. According to the cytotoxicity test, the activity of lung epithelial cells in the experimental group (SM + BMSC-Ex) was significantly enhanced compared to the negative control group (SM) and the positive control group (SM + NAC) (fig. 4).
Preferably, the drug is a drug for improving the expression level or the connection continuity of the protein related to the tight connection of the lung epithelial cells. Immunofluorescence experiments and RT-PCR detection results show that exosomes derived from BMSC obviously improve the expression and positioning of tight junction protein molecules and repair the lung barrier function (fig. 5-7).
Animal model experiments show that the survival rate of the mice injected with the exosome is obviously improved, the survival rate in 7 days is improved from 45% to 75%, and the result is far higher than the result that the survival rate in 7 days of a positive control group (SM + NAC) is 50%.
In a second aspect of the invention, a preparation method of the exosome is provided, which comprises the following steps:
A. bone marrow mesenchymal stem cell culture
Adding a mesenchymal stem cell culture medium into the mesenchymal stem cells, and collecting culture supernatant after 48-72 h, wherein the formula of the mesenchymal stem cell culture medium is as follows: DMEM/F12 medium, 10% fetal bovine serum, 1% penicillin/streptavidin double antibody.
B. Exosome isolation
Collecting the culture supernatant of the mesenchymal stem cells, centrifuging for 10min at 2000g and 4 ℃, collecting the supernatant, centrifuging the supernatant for 30min at 10000g and 4 ℃, and collecting the supernatant again; centrifuging the collected supernatant at 100000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, then re-suspending the precipitate with PBS, repeating the operation, collecting the precipitate, and dissolving the precipitate in PBS to obtain the exosome solution.
The prepared exosome is in a vesicle shape through electron microscope observation, and the diameter of the exosome is about 100 nm. Western blot and CM-Dil detection showed that the marker proteins CD63, CD81, CD9 and TSG101 of exosomes were detected, and that CM-Dil-labeled exosomes were also completely absorbed by lung epithelial cells MLE-12.
In a third aspect of the present invention, a bone marrow mesenchymal stem cell-derived exosome composition is provided, which comprises bone marrow mesenchymal stem cell-derived exosome and a diluent, wherein the diluent is preferably PBS, and the composition is conveniently used as an injection.
In a fourth aspect of the invention, the use of the exosome composition in the preparation of a medicament for treating acute lung injury caused by mustard gas is provided.
In a fifth aspect of the invention, a pharmaceutical composition for treating acute lung injury caused by mustard gas is provided, wherein exosome derived from mesenchymal stem cells of bone marrow is taken as the only active component.
Compared with the prior art, the invention has the following technical effects:
the invention provides application of exosome derived from mesenchymal stem cells in preparation of a medicine for treating acute lung injury caused by mustard gas. Experiments prove that compared with mice exposed by mustard gas, exosomes derived from BMSC can obviously improve the activity of lung epithelial cells, and can also obviously up-regulate the molecular expression and connection continuity of proteins related to tight connection of the lung epithelial cells, so that the lung barrier function is repaired, and the lung injury caused by the mustard gas is relieved. Therefore, the invention provides a new treatment way for acute lung injury caused by mustard gas.
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FIG. 1 shows the identification of exosomes from BMSC, wherein A is exosomes under a transmission electron microscope, B is exosome nanoparticle tracking analysis, C is exosome marker molecule Western blot detection, and D is the uptake of CM-Dil labeled exosomes by MLE-12 cells.
Figure 2 is the effect of BMSC-derived exosomes on survival of mice under SM exposure: CTRL: a control of blank cells without any treatment; SM: a group of SM-infected mice; SM + NAC: a group of SM infected mice to which positive control drug NAC was added; SM + BMSC-Ex: a group of SM infected mice to which BMSC-derived exosomes were added.
FIG. 3 is a pathological analysis of exosomes on lung injury in mice exposed to SM, where A is lung pathology sections of different groups of mice and B is lung pathology scores for each group;
FIG. 4 is a graph of the effect of BMSC-derived exosomes on MLE-12 cell activity under SM exposure.
FIG. 5 is a graph of the effect of exosomes on MLE-12 cell claudin localization under SM exposure.
FIG. 6 is the mRNA level analysis of each claudin in cells exposed to SM, wherein A-D are the mRNA level analysis of each claudin in different groups of cells marked by ZO-1, claudin-1, E-cadherin and occludin antibodies, respectively.
FIG. 7 is a Western blot analysis of the fibronectin of cells exposed to SM, where A is the electrophoresis result and B is the comparison of the fibronectin expression levels of the different groups of cells labeled with different antibodies.
Detailed Description
The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the invention, and the examples do not include detailed descriptions of conventional methods.
Example 1 ultracentrifugation method for obtaining exosome by separating supernatant of mesenchymal stem cell
A. Bone marrow mesenchymal stem cell culture
Adding a mesenchymal stem cell culture medium into the mesenchymal stem cells, and collecting culture supernatant after 48-72 h, wherein the formula of the mesenchymal stem cell culture medium is as follows: DMEM/F12 medium, 10% fetal bovine serum, 1% penicillin/streptavidin double antibody.
B. Exosome isolation
(1) Collecting the culture supernatant of the mesenchymal stem cells at 2000g and 4 ℃ for 10min, and centrifuging to collect the supernatant;
(2)10000g, 4 ℃, 30min, centrifuging and collecting supernatant;
(3)100000g, 4 ℃, 70min, centrifuging, discarding supernatant, collecting precipitate, re-suspending with PBS, and repeating the operation;
(4) the precipitate was collected and dissolved in 100. mu.l PBS to obtain high-purity exosomes.
According to the observation of FIG. 1A and FIG. 1B under a transmission electron microscope, the exosomes obtained by separation are in a vesicle shape, and the diameter is about 100 nm.
Example 2 Western blot identification of exosome-tagged molecules
(1) Using RIPA lysate to lyse the obtained exosomes, centrifuging at 12000rpm and 4 ℃ for 20min, then absorbing supernatant to collect total protein, and using a BCA kit to determine and regulate the protein concentration;
(2) adding the protein loading buffer solution into the protein lysate according to the proportion of 5 Xprotein loading buffer solution to 4:1, shaking and uniformly mixing, and then boiling for 10min at 100 ℃;
(3) adding 10 μ l of protein sample to 10% polyacrylamide gel for electrophoresis;
(4) intercepting the corresponding strip and transferring the strip to the PVDF membrane by using an electrotransfer instrument;
(5) 5% skim milk blocks non-specific binding sites for 1h, then incubated with CD9, CD63, CD81 and TSG101 antibodies overnight at 4 ℃ and washed 3 times with TBST buffer;
(6) HRP-labeled secondary antibody (Millipore) was incubated for 1h at room temperature, followed by 3 washes with TBST buffer;
(7) and adding a developing solution for developing color and photographing for analysis.
According to fig. 1C, the marker proteins CD63, CD81, CD9 and TSG101 of exosomes were all detected.
MLE-12 cellular exosome uptake identification
(1) Adding appropriate amount of CM-Dil stock solution into the obtained exosome to make the final concentration reach 1 μ g/L, and incubating at 37 deg.C for 30 min;
(2) inoculating the exosome on a culture dish for culturing MLE cells;
(3) after incubation at 37 ℃ for 60min, observation was carried out under a fluorescence microscope.
According to FIG. 1D, CM-Dil-labeled exosomes were all taken up by MLE-12 cells.
Example 3 obtaining Lung Pathology sections and analysis of survival curves after SM-infected mice
(1) Selecting ICR mice of 6-8 weeks, randomly distributing the ICR mice to CTRL, SM + NAC and SM + BMSC-Ex groups, and preparing a mustard gas solution of 30mg/kg by subcutaneously injecting 1, 2-propylene glycol into other groups except the CTRL group;
(2) NAC group was given NAC intragastric (200mg/kg, 1 time/d, 7 consecutive days) as positive control group after SM contamination; BMSC-Ex groups are injected into tail veins at 1d and 3d after being infected with virus to serve as experimental groups;
(3) observing the weight, diet and death condition of the mouse every day, and drawing a survival curve;
(4) on day 5, lung tissue was harvested after sacrifice under pentobarbital anesthesia and fixed with 4% paraformaldehyde for HE staining.
According to fig. 2, the survival rate of mice under SM exposure decreased significantly; after the exosome is injected, the survival rate of the mustard gas mice is obviously improved, the survival rate in 7 days is improved from 45% to 75%, and the result is far higher than that of a positive control group (SM + NAC) with the survival rate in 7 days of 50%.
According to fig. 3A, the lung pathology of mice under SM exposure showed blood, edema, inflammation, and a significant impairment of the lung epithelial barrier function; after BMSC-Ex is injected into tail vein, the lung epithelial barrier damage is relieved obviously, and the repair effect is higher than that of NAC in positive group. Pathological lung injury scores showed that the score was also lower in the SM + BMSC-Ex group than in the SM and SM + NAC groups (fig. 3B).
Example 4 cytotoxicity assay
The CCK-8 method is adopted to detect the influence of exosomes derived from BMSC on mustard pneumonocyte damage, and comprises the following specific steps:
MLE-12 cells were collected in the logarithmic growth phase and seeded at a density of 3000 per well in 96-well plates. After cells adhere to the wall overnight, after mustard gas (300 mu M) is infected with toxin for 0.5h, NAC and exosome are respectively added into an NAC group and a BMSC-Ex group, the original culture medium is discarded after 24h of culture, 10 mu l of CCK-8 and 100 mu l of fresh culture solution are added into each hole, and the absorbance value of each hole is detected by a multifunctional microplate reader at the wavelength of 450nm after 2h of culture. Repeat 3 times and calculate the average.
According to FIG. 4, the MLE-12 cells in the SM + BMSC-Ex group were most active, and as the concentration of exosomes was increased, the MLE-12 cells were also in an increased state.
Example 5 detection of MLE-12 cell tight junction-associated protein distribution by immunofluorescence assay
(1) MLE-12 cells in logarithmic growth phase were collected at 3X 10 per well5The density of cells was plated in 6-well plates and mustard gas (300. mu.M) was poisoned 0 after cells were attached overnight.After 5h, adding NAC and exosome into the NAC group and the BMSC-Ex group respectively;
(2) after 24h of culture, PBS is washed once, and a methanol permeable membrane precooled at-20 ℃ is added;
(3) sealing by immunofluorescence staining sealing liquid for 1h, and adding occludin, claudin-1, E-cadherin and ZO-1 antibodies respectively for incubation for 3 h;
(4) washing the plate for 3 times, adding a fluorescent secondary antibody, incubating for 1h, and washing with PBS for 1 time;
(5) the DAPI reagent stains cell nucleus, after incubation for 10min, PBS washes 2 times, and observes under the fluorescence microscope.
According to FIG. 5, the tight junction between MLE-12 cells in mice exposed to SM was not continuous, and the discontinuous junction between MLE-12 cells was relieved after NAC administration in the positive control group, but the effect was far less than that in the BMSC-Ex group administered with exosomes.
Example 6 RT-PCR detection of mRNA levels of the tight junction associated protein of MLE-12 cells
(1) TRIzol (Takara) extracts total RNA of four groups of cells respectively, and the specific steps are as follows:
MLE-12 cells in logarithmic growth phase were collected at 3X 10 per well5Inoculating the cells in a 6-well plate at a density, attaching the cells to the wall overnight, and adding NAC and exosomes into an NAC group and a BMSC-Ex group respectively after mustard gas (300 mu M) is infected for 0.5 h; and after 24 hours, washing with PBS, adding 500ul TRIzol into each hole, fully mixing, and standing at room temperature for 3-5 min. Adding 1/5 volumes of chloroform, reversing and uniformly mixing for 5-10 times, and standing for 3 min; centrifuging at 4 deg.C and 12500rpm for 15min, collecting upper water phase 200ul, adding equal volume of isopropanol, mixing, and standing at room temperature for 10 min; centrifuging at 4 deg.C and 12000rpm for 15min, discarding supernatant to obtain white precipitate, and adding 1ml precooled 75% ethanol; centrifuging at 4 ℃ and 12500rpm for 15min, removing the supernatant, air-drying the RNA precipitate at room temperature, adding 20 mu l of DEPC (diethyl phthalate) treatment water to dissolve the precipitate to obtain total RNA, and simultaneously detecting the RNA concentration by using an enzyme-linked immunosorbent assay;
(2) by using
All-in-One First-Strand cDNA Synthesis Supermix for qPCR (One-Step gDNAremove) (AE341-02) to obtain each group of occludin, claudin-1, E-cadherin,The cDNA of ZO-1 comprises the following steps:
the following reaction systems were added to the PCR tubes:
mixing gently, reacting at 42 deg.C for 15min, and heating at 85 deg.C for 5s to inactivate reverse transcriptase;
(3) fluorescent quantitative PCR detection
By using
The Green qPCR SuperMix (AQ101-01) kit is used for reaction, the reaction system is as follows,
cDNA Variable
(the total amount of cDNA was controlled to 10 pg-1. mu.g based on the template concentration)
Two-step amplification was performed using an ABI Prism 7000 instrument with pre-denaturation at 94 ℃ for 30s, 40 PCR cycles at 94 ℃ for 5s and 60 ℃ for 30 s.
According to FIG. 6, the mRNA level of the tight junction-associated protein in MLE-12 cells in the SM + NAC group was comparable to that in the SM group, and was much lower than that in the SM + BMSC-Ex group.
Example 7 Western blot detection of expression level of MLE-12 cell-tight junction-associated protein
(1) MLE-12 cells in logarithmic growth phase were collected at 3X 10 per well5Inoculating the cells in a 6-well plate at a density, attaching the cells to the wall overnight, and adding NAC and exosomes into an NAC group and a BMSC-Ex group respectively after mustard gas (300 mu M) is infected for 0.5 h; washing with PBS after 24h, adding 200 mul of RIPA lysate into each well for lysis, collecting into a centrifuge tube, centrifuging at 12000rpm and 4 ℃ for 20min, absorbing supernatant to collect total protein, and measuring and regulating the protein concentration by using a BCA kit;
(2) adding the protein loading buffer solution into the protein lysate according to the proportion of 5 Xprotein loading buffer solution to 4:1, shaking and uniformly mixing, and then boiling for 10min at 100 ℃;
(3) adding 10 μ l of protein sample to 10% polyacrylamide gel for electrophoresis;
(4) intercepting the corresponding strip and transferring the strip to the PVDF membrane by using an electrotransfer instrument;
(5) 5% skimmed milk blocks non-specific binding sites for 1h, and then incubate with occludin, claudin-1, E-cadherin, ZO-1 antibodies overnight at 4 ℃, and wash with TBST buffer solution for 3 times;
(6) HRP-labeled secondary antibody (Millipore) was incubated for 1h at room temperature, followed by 3 washes with TBST buffer;
(7) and adding a developing solution for developing color and photographing for analysis.
The results are shown in FIG. 7, the expression level of the tight junction related protein molecules in the SM group is reduced, and the expression level of the tight junction related protein molecules in the SM + NAC group is almost not different from that in the SM group, and is far lower than that in the SM + BMSC-Ex group. Exosomes derived from BMSC obviously relieve lung injury caused by mustard gas, improve the expression and positioning of tight junction protein molecules and restore the lung barrier function.
The undescribed parts of the present invention are the same as or implemented using prior art. The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (8)
1. Application of exosome derived from bone marrow mesenchymal stem cells in preparation of medicine for treating acute lung injury caused by mustard gas.
2. Use of bone marrow mesenchymal stem cell-derived exosomes according to claim 1 in the preparation of a medicament for treating acute lung injury caused by mustard gas, characterized in that:
wherein the drug is a drug that enhances the activity of lung epithelial cells.
3. Use of bone marrow mesenchymal stem cell-derived exosomes according to claim 1 in the preparation of a medicament for treating acute lung injury caused by mustard gas, characterized in that:
wherein the medicament is a medicament for improving the expression level or the connection continuity of the tight junction related protein of the lung epithelial cells.
4. The use of exosome derived from mesenchymal stem cells of bone marrow according to claim 1 in the preparation of a medicament for treating acute lung injury caused by mustard gas, characterized in that the exosome is prepared by the following method:
A. bone marrow mesenchymal stem cell culture
Adding a mesenchymal stem cell culture medium into bone marrow mesenchymal stem cells, and collecting culture supernatant after 48-72 h, wherein the formula of the mesenchymal stem cell culture medium is as follows: DMEM/F12 medium, 10% fetal bovine serum, 1% penicillin/streptomycin double antibody;
B. exosome isolation
Collecting the culture supernatant of the mesenchymal stem cells, centrifuging for 10min at 2000g and 4 ℃, collecting the supernatant, centrifuging the supernatant for 30min at 10000g and 4 ℃, and collecting the supernatant again; centrifuging the collected supernatant at 100000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, then re-suspending the precipitate with PBS, repeating the operation, collecting the precipitate, and dissolving the precipitate in PBS to obtain the exosome solution.
5. The exosome composition derived from the bone marrow mesenchymal stem cells is characterized by comprising exosomes derived from the bone marrow mesenchymal stem cells and a diluent.
6. The mesenchymal stem cell-derived exosome composition of claim 5, characterized in that:
wherein, the composition is an injection.
7. Use of the mesenchymal stem cell-derived exosome composition of claim 5 or 6 in the preparation of a medicament for treating acute lung injury caused by mustard gas.
8. A pharmaceutical composition for treating acute lung injury caused by mustard gas is characterized in that exosomes derived from mesenchymal stem cells of bone marrow are taken as the only active ingredients.
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| CN111346110A (en) * | 2020-03-17 | 2020-06-30 | 遵义医学院附属医院 | Application of mesenchymal stem cell supernatant in preparation of preparation for treating lung cell injury |
| CN115120615A (en) * | 2022-06-10 | 2022-09-30 | 中国人民解放军海军军医大学 | Application of miR-146a-5p overexpression engineering stem cell exosome in preparation of medicine for treating mustard seed lung injury caused by qi |
| CN115120615B (en) * | 2022-06-10 | 2024-02-06 | 中国人民解放军海军军医大学 | Application of miR-146a-5p overexpression engineering stem cell exosome in preparation of medicines for treating mustard gas-induced lung injury |
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