CN115120616B - Application of miR-199a-5p over-expression engineering stem cell exosome in preparation of medicines for treating mustard gas-induced lung injury - Google Patents
Application of miR-199a-5p over-expression engineering stem cell exosome in preparation of medicines for treating mustard gas-induced lung injury Download PDFInfo
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- CN115120616B CN115120616B CN202210650729.8A CN202210650729A CN115120616B CN 115120616 B CN115120616 B CN 115120616B CN 202210650729 A CN202210650729 A CN 202210650729A CN 115120616 B CN115120616 B CN 115120616B
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Abstract
The invention provides an application of miR-199a-5p over-expression engineering stem cell exosome in preparation of a medicament for treating mustard gas-induced lung injury. Aiming at the characteristics that the mustard is a typical representation of refractory chemical warfare agents, no specific antitoxic drug exists, the lung is an important target organ for the damage of the mustard, and the oxidative stress reaction plays an important role in the lung damage of the mustard, experiments prove that the miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosome can effectively improve the lung damage caused by the mustard and obviously improve the activity of lung epithelial cells at both the cellular level and the animal level. Further analysis shows that the method relieves lung injury caused by mustard gas by inhibiting oxidative stress reaction, and the treatment effect of miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosomes is obviously better than that of human umbilical cord mesenchymal stem cell exosomes, so that a new thought is provided for treating the mustard gas injury, and the method has a prospect of being used for preparing a medicine for treating the mustard gas injury.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of miR-199a-5p over-expression engineering stem cell exosomes derived from human umbilical cord mesenchymal stem cells in preparation of a medicament for treating acute lung injury caused by mustard gas, and a pharmaceutical composition containing the exosomes.
Background
Mustard gas (sulfur mustard, SM) is a typical representative of an erosive agent and is used in various collisions such as one war, japanese invading war, two-way war, and syrian war, resulting in significant casualties including a large number of civilians. Mustard gas has a simple structure, is easy to synthesize and produce, is easy to master and utilize by terrorists, and is one of the most likely toxicants used in chemical terrorist activities. Mustard gas is also one of main chemical warfare agents abandoned in Huachengzhangquan in Japan, and constitutes a great threat to the environmental safety and the health of people in China. The lung is one of main target organs of the mustard gas injury, when the mustard gas is exposed rapidly, a poisoning person can generate acute respiratory distress syndrome, even pulmonary edema respiratory failure or death caused by secondary pulmonary infection, so the reduction of acute lung injury is the key of treatment, and the injury caused by oxidative stress is the key link of acute and chronic SM injury (Beigi Harchegani A,Khor A,Tahmasbpour E,et al.Role of oxidative stress and antioxidant therapy in acute and chronic phases of sulfur mustard injuries:a review.Cutaneous and Ocular Toxicology,2018,38(1):9-17.)
At present, no specific antitoxic drug is used for preventing and treating mustard injury, chemical drugs are mainly used for symptomatic treatment, but the effect is not ideal. The bone marrow mesenchymal stem cells (BMSCs) can regulate and control inflammatory reaction to improve mustard gas lung injury (Feng Y,Xu Q,Yang Y,Shi W,Meng W,Zhang H,et al.The therapeutic effects of bone marrow-derived mesenchymal stromal cells in the acute lung injury induced by sulfur mustard.Stem cell research&therapy 2019;10:90),, but the sources of the BMSCs are limited, and the human umbilical cord mesenchymal stem cells (Human umbilical cord MESENCHYMAL STEM CELLS, hucMSC) are rich in sources, have little ethical disputes, strong proliferation capacity and are easy to collect and can be used as substitutes of the BMSCs. However, hucMSC is still involved in rejection and poor differentiation after long-term implantation, and the exosomes (Human umbilical cord MESENCHYMAL STEM CELLS-derived exosomes, hucMSC-Exo) can avoid these risks .(Lu LL,Liu YJ,Yang SG,Zhao QJ,Wang X,Gong W,et al.Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.Haematologica.2006;91(8):1017-26.)
HucMSC-Exo is a hucMSC actively secreted lipid bilayer structure vesicle with a diameter of about 40-100nm, contains various functional biomolecules, can be fused with cell membranes of adjacent cells through exocytosis, so as to transfer information, regulate intercellular signal transduction, and play various biological functions of antioxidation, anti-inflammation, anti-apoptosis and the like. microRNA (miRNA) is the main component carried by exosomes and plays an important role in the process of bringing hucMSC-Exo into biological function. We have found that hucMSC-Exo can ameliorate mustard-induced lung injury. Information retrieval and experimental research show that miR-199a-5p and miR-146a-5p play an important role in hucMSC-Exo improving mustard gas-induced lung injury; further experimental study shows that miR-199a-5p over-expression engineering hucMSC-Exo can effectively improve mustard gas-induced lung injury by inhibiting oxidative stress reaction, and the treatment effect is better than hucMSC-Exo. At present, the effect of miR-199a-5p over-expression engineering exosomes in improving mustard gas lung injury is not reported in related documents.
Disclosure of Invention
The invention is carried out to solve the technical problems, and aims to provide a novel application of an exosome derived from human umbilical cord mesenchymal stem cells, a preparation method of the exosome and a pharmaceutical composition containing the exosome.
The invention discovers that hucMSC derived exosomes can treat acute lung injury caused by mustard gas. The method comprises the steps of obtaining hucMSC-derived exosomes by an ultracentrifugation method, analyzing components which play a role in human umbilical cord mesenchymal stem cell exosomes by combining a bioinformatics method, and finally confirming miR-199a-5p as a key component by a cell experiment.
MiR-199a-5p over-expression engineered human umbilical cord mesenchymal stem cell exosomes (miR-199 a-hucMSC-Exo) are obtained by transfecting miR-199a-5p mimics in human umbilical cord mesenchymal stem cells. Administration of miR-199a-5p over-expression engineered human umbilical cord mesenchymal stem cell exosomes in mustard gas-injured human lung epithelial cells (BEAS-2B; ATCC, CRL-9609); a mouse model of mustard-induced lung injury was then constructed and the exosomes described above were administered. The results show that the exosome can improve the lung injury caused by mustard gas and improve the cell activity. Further studies have found that the exosomes exert an effect of inhibiting oxidative stress damage caused by mustard contamination by activating expression of oxidative stress related proteins such as NRF 2.
The invention provides an application of miR-199a-5p over-expression engineering stem cell exosomes in preparation of medicines for treating mustard gas-induced lung injury.
Preferably, the medicament is a medicament for improving the activity of lung epithelial cells and improving oxidative stress injury. The detection result of an oxygen free Radical (ROS) fluorescent probe method shows that miR-199a-5p over-expression engineering stem cell exosomes can obviously reduce the ROS content in mustard gas contaminated BEAS-2B cells, other experiments indicate that the exosomes can reduce the expression quantity of intracellular lipid peroxidation products Malondialdehyde (MDA), improve the expression quantity of intracellular superoxide dismutase (SOD), the ratio of intracellular glutathione to oxidized glutathione (GSH/GSSG) and promote the expression of oxidative stress related proteins such as NRF2, HO1, NQO1 and the like in the mustard gas contaminated BEAS-2B cells (figures 3C-3G).
Preferably, the drug is a drug for improving mustard-induced lung tissue injury, reducing ROS content in mustard-exposed lung tissue, and promoting expression of oxidative stress-related proteins such as NRF2, HO1, NQO1 and the like in mustard-exposed mouse lung tissue (FIGS. 4A-4D).
In a second aspect of the present invention, there is provided a method for preparing the exosome, comprising:
A. Human umbilical cord mesenchymal stem cell culture
The mesenchymal stem cell culture medium was added to human umbilical cord mesenchymal stem cells, and after 48 hours, the culture supernatant was collected.
The formula of the mesenchymal stem cell culture medium is as follows: 5% of ELITECELL animal serum free cell culture supplement was added to Dakewe's mesenchymal stem cell basal medium.
B. Exosome separation
Collecting human umbilical cord mesenchymal stem cell culture supernatant, centrifuging at 300g and 4deg.C for 10min, discarding precipitate, centrifuging at 2000g and 4deg.C for 10min, discarding precipitate, centrifuging at 10000g and 4deg.C for 30min, and collecting supernatant; supernatant is centrifuged for 70min at 120000g and 4 ℃, and PBS is collected for resuspension after precipitation, supernatant is centrifuged for 70min at 120000g and 4 ℃ again, and precipitation is collected and dissolved in 200 mu LPBS, and finally the exosome solution is obtained. The exosomes prepared were vesicle-shaped, about 40-100nm in diameter, and the marker proteins CD9, CD63, CD81, HSP70 and Cav1 of the exosomes were all detected by electron microscopy (fig. 1A-1D).
C. engineering human umbilical cord mesenchymal stem cell exosome
Sequentially adding the Exo-Fect solution, the miR-199a-5p analogue, the PBS solution and the 1X 10 7 part exosome solution into a centrifuge tube according to the volume ratio of 1:2:7:5, reversing the solution vertically for three times to uniformly mix, taking notice of incapability of vortex oscillation, vibrating and mixing at 37 ℃ for 10 minutes, and then immediately transferring the mixture onto ice; adding ExoQuick-TC solution with the volume of being three times that of Exo-Fect, and uniformly mixing for six times upside down to stop the reaction, wherein the vortex oscillation cannot be noticed; standing at 4 ℃ for 30 minutes, centrifuging at 13000rpm for 3 minutes at 4 ℃, discarding the supernatant, and adding a PBS solution with the volume 300 times of the Exo-Fect solution volume into the precipitate for resuspension to obtain miR-199a-5p over-expression engineering exosome suspension.
The exosomes prepared are in a vesicle shape and have diameters of about 40-100nm, and marker proteins CD9, CD63, CD81, HSP70 and Cav1 of the exosomes are detected by observation through an electron microscope.
In a third aspect of the invention, an exosome composition derived from human umbilical mesenchymal stem cells is provided, comprising miR-199a-5p over-expression engineering stem cell exosomes and a diluent, wherein the diluent is preferably PBS, so that the composition can be conveniently used as an injection.
In a fourth aspect of the invention, there is provided the use of the exosome composition in the manufacture of a medicament for the treatment of mustard-induced acute lung injury.
In a fifth aspect of the invention, a pharmaceutical composition for treating acute lung injury caused by mustard gas is provided, wherein miR-199a-5p over-expression engineering stem cell exosomes are taken as the only active components.
Compared with the prior art, the invention has the following technical effects:
Aiming at the characteristics that no specific antitoxic drug is used for preventing and treating mustard gas injury and reducing acute lung injury is a key for curing, and oxidative stress reaction plays an important role in the mustard gas lung injury, the invention provides miR-199a-5p overexpression engineering stem cell exosomes. Experiments prove that miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosome can obviously improve lung epithelial cell activity at both a cellular level and an animal level, relieve lung injury caused by mustard gas, and exert the effects by improving the oxidative stress of the mustard gas-induced lung injury, the treatment effect of the miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosome is obviously superior to that of the human umbilical cord mesenchymal stem cell exosome, a new thought is provided for treating the mustard gas injury, and the method has a prospect of preparing a medicine for treating the mustard gas injury.
Drawings
FIG. 1 shows the isolation and identification of exosomes.
FIG. 2 is the effect on mustard exposure BEAS-2B cell viability following transfection of different miRNA inhibitors.
FIG. 3A is a graph showing the effect of miR-199a-5p over-expression on miR-199a-5p expression in mustard gas-exposed cells from an extracellular body of an engineered human umbilical cord mesenchymal stem cell.
FIG. 3B is the effect of miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosomes on mustard exposure cell viability.
FIG. 3C is the effect of miR-199a-5p over-expression on the intracellular oxygen Radical (ROS) content of mustard gas-exposed cells of engineered human umbilical cord mesenchymal stem cell exosomes.
FIG. 3D is a graph showing the effect of miR-199a-5p over-expression on the expression level of Malondialdehyde (MDA), a lipid peroxidation product of mustard-exposed cells, by engineering human umbilical cord mesenchymal stem cell exosomes.
FIG. 3E is the effect of miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosomes on the expression amount of mustard gas exposed cell superoxide dismutase (SOD).
FIG. 3F is the effect of miR-199a-5p over-expression on the expression level of Glutathione (GSH) in mustard gas-exposed cells by engineering human umbilical cord mesenchymal stem cell exosomes.
FIG. 3G is the effect of miR-199a-5p over-expression on expression of oxidative stress-related proteins in mustard gas-exposed cells by engineering human umbilical cord mesenchymal stem cell exosomes.
FIG. 4A is a graph showing the effect of miR-199a-5p over-expression on miR-199a-5p expression level in lung tissue of a mustard-exposed mouse by engineering human umbilical cord mesenchymal stem cell exosomes.
FIG. 4B is the effect of miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosomes on lung tissue pathological sections and scores of mustard-exposed mice.
FIG. 4C is the effect of miR-199a-5p over-expression engineering human umbilical cord mesenchymal stem cell exosomes on oxygen free Radical (ROS) content in lung tissue of mustard-exposed mice.
FIG. 4D is the effect of miR-199a-5p over-expression of engineered human umbilical cord mesenchymal stem cell exosomes on expression of oxidative stress-related proteins in mustard gas-exposed mouse lung tissue.
Detailed Description
The nucleotide sequence of miR-199a-5p is as follows: 5'-CCCAGUGUUCAGACUACCUGUUC-3' (SEQ ID NO. 1).
Example 1 cell experiments confirm that miR-199a-5p acts as a key component in human umbilical cord mesenchymal stem cell exosomes
1. Exosome separation
A. Human umbilical cord mesenchymal stem cell culture
The mesenchymal stem cell culture medium was added to human umbilical cord mesenchymal stem cells, and after 48 hours, the culture supernatant was collected. Wherein, the formula of the mesenchymal stem cell culture medium is as follows: 5% of ELITECELL animal serum free cell culture supplement was added to Dakewe's mesenchymal stem cell basal medium.
B. exosome isolation and identification
Collecting human umbilical cord mesenchymal stem cell culture supernatant, centrifuging at 300g and 4deg.C for 10min, discarding precipitate, centrifuging at 2000g and 4deg.C for 10min, discarding precipitate, centrifuging at 10000g and 4deg.C for 30min, and collecting supernatant; supernatant was centrifuged at 120000g and 4℃for 70min, the supernatant was collected and precipitated and resuspended in PBS, and the supernatant was centrifuged again at 120000g and 4℃for 70min, and the precipitate was collected and dissolved in 200. Mu.L of PBS to give an exosome solution.
The exosomes prepared were vesicle-shaped (fig. 1A), approximately 40-100nm in diameter (fig. 1B), and all of the marker proteins CD9, CD63, CD81, HSP70, and Cav1 (fig. 1C) of the exosomes were detected by electron microscopy.
According to the analysis of the miRNA components of the human umbilical cord mesenchymal stem cell exosomes and the sequencing analysis of the transcriptome, and by combining with a bioinformatics method, miRNA molecules possibly participating in the action in the human umbilical cord mesenchymal stem cell exosomes are screened out: miR-100-5p, miR-199a-5p, miR-23a-3p, let-7a,22-3p, miR-424-5p, miR-221-3p, miR-15a-5p, miR-145-5p and the like.
BEAS-2B cells were diluted to a cell suspension at a concentration of 1X 10 6 cells/mL and plated in 96-well plates with 100. Mu.L of cell suspension per well.
After 12 hours, the inhibitors of the above miRNA molecules were transfected using lipofectamine RNAIMAX kit. And respectively diluting the miRNA molecular inhibitor and lipofectamine RNAIMAX with a serum-free Opti-MEM culture medium, uniformly mixing, standing for 5 minutes, adding into a cell culture hole, and gently shaking to uniformly distribute.
After 24 hours of transfection, the cells were treated with mustard gas contamination. The mustard stock solution was diluted to 12.5 μm dilution with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, fresh culture medium is replaced, human umbilical mesenchymal stem cell exosomes are added, after continuous culture for 24 hours, 10 mu L of CCK-8 solution is added into each well, after incubation for 1 hour in an incubator, absorbance of each well at 450nm is measured by an enzyme-labeled instrument, and cell viability is calculated.
As shown in figure 2, after miR-199a-5p is inhibited, the protection effect of human umbilical cord mesenchymal stem cell exosomes on cell viability can be obviously reduced, while the effects of other components are not obvious enough, and the key components of miR-199a-5p acting in human umbilical cord mesenchymal stem cell exosomes are confirmed.
EXAMPLE 2 preparation of engineered human umbilical mesenchymal Stem cell exosomes
And transfecting the exosome by using an Exo-Fect kit of System Biosciences company to obtain the miR-199a-5p over-expression engineering exosome. 10. Mu.L of Exo-Fect solution, 20. Mu.L (20 pmol) of miR-199a-5p mimetic, 70. Mu.L of PBS solution and 50. Mu.L of exosome solution (1X 10 7 part) are sequentially added into a 1.5mL centrifuge tube according to the use instructions of the kit, and the mixture is turned upside down for 3 times to ensure uniform mixing, and the vortex oscillation cannot be noticed. Mix for 10 minutes with shaking at 37℃and then immediately transpose to ice. 30 mu LExoQuick-TC solution was added and mixed well upside down 6 times to stop the reaction, taking care that vortex was not possible. Standing at 4 ℃ for 30 minutes, centrifuging at 13000rpm at 4 ℃ for 3 minutes, discarding the supernatant, adding 300 mu LPBS solution into the precipitate, and re-suspending to obtain miR-199a-5p over-expression engineering exosome suspension.
Example 3 miR-199a-5p overexpression engineering human umbilical cord mesenchymal stem cell exosomes improved mustard gas induced cellular oxidative stress.
Further, miR-199a-5p over-expression engineering human umbilical mesenchymal stem cell exosome is given to the mustard injured BEAS-2B cells (the concentration of the mustard contamination is 12.5 mu M).
(1) Detecting miR-199a-5p expression quantity in cells by using an RT-PCR method: BEAS-2B cells were diluted to a cell suspension at a concentration of 5X 10 4 cells/mL, and 1mL of cell suspension was plated per well in a 24-well plate. After 24 hours, the cells were treated with mustard contamination and the mustard stock was diluted to 50 μm with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, the fresh medium was changed and miR-199a-5p over-expressed engineered exosomes were added, and after further incubation for 24 hours, the supernatant was collected for subsequent detection.
After the cells are washed 3 times by PBS solution, 200 mu L of TRIzol reagent is added into each hole, and after the cells are stood for 5 minutes, the cells are blown into a centrifuge tube; 40. Mu.L of chloroform was added thereto, vortexed for 15 seconds, allowed to stand at room temperature for 3 minutes, and then centrifuged at 12000rpm at 4℃for 15 minutes; transferring the supernatant to a new centrifuge tube, adding equal volume of isopropanol (about 100 μl), mixing upside down, and standing at room temperature for 10 min; centrifuge at 12000rpm at 4℃for 15 min. Carefully remove the supernatant, add 1mL of pre-chilled 75% ethanol to the pellet, reverse the pellet upside down to float the pellet; centrifuge at 12000rpm for 15 min at 4℃and carefully discard the supernatant ethanol, then continue to add 1mL of pre-chilled 75% ethanol, reverse upside down to float the pellet, centrifuge at 12000rpm for 15 min at 4℃and carefully discard the supernatant ethanol. The precipitate was dried at room temperature for 3 minutes, and an appropriate amount of DEPC water (about 20. Mu.l) was added as the case may be, to thereby obtain an RNA sample. OD 260 and OD 280 and their ratios were determined, the RNA sample purity was analyzed, and the RNA sample concentration was calculated.
RNA samples were reverse transcribed to cDNA using TRANSSCRIPT MIRNA FIRST-STRAND CDNA SYNTHESIS Supermix kit. 1. Mu. L TRANSSCRIPT MIRNA RT Enzyme Mix and 10. Mu.l 2X TS miRNA Reaction Mix were added to 1. Mu.g of RNA sample and made up to 20. Mu.l with RNase-free ddH2O according to the instructions. The mixture was gently swirled with a pipette and incubated at 37℃for 1 hour, heated at 85℃for 5 seconds, and the product was used for qPCR.
QPCR reactions were performed using SYBR Green chimeric fluorescence: 10. Mu.L of 2 XSYBR QPCR MASTER Mix, 0.4. Mu.L of forward primer, 0.4. Mu.L of reverse primer, 2. Mu.L of cDNA sample, and make up to 20. Mu.L with RNase-free ddH 2 O were added to the reaction wells; pre-denaturation at 95 ℃ for 30 seconds; cycling 40 times at 95 ℃ for 10 seconds and 60 ℃ for 30 seconds; the melting curve was 95℃for 15 seconds, 60℃for 60 seconds, and 95℃for 15 seconds. And calculating the relative expression according to the CT value.
TCAAGAGCAATAACGAAAAATGT (SEQ ID NO. 2) as miR-199a-5p forward primer (5 '-3'); reverse primer (5 '-3'): GCTGTCAACGATACGCTACGT (SEQ ID NO. 3); ATTGGAACGATACAGAGAAGATT (SEQ ID NO. 4), reverse primer (5 '-3'): GTCCTTGGTGCCCGAGTG (SEQ ID NO. 5), which is a universal primer.
As shown in FIG. 3A, the miR-199a-5p over-expression level in mustard gas damage BEAS-2B cells is obviously reduced. The human umbilical cord mesenchymal stem cell exosome can increase the miR-199a-5p over-expression amount in the mustard gas injury BEAS-2B cell, and the miR-199a-5p over-expression engineering exosome has more obvious effect.
(2) Cell viability was measured by CCK8 method: BEAS-2B cells were diluted to a cell suspension at a concentration of 1X 10 7 cells/mL and plated in 96-well plates with 100. Mu.L of cell suspension per well. After 24 hours, the cells were treated with mustard contamination and the stock solution of mustard was diluted to 12.5 μm with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, the fresh culture medium is replaced, miR-199a-5p over-expression engineering exosome is added, after continuous culture for 24 hours, 10 mu L of CCK-8 solution is added into each hole, after incubation for 1 hour in an incubator, absorbance of each hole at 450nm is measured by an enzyme-labeled instrument, and cell viability is calculated.
The results are shown in FIG. 3B, and miR-199a-5p over-expression engineering exosomes can significantly increase mustard damage BEAS-2B cell viability compared to the mustard contamination group. The homotypic control miR-NC exosomes used as miR-199a-5p have no obvious difference compared with the common exosome group. The result shows that miR-199a-5p over-expression engineering exosome can relieve mustard gas-induced cytotoxicity and promote the recovery of BEAS-2B cell viability.
(3) The effect of oxygen Radical (ROS) production in cells was detected using DCFH-DA probes.
Cells were seeded one day in 6-well plates in advance and treated with mustard contamination. The mustard stock solution was diluted to 12.5 μm dilution with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, fresh medium was changed and either human umbilical mesenchymal stem cell exosomes or miR-199a-5p over-expression engineered exosomes were added and incubated in an incubator for 24h. DCFH-DA was dissolved in serum-free medium, diluted to a 1:1000 ratio and the final concentration was 10. Mu.M. The medium was removed from each well and 2mL of DCFH-DA diluent was added to each well. Incubate for 20min at 37℃in the dark, wash away residual liquid with PBS, observe and capture fluorescence images of cells using fluorescence microscopy.
The results are shown in fig. 3C, where the fluorescence intensity showing ROS content in mustard gas group cells is significantly increased compared to normal group; the administration of miR-199a-5p over-expressed engineered exosome cells has significantly reduced ROS content compared to mustard gas group cells. Expression of ROS content in miR-199a-5p over-expression engineered exosome cells is significantly reduced compared to exosome cells. The result shows that miR-199a-5p over-expression engineering exosome can improve oxidative stress of mustard damaged cells.
(4) The expression level of Malondialdehyde (MDA), a lipid peroxidation product, in cells was detected using a colorimetric method.
① Cell treatment: cells were seeded one day in 6-well plates in advance and treated with mustard contamination. The mustard stock solution was diluted to 12.5 μm dilution with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, fresh medium was changed and either human umbilical mesenchymal stem cell exosomes or miR-199a-5p over-expression engineered exosomes were added and incubated in an incubator for 24h. Washing with PBS for 3 times, sucking, adding 100 mu L of cell lysate into each hole, standing in a refrigerator at 4 ℃ for 15min, collecting samples by cell scraping and shaking with intense vortex, centrifuging for 20min by using a precooled high-speed centrifuge, setting the rotating speed to 12000r/min, and taking out supernatant to be tested.
② According to the number of the detection samples, a TBA storage solution is prepared according to the proportion of adding 6.76mL of preparation solution to 25mg of TBA, so that the concentration of the TBA storage solution is 0.37%, and the preparation solution needs to be fully dissolved for use after heating.
③ The MDA working solution is prepared from TBA diluent, TBA storage solution and antioxidant according to the proportion (150:50:3).
④ The standard was diluted with distilled water to give concentrations of 100, 50, 20, 10, 5, 2, 1. Mu.M.
⑤ Taking 100 mu L of prepared supernatant to be tested, standard substances and blank control (lysate) of a sample, respectively adding the supernatant to be tested, the standard substances and the blank control (lysate) into a centrifuge tube, respectively and uniformly mixing the supernatant with 200 mu L of MDA working solution, heating the mixture at 100 ℃ for 15min, centrifuging the mixture in a centrifuge for 10min after the mixture is cooled to room temperature, setting the rotating speed to 1000g, and measuring the absorbance value of the supernatant by using an enzyme-labeled instrument (setting the detection wavelength to 532 nm).
⑥ Cell supernatant protein concentration was determined according to BCA kit instructions. And (3) fully and uniformly mixing the BCA reagent A and the reagent B according to the volume ratio of 50:1, and preparing a proper amount of BCA working solution. And (3) diluting the protein standard substance into a protein standard substance solution with the concentration of 0.5mg/mL, and preparing standard yeast by double-ratio dilution. Samples or standard solutions were added to the sample wells, respectively, 200 μl of BCA working solution was added to each well, and left at 37 ℃ for 30 minutes. And detecting the absorbance of each hole at 562nm wavelength by using an enzyme-labeled instrument, and drawing a standard curve by taking the concentration of a protein standard substance as an ordinate and the OD value as an abscissa. Substituting the OD value of the sample to be measured into a standard curve, and calculating the protein concentration of the sample.
⑦ MDA content was calculated from standard curve readings and sample concentrations.
As shown in fig. 3D, the intracellular lipid peroxidation product MDA content of the mustard gas group was significantly increased compared to the normal group; the administration of miR-199a-5p over-expression engineering exosome treatment significantly reduced MDA content compared to mustard gas group cells. Compared with exosome, the MDA content in the miR-199a-5p over-expression engineering exosome cell is obviously reduced. The result shows that the miR-199a-5p over-expression engineering exosome can effectively reduce the generation of oxidation products caused by SM contamination, and can inhibit oxidative stress injury of cells caused by mustard contamination.
(5) The expression level of superoxide dismutase (SOD) in the cells was measured using a WST-8-based color reaction.
① Cell treatment: the method is as in part ① of (4).
② According to the number of detection samples, preparing WST-8/enzyme working solution according to the proportion of 151 mu L of SOD buffer solution to be detected, 8 mu L of WST-8 solution and 1 mu L of enzyme solution according to the number of samples per hole, taking care of light-shielding and storing at 4 ℃.
③ The concentration of the stock solution of the reaction initiator was 40×, and dilution was performed with SOD assay buffer.
④ Taking 20 mu L of prepared supernatant to be tested of a sample and a blank control, respectively adding the supernatant to be tested and the blank control into a 96-well plate, respectively and uniformly mixing with 160 mu L of WST-8/enzyme working solution and 20 mu L of starting working solution, adding 20 mu L of SOD buffer into the blank control, and incubating for 30min at 37 ℃. Note that the starting working fluid is added last with a gun to reduce errors. And (5) setting the detection wavelength of the enzyme label instrument to 450nm, and then measuring the absorbance value of the sample.
⑤ Determination of cell supernatant protein concentration: the method is as in part ⑥ of (4).
⑥ And calculating SOD activity according to an enzyme activity calculation formula and the concentration of the sample.
The results are shown in FIG. 3E, where the intracellular antioxidant enzyme SOD content of the mustard gas group is significantly reduced compared to the normal group; the administration of exosome-treated cells had an elevated SOD content compared to mustard gas-treated cells. Compared with exosome, the expression of the SOD content in the miR-199a-5p over-expression engineering exosome cell is obviously improved. The result shows that miR-199a-5p over-expression engineering exosome can effectively improve the activity of antioxidant enzyme, can improve oxidative stress of mustard injured cells, and plays an important role in reducing oxidative stress injury caused by SM.
(6) The amount of expression of antioxidant enzyme Glutathione (GSH) in cells was detected using a colorimetric method.
① According to the requirements of the specification, each reagent is diluted and dissolved in proportion in advance for standby.
② Cell treatment: the method is the same as in part ① of (4), but note that in sample preparation, reagent IV in the kit is added for homogenization cleavage during cleavage.
③ GSH and GSSG standards (1 mmol/L) were diluted with distilled water and formulated with reagent IV at concentrations of 100, 50, 25, 12.5, 0. Mu.M.
④ Determination of T-GSH value: taking 10 mu L of sample supernatant to be detected and 10 mu L of GSH standard substance with each concentration, respectively adding the supernatant and the GSH standard substance into a 96-well plate, respectively and uniformly mixing with 100 mu L of the first reagent and 10 mu L of the second reagent, standing for 2min, adding 50 mu L of the third reagent, measuring absorbance A1 at 405nm under an enzyme-labeling instrument at 30s, and reading absorbance A2 at 10min for 30 s.
⑤ Determination of GSSH value: taking 100 mu L of sample supernatant to be tested and 100 mu L of GSSH standard substance with each concentration, respectively adding the supernatant to be tested and the 100 mu L of GSSH standard substance into a 96-well plate, respectively mixing the supernatant with 2 mu L of reagent five and 5 mu L of reagent six uniformly, carrying out vortex mixing, reacting for 30min at 37 ℃, taking 10 mu L of sample, adding 100 mu L of reagent one and 10 mu L of reagent two uniformly, standing for 2min, adding 50 mu L of reagent three, measuring absorbance A1 at 405nm under an enzyme-labeling instrument at 30s, and reading absorbance A2 at 10min for 30 s.
⑥ And respectively calculating the contents of the T-GSH and the GSSG according to a calculation formula, wherein the content of the reduced Glutathione (GSH) is = the content of the T-GSH and the content of the GSSG is-2 multiplied by the content of the GSSG. And finally calculating GSH/GSSG ratio of each group.
The results are shown in FIG. 3F, and the intracellular antioxidant enzyme GSH/GSSG content of the mustard gas group is remarkably reduced compared with the normal group; compared with mustard gas group cells, the ratio of GSH/GSSG in cells after miR-199a-5p over-expression engineering exosome is obviously increased. Compared with the exosome group, the expression of the GSH/GSSG ratio in the miR-199a-5p over-expression engineering exosome group cell is obviously improved. The result shows that miR-199a-5p over-expression engineering exosome can effectively improve the activity of antioxidant enzyme, can inhibit oxidative stress of mustard injured cells, and plays an important role in reducing oxidative stress injury caused by SM.
(7) Western Blot (Western Blot) for determining oxidative stress related target proteins in cell samples
① Preparation of cell total protein samples: cells were seeded one day in 6-well plates in advance and treated with mustard contamination. The mustard stock solution was diluted to 12.5 μm dilution with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, fresh medium was changed and either human umbilical mesenchymal stem cell exosomes or miR-199a-5p over-expression engineered exosomes were added and incubated in an incubator for 24h. Gently washing the adherent cells subjected to experimental treatment with precooled PBS for three times, drying by suction, adding RIPA buffer solution containing protease inhibitor, standing at 4 ℃ for cracking, taking out after 15min, scraping the adherent cells, collecting the adherent cells in a 1.5mL centrifuge tube, fully shaking, putting the adherent cells back into the 4 ℃ refrigerator after vortex, cracking for 10min, and repeating the vortex and the cracking for three times to ensure full cracking. Centrifuging in a pre-cooled centrifuge at 4deg.C for 20min at 12000rpm/min, and collecting supernatant.
② Preparation of a nuclear protein sample: after cell collection, the cells were washed with PBS and discarded, and plasma protein extraction reagent I (CPEB I) containing protease inhibitor was added to the pellet, thoroughly mixed, vigorously vortexed for 15 seconds, and incubated on ice for 2 minutes, and repeated 5 times. Adding plasma protein extraction reagent II (CPEB II), shaking for 5 seconds, incubating on ice for 1 minute, centrifuging in a high-speed centrifuge with pre-cooling at 4deg.C for 15min, and collecting supernatant as cytoplasmatic protein and its precipitate as cytoplasmatic protein. Plasma protein extraction reagent I (CPEB I) was added to the cell pellet and the supernatant was discarded after high speed vortexing (to further remove residual plasma protein in the nucleoprotein). The nucleoprotein extraction reagent NPEB (NPEB) containing protease inhibitors was added to the cell pellet, and the pellet was resuspended and vortexed for 15 seconds before incubation on ice for 5 minutes, repeated 6 times, to fully lyse the nucleoprotein. The pellet suspension was centrifuged in a pre-chilled high speed centrifuge for 10min, the supernatant of which was the desired nuclear protein.
③ Determining the protein concentration of a total cellular protein sample or a nuclear protein sample: the method is as in part ⑥ of (4).
④ The WB method determines the protein content of NRF2 and related molecules. The cell samples were added to 4 x protein loading buffer and subjected to subsequent experiments by denaturation at 100 ℃ in a constant temperature metal bath. Samples were added to the electrophoresis apparatus and the loading volume was determined based on the measured protein concentration, ensuring that the loading amount of each group of samples was 20. Mu.g protein. Adding freshly prepared electrophoresis liquid for electrophoresis, and adjusting the parameters to be: electrophoresis was performed for 30min at a constant voltage of 80V, and then for 60min at a voltage of 120V. Cutting PVDF film with the same size as gel, adding methanol for activation, covering the gel after electrophoresis, placing double-layer filter paper up and down, placing a film transferring clamp, performing wet transfer in a film transferring instrument for 90min, and setting constant current of 280mA. After transfer, blocking with 3% bsa solution was performed for at least 1h. The PVDF membrane was washed 3 times with TBST. The bands were cut according to the positions of the target bands indicated by the markers, diluted primary antibodies were added, and incubated overnight at 4 ℃. The primary antibody on the residual film was washed 3 times with TBST. The secondary antibody was added and incubation was continued for 1h, and the residual membrane was washed 3 times with TBST. The membrane washing time of the above experiment was 10min each time. Finally, chemiluminescent developer is added, the strips are exposed and scanned in a developing instrument, and the strips are quantified by imageJ. The experiment was repeated three times.
NRF2 is an intracellular signaling molecule belonging to the family of activating transcription factors, which is necessary for the cell to resist various stress injuries. Nuclear translocation of NRF2 results in induction of antioxidant enzyme genes, such as HO1 and NQO1, playing an important role in scavenging ROS, thereby protecting cells from oxidative damage. The results are shown in fig. 2G, where the NRF2 content in the mustard group nuclei was significantly reduced compared to the normal group; compared with mustard gas group cells, the nuclear content of cells NRF2 is increased after miR-199a-5p over-expression engineering exosome is given. Compared with exosome, the expression of the NRF2 nuclear content in the cells of the miR-199a-5p over-expression engineering exosome is more obvious. Meanwhile, compared with a mustard gas group contamination group, the expression level of cells and endogenous antioxidant enzymes HO1 and NQO1 proteins is obviously increased after miR-199a-5p over-expression engineering exosomes are given. The result shows that the miR-199a-5p over-expression engineering exosome can effectively improve the antioxidant enzyme activity by activating the NRF2 signal path, so that the oxidative stress damage of cells caused by mustard gas is inhibited.
EXAMPLE 4 improvement of oxidative stress in mice with mustard gas-induced lung injury by human umbilical cord mesenchymal Stem cell exosomes overexpressed by miR-199a-5p
The first and third days after mustard contamination, mice with mustard damage were given miR-199a-5p over-expression engineered exosomes by i.v. injection. On the fifth day after the mustard gas is infected, the expression quantity of miR-199a-5p in the lung tissue of the mouse is detected, the HE staining slice of the lung tissue is observed, the total protein concentration of alveolar lavage fluid and the lung wet/dry weight ratio are measured, and the ROS expression quantity in the lung tissue is detected to evaluate the improvement effect of the miR-199a-5p over-expression engineering exosome on the oxidative stress of the mustard gas-induced lung injury.
(1) Expression level of miR-199a-5p in lung tissue: the lung tissue of the mouse was weighed, 9 volumes of TRIzol reagent was added thereto, and after low-temperature grinding, the mixture was centrifuged at 12000rpm at 4℃for 15 minutes, and the supernatant was collected, followed by the same procedure as in (1) of example 3.
As shown in FIG. 4A, compared with a mustard gas contamination group, the gene expression level of miR-199a-5p in the lung tissue of the mice in the miR-NC exosome group is obviously increased, and the gene expression level of miR-199a-5p in the lung tissue of the mice in the miR-NC exosome group is not obviously different from that in the common exosome group.
(2) Lung tissue HE stained sections: the lung tissue was fixed with 4% paraformaldehyde tissue fixative for 24h. And (5) embedding paraffin after dehydration. Slicing and dewaxing. After hematoxylin-eosin staining, the sample was observed under a microscope and photographed. Two pathologists scored blindly for inflammatory cell infiltration of lung tissue, alveolar wall thickening, alveolar hemorrhage edema, etc. The degree of lung tissue injury was classified into 5 classes from light to heavy. 0 point: normal; 1, the method comprises the following steps: the lung interstitium is infiltrated by a small amount of inflammatory cells, and the alveolar structure is not obviously changed; 2, the method comprises the following steps: the lung interstitium has mild to moderate inflammatory changes, and the lung structure is not obviously damaged; 3, the method comprises the following steps: the lung interstitium has moderate to severe inflammatory injury, the alveolar space is thickened, and the alveolar structure is obviously destroyed; 4, the following steps: severe inflammatory injury and alveolar collapse. Finally, the scores are summarized to represent the lung injury degree.
As shown in FIG. 4B, the mice with mustard group had serious lung tissue injury, obvious structural damage, diffuse inflammatory cells in the alveolar space, a large amount of exudates and obviously thickened lung interval. Compared with mustard gas, the lung injury symptom of exosome group is reduced, a few alveolus structures are damaged, partial inflammatory cells infiltrate, a small amount of exudates are absorbed, and the alveolus walls are slightly thickened. The lung tissue injury symptom of mice in the miR-199a-5p over-expression engineering exosome group is obviously reduced compared with that of mice in the mustard gas group. The result shows that miR-199a-5p over-expression engineering exosome can improve mice lung tissue injury caused by mustard gas.
(3) The effects of oxygen Radical (ROS) production in lung tissue were detected using a Dihydroethidium (DHE) staining method.
24 Hours after the administration of each group of mice, fresh lung tissue was taken and 5 μm thick sections were prepared. DHE staining solution diluted with dimethyl sulfoxide (DMSO) was applied to tissue sections, and after incubation in a dark room for 30min, the red fluorescence signal was detected with a fluorescence microscope. The optical density value per unit area was calculated using ImageJ software.
The results are shown in fig. 4C, where the expression of ROS levels in the lung tissue of mice in the mustard group was elevated compared to the normal group. Compared to the mustard gas group, ROS levels in lung tissue of mice given miR-199a-5p over-expression engineered exosomes group were significantly reduced. The reduction in expression of the ROS levels in miR-199a-5p over-expression engineered exosomes is more pronounced compared to exosomes. The result shows that miR-199a-5p over-expression engineering exosome can reduce superoxide generation and improve oxidative stress of mice with mustard-induced lung injury.
(4) Western Blot (Western Blot) was used to determine the expression of oxidative stress-related proteins in mouse lung tissue.
① Preparation of a lung tissue total protein sample: weighing the tissues, respectively placing into a centrifuge tube, adding 9 times of RIPA lysate (for preparation) containing protease inhibitor and phosphatase inhibitor, adding small magnetic beads into the centrifuge tube, fully grinding and cracking in a full-automatic rapid grinding instrument, taking out the small magnetic beads, and centrifuging in a high-speed centrifuge for 20min. Collecting the supernatant to be tested.
② Preparation of a tissue nucleoprotein sample: cutting lung tissue into small pieces, washing with PBS, sucking off PBS, adding protein extract reagent I (CPEB I) containing protease inhibitor, mixing thoroughly, and homogenizing. The homogenate was vortexed for 15 seconds and incubated on ice for 2 minutes and repeated 5 times. Adding plasma protein extraction reagent II (CPEB II), shaking for 5 seconds, incubating on ice for 1 minute, centrifuging in a high-speed centrifuge with pre-cooling at 4deg.C for 15min, and collecting supernatant as cytoplasmatic protein and its precipitate as cytoplasmatic protein. Plasma protein extraction reagent I (CPEB I) was added to the cell pellet and the supernatant was discarded after high speed vortexing (to further remove residual plasma protein in the nucleoprotein). The nucleoprotein extraction reagent NPEB (NPEB) containing protease inhibitors was added to the cell pellet, and the pellet was resuspended and vortexed for 15 seconds before incubation on ice for 5 minutes, repeated 6 times, to fully lyse the nucleoprotein. The pellet suspension was centrifuged in a pre-chilled high speed centrifuge for 10min, the supernatant of which was the desired nuclear protein.
③ Determining the protein concentration of a total cellular protein sample or a nuclear protein sample: the procedure is as in part ⑥ of example 3, part (4).
④ Protein content of NRF 2and related molecules measured by WB method: the procedure is as in part ④ of example 3, part (7).
As shown in fig. 4D, the NRF2 content in the lung tissue nuclei was significantly reduced in the mustard group mice compared to the normal group; compared with the mustard gas group, the nuclear content of NRF2 in lung tissues is increased after miR-199a-5p over-expression engineering exosome is given. Compared with the exosome group, the expression of the NRF2 nuclear content of the miR-199a-5p over-expression engineering exosome group is more obvious. At the same time, the expression level of endogenous antioxidant enzymes HO1 and NQO1 protein is obviously increased. The result shows that miR-199a-5p over-expression engineering exosome plays an important role in activating an NRF2 signal pathway, and the oxidation stress response of lung tissues of a mustard injured mouse is reduced by improving the antioxidant enzyme activity.
In conclusion, the invention provides application of miR-199a-5p over-expressed engineering human umbilical mesenchymal stem cell exosome in preparation of medicines for treating mustard gas-induced lung injury.
The foregoing description of the embodiments is provided to illustrate the present invention so that those skilled in the art may readily understand and use the present invention, and is not limited thereto. The present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present invention, can make improvements and modifications without departing from the scope of the present invention.
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Claims (3)
- The application of miR-199a-5p over-expression engineering stem cell exosome in preparation of a medicament for treating mustard gas-induced lung injury is characterized in that: the nucleotide sequence of the gene encoding miR-199a-5p is shown as SEQ ID NO. 1;The medicine is used for improving the oxidative stress reaction of mustard-induced lung tissues and cells, improving the damage of the mustard-induced lung tissues of mice and improving the activity of lung epithelial cells;the stem cell exosomes are derived from human umbilical cord mesenchymal stem cells.
- 2. The use according to claim 1, characterized in that:Wherein the drug for improving oxidative stress of mustard gas-induced lung tissue and cells is a drug for reducing the content of oxygen free radicals in lung tissue and cells and the expression level of malondialdehyde which is a lipid peroxidation product in cells, a drug for improving the expression level of superoxide dismutase in cells and the ratio of glutathione to oxidized glutathione in cells, or a drug for promoting the expression of oxidative stress related proteins in the lung tissue and cells of mice exposed by the mustard gas,The medicine for improving the activity of the lung epithelial cells is a medicine for improving the activity of mustard gas exposed BEAS-2B cells.
- 3. The use according to claim 1, wherein the exosome is prepared by the following method:A. Human umbilical cord mesenchymal stem cell cultureCulturing human umbilical cord mesenchymal stem cells by adopting a mesenchymal stem cell culture medium, and collecting culture supernatant after 48 hours, wherein the formula of the mesenchymal stem cell culture medium is as follows: adding 5% ELITECELL animal serum free cell culture supplement to Dakewe's mesenchymal stem cell basal medium;B. Exosome separationCollecting human umbilical cord mesenchymal stem cell culture supernatant, centrifuging at 300 g and 4 ℃ for 10 min, discarding the precipitate, centrifuging at 2000 g and 4 ℃ for 10 min, discarding the precipitate, centrifuging at 10000 g and 4 ℃ for 30min, and collecting the supernatant; supernatant is subjected to 120000 g and 4 ℃, 70: 70 min is centrifuged, PBS is used for resuspension after precipitation is collected, supernatant is subjected to 120000 g and 4 ℃ and 70: 70 min is centrifuged again, precipitation is collected and dissolved in 200 mu LPBS, and an exosome solution is obtainedC. engineering human umbilical cord mesenchymal stem cell exosomeSequentially adding the Exo-Fect solution, the miR-199a-5p analogue, the PBS solution and the 1X 10 7 part exosome solution into a centrifuge tube according to the volume ratio of 1:2:7:5, reversing the solution vertically for three times to uniformly mix, taking notice of incapability of vortex oscillation, vibrating and mixing at 37 ℃ for 10 minutes, and then immediately transferring the mixture onto ice; adding ExoQ uick-TC solution with the volume of being three times that of Exo-Fect, and uniformly mixing for six times upside down to stop the reaction, wherein the vortex oscillation cannot be noticed; standing at 4 ℃ for 30 minutes, centrifuging at 13000rpm for 3 minutes at 4 ℃, discarding the supernatant, and adding a PBS solution with the volume 300 times of the Exo-Fect solution volume into the precipitate for resuspension to obtain miR-199a-5p over-expression engineering exosome suspension.
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| WO2019168364A1 (en) * | 2018-03-02 | 2019-09-06 | 사회복지법인 삼성생명공익재단 | Method for upregulation of thioredoxin expression in stem cells |
| CN113717944A (en) * | 2021-09-03 | 2021-11-30 | 江苏大学 | miRNA13896 over-expressed engineered human umbilical cord mesenchymal stem cell-derived exosome and preparation method and application thereof |
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| WO2019168364A1 (en) * | 2018-03-02 | 2019-09-06 | 사회복지법인 삼성생명공익재단 | Method for upregulation of thioredoxin expression in stem cells |
| CN113717944A (en) * | 2021-09-03 | 2021-11-30 | 江苏大学 | miRNA13896 over-expressed engineered human umbilical cord mesenchymal stem cell-derived exosome and preparation method and application thereof |
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| 脂多糖诱发小鼠急性肺损伤microRNA-199a表达及其对基因表达的调控;才志刚;张绍明;张珩;周宜勇;吴海波;徐小平;;华南国防医学杂志(第04期);摘要 * |
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