CN115120615A - Application of miR-146a-5p overexpression engineering stem cell exosome in preparation of medicine for treating mustard seed lung injury caused by qi - Google Patents
Application of miR-146a-5p overexpression engineering stem cell exosome in preparation of medicine for treating mustard seed lung injury caused by qi Download PDFInfo
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Abstract
The invention provides application of miR-146a-5p overexpression engineering stem cell exosomes in preparation of a medicine for treating mustard air-induced lung injury. Aiming at the characteristic that mustard gas is a typical representative of a chemical warfare agent difficult to prevent and treat and has no specific anti-toxic drug, the lung is an important target organ damaged by the mustard gas, and the inflammatory reaction plays an important role in the mustard gas lung injury, experiments prove that miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosome can effectively improve the mustard gas-induced lung injury inflammatory reaction on both cell level and animal level, obviously improve the activity of lung epithelial cells, relieve the mustard gas-induced lung injury, has a treatment effect obviously superior to that of the human umbilical cord mesenchymal stem cell exosome, provides a new thought for treating the mustard gas injury, and has a prospect of preparing a mustard gas injury treating and treating drug.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of miR-146a-5p overexpression engineering stem cell exosomes derived from human umbilical cord mesenchymal stem cells in preparation of a medicine for treating acute lung injury caused by mustard gas and a medicine composition containing the exosomes.
Background
Mustard gas (SM), which is a typical representative of blister agents, is used in a variety of conflicts, such as the first battle, the japan aggression war, the bisi war, and the 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 toxic agents used in chemical terrorist activities. Mustard gas is also one of the main chemical warfare agents abandoned in China chemical weapons in Japan, and poses a significant threat to the environmental safety and the health of people in China. The lung is one of the major target organs of mustard injury, and acute exposure of mustard can lead to acute respiratory distress syndrome of poisons, even pulmonary edema, respiratory failure or secondary lung infection leading to death, so that the alleviation of acute lung injury is the key to treatment (Eteman L, Moshiri M, Balai-food M. Advances in therapy of acute sulfurous lung infection-a clinical review. critical Reviews in clinical study.2019; 49(3): 191-214).
At present, no specific antibacterial medicament is used for preventing and treating mustard gas damage, and the effect is not ideal when chemical medicaments are mainly applied to symptomatic treatment. Bone marrow mesenchymal stem cells (BMSCs) can regulate inflammatory response to improve mustard pneumolung injury (FengY, Xu Q, Yang Y, Shi W, Meng W, Zhang H, et al. the thermal effects of bone marrow stromal cells in the acid lung expressed by bone marrow cell tissue, Stem cell research & thermal, 2019; 10:90), but the source of BMSCs is limited, while Human umbilical cord mesenchymal stem cells (HucMSC) have abundant sources, less ethical controversy, strong proliferation capacity and easy collection, and can be used as a substitute for BMSCs. However, there are still risks of rejection and poor differentiation after long-term transplantation of HucMSCs into the body, and the 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 murine cardiac stem cells with hematology-reporting function and other capabilities. Haematologic.2006; 91(8): 1017-26).
HucMSC-Exo is a lipid bilayer structure vesicle actively secreted by HucMSC, has a diameter of about 40-100nm, contains various functional biomolecules, can be fused with cell membranes of adjacent cells through exocytosis, thereby performing information transmission, regulating intercellular signal conduction, and playing various biological functions such as anti-inflammation, anti-oxidation and the like. miRNA is a main component carried by exosome and plays an important role in the process of biological function of HucMSC-Exo. We found that HucMSC-Exo improved the inflammatory response of mustard induced lung injury. Information retrieval and experimental research show that miR-146a-5p and miR-199a-5p play an important role in improving mustard gas-induced lung injury by HucMSC-Exo; further experimental research shows that miR-146a-5p overexpression engineering HucMSC-Exo can effectively improve the lung injury inflammatory response caused by mustard gas, and the treatment effect is superior to that of the HucMSC-Exo. At present, the function of the miR-146a-5p overexpression engineering exosome in improving mustard gas lung injury is not reported in related documents.
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 human umbilical cord mesenchymal stem cells, a preparation method of the exosome, and a pharmaceutical composition comprising the exosome.
The research of the invention discovers that the HucMSC-derived exosome can treat acute lung injury caused by mustard gas. Obtaining an exosome from HucMSC by an ultracentrifugation method, analyzing components playing a role in the exosome of the human umbilical cord mesenchymal stem cells by combining a bioinformatics method, and confirming that miR-146a-5p is a key component by a cell experiment.
miR-146a-5p overexpression engineering human umbilical cord mesenchymal stem cell exosomes (miR-146a-5 p) are obtained by transfecting miR-146a-5p mimics in human umbilical cord mesenchymal stem cell exosomes + -Exo). Giving mustard gas damage RAW264.7 cell miR-146a-5p overexpression engineering human umbilical cord mesenchymal stem cell exosome; and then constructing a mustard air-induced lung injury mouse model, and administering the exosome, wherein the result shows that the exosome can improve the mustard air-induced lung injury inflammatory reaction and the cell inflammatory reaction, reduce the mustard air-induced lung tissue injury condition, reduce the total protein concentration of mustard air-exposed alveolar lavage fluid, and reduce the wet-dry-weight ratio of the mustard air-induced lung tissue.
The invention provides application of miR-146a-5p overexpression engineering stem cell exosomes in preparation of a medicine for treating mustard air-induced lung injury.
Preferably, the medicine is used for improving the inflammatory response of the lung injury caused by mustard gas and the inflammatory response of cells. The detection result of the ELISA method shows that miR-146a-5p overexpression engineering stem cell exosome can obviously reduce the expression quantity of tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6 in cells, improve the expression quantity of interleukin-10 in the cells and improve inflammatory reaction (figures 3C-3F and 4E-4H).
Preferably, the drug is one that ameliorates mustard air-induced lung tissue damage, reduces mustard air exposure to total protein concentration in alveolar lavage fluid, or reduces the wet-to-dry weight ratio of mustard air-induced lung tissue (FIGS. 4B-4D).
In a second aspect of the invention, a preparation method of the exosome is provided, which comprises the following steps:
A. human umbilical cord mesenchymal stem cell culture
Adding the mesenchymal stem cell culture medium into the human umbilical cord mesenchymal stem cells, and collecting culture supernatant after 48 hours. The formula of the mesenchymal stem cell culture medium is as follows: the basic mesenchymal stem cell medium of Dakewe was supplemented with 5% EliteCell animal serum free cell culture supplement.
B. Exosome isolation
Collecting human umbilical cord mesenchymal stem cell culture supernatant, centrifuging for 10min at 300g and 4 ℃, removing precipitate, centrifuging for 10min at 2000g and 4 ℃, removing precipitate, centrifuging for 30min at 10000g and 4 ℃ for supernatant, and collecting supernatant; centrifuging the supernatant at 120000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, then re-suspending the precipitate with PBS, centrifuging the supernatant at 120000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, and dissolving the precipitate in 200 mu l of PBS to obtain the exosome solution. The prepared exosome is in a vesicle shape through electron microscope observation, the diameter of the exosome is about 40-100nm, and the marker proteins CD9, CD63, CD81, HSP70 and Cav1 of the exosome are detected.
C. Engineered human umbilical cord mesenchymal stem cell exosome
Sequentially mixing an Exo-Fect solution, a miR-146a-5p simulant, a PBS solution and 1 × 10 7 Adding the particle exosome solution into a centrifuge tube according to the volume ratio of 1:2:7:5, turning upside down for three times to mix uniformly, taking notice that vortex oscillation cannot be carried out, vibrating and mixing for 10 minutes at 37 ℃, and immediately transferring the mixture onto ice; adding an ExoQuick-TC solution with the volume being three times that of the Exo-Fect solution, reversing the solution up and down for six times, and uniformly mixing to stop the reaction, wherein the vortex oscillation cannot be carried out; standing at 4 ℃ for 30 minutes, then centrifuging at 4 ℃ and 13000rpm for 3 minutes, removing the supernatant, adding a PBS solution with the volume 300 times that of the Exo-Fect solution into the precipitate, and re-suspending to obtain the miR-146a-5p overexpression engineering exosome suspension.
In a third aspect of the invention, the exosome composition derived from the human umbilical cord mesenchymal stem cells comprises miR-146a-5p overexpression engineering stem cell exosomes and diluent, wherein the diluent is preferably PBS, and the composition is convenient to use as an injection.
In a fourth aspect, the invention provides the use of the exosome composition in the preparation of a medicament for treating acute lung injury caused by mustard gas.
In the fifth aspect of the invention, the pharmaceutical composition for treating acute lung injury caused by mustard gas is provided, and miR-146a-5p overexpression engineering stem cell exosomes are used as the only active components.
Compared with the prior art, the invention has the following technical effects:
aiming at the special-effect antiviral drug for preventing and treating mustard gas injury, the key of treatment and relief of acute lung injury is to reduce the acute lung injury, and the characteristic that inflammatory reaction plays an important role in mustard gas lung injury is provided, and the miR-146a-5p overexpression engineering stem cell exosome is provided. Experiments prove that the miR-146a-5p overexpression engineering human umbilical cord mesenchymal stem cell exosome can effectively improve the lung injury inflammatory response caused by mustard gas at both the cell level and the animal level, obviously improves the activity of lung epithelial cells, relieves the lung injury caused by mustard gas, has a treatment effect obviously superior to that of the human umbilical cord mesenchymal stem cell exosome, provides a new idea for treating the mustard gas injury, and has a prospect of preparing a mustard gas injury treatment medicine.
Drawings
FIG. 1 shows the isolation and characterization of exosomes.
Figure 2 is a graph of the effect on mustard gas-exposed cell viability after transfection of different miRNA inhibitors.
FIG. 3A is a graph showing the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on miR-146a-5p expression amount in mustard gas exposed cells.
FIG. 3B is a graph of the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosomes on mustard gas-exposed cell viability.
FIG. 3C shows the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on the expression level of tumor necrosis factor-alpha (TNF-alpha) of mustard gas-exposed cells.
FIG. 3D is a graph of the effect of miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosome on the expression level of mustard gas-exposed cell interleukin-1 beta (IL-1 beta).
FIG. 3E shows the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on the expression amount of interleukin-6 (IL-6) in mustard gas-exposed cells.
FIG. 3F is a graph showing the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on the expression amount of interleukin-10 (IL-10) in mustard gas-exposed cells.
FIG. 4A is a graph showing the effect of miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosome on miR-146a-5p expression amount in lung tissue of mustard gas-exposed mice.
FIG. 4B is a graph of the effect of miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosomes on pathological section and scoring of mustard gas-exposed mouse lung tissue.
FIG. 4C is a graph of the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosomes on total protein concentration of alveolar lavage fluid of mustard gas-exposed mice.
FIG. 4D is a graph of the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on the wet/dry weight ratio of mustard gas exposed mouse lung tissue.
FIG. 4E shows the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on the expression level of tumor necrosis factor-alpha (TNF-alpha) in serum of mustard gas-exposed mice.
FIG. 4F is a graph showing the effect of miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosome on interleukin-1 beta (IL-1 beta) expression amount in serum of mustard gas-exposed mice.
FIG. 4G is a graph showing the effect of miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosome on interleukin-6 (IL-6) expression level in serum of mustard gas-exposed mice.
FIG. 4H shows the effect of miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosome on the expression level of interleukin-10 (IL-10) in serum of mustard gas-exposed mice.
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 cell experiment confirms that miR-146a-5p is a key component playing a role in exosomes of human umbilical cord mesenchymal stem cells
1. Exosome isolation
A. Human umbilical cord mesenchymal stem cell culture
Adding the mesenchymal stem cell culture medium into the human umbilical cord mesenchymal stem cells, and collecting culture supernatant after 48 hours. The formula of the mesenchymal stem cell culture medium is as follows: the supplement for elitec serum free cell culture was added at 5% to the mesenchymal stem cell basal medium of Dakewe.
B. Exosome isolation and identification
Collecting culture supernatant of human umbilical cord mesenchymal stem cells, centrifuging for 10min at 300g and 4 ℃, removing precipitate, centrifuging for 10min at 2000g and 4 ℃, removing precipitate, centrifuging for 30min at 10000g and 4 ℃ for supernatant, and collecting supernatant; centrifuging the supernatant at 120000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, then re-suspending the precipitate with PBS, centrifuging the supernatant at 120000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, and dissolving the precipitate in 200 mu l of PBS to obtain the exosome solution.
The prepared exosomes are in a vesicular shape (figure 1A) and have the diameter of about 40-100nm (figure 1B) through electron microscope observation, and the marker proteins of the exosomes, such as CD9, CD63, CD81, HSP70, Cav1 and the like (figure 1C), are detected.
According to the miRNA component analysis and transcriptome sequencing analysis of the exosomes of the human umbilical cord mesenchymal stem cells and the bioinformatics method, the miRNA molecules which are possibly involved in the human umbilical cord mesenchymal stem cells exosomes and play a role are screened out: miR-100-5p, miR-146a-5p, miR-23a-3p, let-7a, 22-3p, miR-424-5p, miR-221-3p, miR-15a-5p, miR-199a-5p, miR-145-5p and the like.
Human lung epithelial cells (BEAS-2B, ATCC: CRL-9609) were diluted to a concentration of 1X 10 6 one/mL cell suspension, 100 μ l cell suspension per well was plated in 96-well plates.
After 12 hours, the inhibitor of the miRNA molecule was transfected using lipofectamine RNAIMAX kit. Respectively diluting the miRNA molecular inhibitor and lipofectamine RNAIMAX by using 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.
24 hours after transfection, the cells were infected with mustard gas. Mustard gas stock was diluted to 50 μ M dilution with PBS and serum-free medium, replacing the conventional medium in the cell culture wells. After 30 minutes, replacing the fresh culture medium, adding the human umbilical cord mesenchymal stem cell exosome, continuously culturing for 24 hours, adding 10 mu l of CCK-8 solution into each hole, incubating in an incubator for 1 hour, measuring the absorbance of each hole under 450nm by using an enzyme-labeling instrument, and calculating the cell activity.
The result is shown in figure 2, after the miR-146a-5p is inhibited, the protection effect of the human umbilical cord mesenchymal stem cell exosome on the cell viability can be obviously reduced, the effect of other components is not obvious enough, and the miR-146a-5p is confirmed to be a key component playing a role in the human umbilical cord mesenchymal stem cell exosome.
The nucleotide sequence of miR-146a-5p is as follows: 3 '-UUGGGUACCUUAAGUCAAGAGU-5' (SEQ ID NO. 1).
Example 2 preparation of engineered human umbilical cord mesenchymal stem cell exosomes
The Exo-Fect kit of System Biosciences company is applied to transfect the exosome to obtain the miR-146a-5p overexpression engineering exosome. According to the kit instructions, 10 ul of Exo-Fect solution, 20 ul (20pmol) of miR-146a-5p simulant or miR-146a-5p inhibitor, 70 ul of PBS solution and 50ul of exosome solution (1 × 10) 7 particle) was added to a 1.5mL centrifuge tube and the tube was inverted 3 times to mix well, noting that vortex oscillation was not possible. Mix with shaking at 37 ℃ for 10 minutes and immediately transpose on ice. Add 30. mu.l ExoQuick-TC solution and mix well by turning upside down 6 times to stop the reaction, taking care that vortex was not possible. Standing at 4 ℃ for 30 minutes, then centrifuging at 4 ℃ and 13000rpm for 3 minutes, removing the supernatant, adding 300 mu l of PBS solution into the precipitate for resuspension, and obtaining miR-146a-5p overexpression or miR-146a-5p knock-down engineered exosome suspension.
Example 3 miR-146a-5p overexpression of engineered human umbilical cord mesenchymal stem cell exosomes improves mustard gas-induced cell inflammatory response
The miR-146a-5p is given to mustard gas damaged macrophage (RAW264.7, ATCC: TIB-71) (mustard gas contamination concentration is 50 mu M) to over-express engineered human umbilical cord mesenchymal stem cell exosomes.
(1) Detecting the expression quantity of miR-146a-5p in cells by an RT-PCR method: mixing RAW264.7 dilution of cells to a concentration of 5X 10 4 one/mL cell suspension, and plating with 1mL cell suspension per well in a 24-well plate. After 24 hours the cells were infected with mustard gas and the mustard gas stock was diluted to 50 μ M dilution with PBS and serum-free medium to replace the conventional medium in the cell culture wells. And after 30 minutes, replacing the fresh culture medium, adding the miR-146a-5p overexpression engineering exosome, continuously culturing for 24 hours, and collecting the supernatant for subsequent detection.
After washing the cells with PBS solution 3 times, 200. mu.l TRIzol reagent was added to each well, and after standing for 5 minutes, the cells were transferred to a centrifuge tube by pipetting. Add 40. mu.l chloroform, vortex for 15s, and let stand at room temperature for 3 minutes. Then, the mixture was centrifuged at 12000rpm at 4 ℃ for 15 minutes. The supernatant was transferred to a new centrifuge tube, an equal volume of isopropanol (about 100. mu.l) was added thereto, mixed by inversion, and allowed to stand at room temperature for 10 minutes. Centrifuge at 12000rpm for 15 minutes at 4 ℃. The supernatant was carefully removed and the pellet was added to 1mL of pre-cooled 75% ethanol and the pellet was allowed to float by turning upside down. Centrifuge at 12000rpm for 15 minutes at 4 ℃ and carefully discard the supernatant ethanol. 1mL of pre-cooled 75% ethanol was added and the pellet was allowed to float by turning upside down. Centrifuge at 12000rpm for 15 minutes 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 appropriate to obtain an RNA sample. Determination of OD 260 And OD 280 And the ratio thereof, analyzing the purity of the RNA sample, and calculating the concentration of the RNA sample.
RNA samples were reverse transcribed into cDNA using the TransScript miRNAFirst-Strand cDNA Synthesis SuperMix kit. Mu.l TransScript mirnarart Enzyme Mix and 10. mu.l 2 XTS miRNAReaction Mix were added to 1. mu.g of RNA sample according to the instructions and RNase-free ddH was used 2 Make up to 20. mu.l of O. And (3) lightly pipetting and beating by using a pipette, uniformly mixing, incubating for 1 hour at 37 ℃, heating for 5 seconds at 85 ℃, and using the product for qPCR reaction.
qPCR reaction was performed using SYBR Green chimeric fluorescence method. Mu.l of 2 XSSYBR qPCR Master Mix, 0.4. mu.l of forward primer, 0.4. mu.l of reverse primer, 2. mu.l of cDNA sample were added to the reaction wells and RNase-free ddH was used 2 O make up to 20. mu.l. Pre-denaturation at 95 ℃ for 30 seconds. The temperature of 95 ℃ is 10 seconds, and the temperature of 60 ℃ is 30 seconds, and the cycle is 40 times. The melting curve is 95 ℃ for 15 seconds, 60 ℃ for 60 seconds and 95 ℃ for 15 seconds. The relative expression level was calculated from the CT value.
Wherein, the miR-146a-5p forward primer (5 '-3'): TGAGAACTGAATTCCATGGGTT (SEQ ID NO. 2); u6 forward primer (5 '-3'): CTCGCTTCGGCAGCACA (SEQ ID NO. 3); the reverse primer is a universal primer.
The result is shown in figure 3A, and the miR-146a-5p overexpression level in the mustard gas damage RAW264.7 cell is obviously reduced. The human umbilical cord mesenchymal stem cell exosome can increase the miR-146a-5p overexpression amount in a mustard gas injury RAW264.7 cell, and the miR-146a-5p overexpression engineering exosome has more obvious effect of increasing. Human lung fibroblast exosomes as isotype controls had no significant effect.
(2) Cell viability by the CCK8 method: RAW264.7 cells were diluted to a concentration of 1X 10 7 one/mL cell suspension, 100 μ l cell suspension per well was plated in 96-well plates. After 24 hours the cells were infected with mustard gas and the mustard gas stock was diluted to 50 μ M dilution with PBS and serum-free medium to replace the conventional medium in the cell culture wells. And after 30 minutes, replacing a fresh culture medium, adding the miR-146a-5p overexpression engineering exosome, continuously culturing for 24 hours, adding 10 mu l of CCK-8 solution into each hole, incubating for 1 hour in an incubator, measuring the absorbance of each hole under 450nm by using a microplate reader, and calculating the cell activity.
The result is shown in fig. 3B, the activity of RAW264.7 cells damaged by mustard gas can be obviously improved by the human umbilical cord mesenchymal stem cell exosome, the improving effect of miR-146a-5p overexpression engineering exosome is more obvious, and the improving effect of exosome can be inhibited by knocking down miR-146a-5 p. Human lung fibroblast (HFL-1) exosomes as isotype controls had no significant improvement. Results show that the miR-146a-5p overexpression engineered exosome can reduce mustard gas-induced cytotoxicity and promote recovery of RAW264.7 cells.
(3) The ELISA method is used for detecting the expression quantity of inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-10 (IL-10) and the like in cell supernatant.
The cell supernatant collected in (1) above was centrifuged at 3000rpm at 4 ℃ for 10 minutes to remove the precipitate. And detecting the expression quantity of the inflammatory factor by adopting a double-antibody sandwich ABC-ELISA method. The specific operation method comprises the following steps:
preparing a specimen diluent: the 10 Xspecimen dilution was diluted with distilled water at a ratio of 1: 10.
Preparing a standard solution: 8 centrifuge tubes of 1.5mL are taken, 900ul of specimen diluent is added into the first tube, and 500ul of specimen diluent is added into the second tube to the eighth tube. 100ul of 5ng/mL standard solution was added to the first tube, mixed on a vortex mixer, 500ul was aspirated with a sample applicator, and transferred to the second tube. This was repeated to make a double dilution, and 500ul of the solution was aspirated from the seventh tube and discarded. The eighth tube is blank.
Preparing a washing solution: diluting with double distilled water at a ratio of 1: 20.
100ul of standard substance or sample to be detected is added into the reaction hole, and is placed for 40 minutes at 37 ℃ after being fully and uniformly mixed. The reaction plate was washed thoroughly 4-6 times with washing solution and printed dry on filter paper. Each well was charged with 50ul of each of distilled water and primary antibody working solution (except blank). The reaction plate was mixed well and left at 37 ℃ for 20 minutes. And (5) washing the plate. 100ul of the enzyme-labeled antibody working solution was added to each well. The reaction plate was left at 37 ℃ for 10 minutes. And (5) washing the plate. 100ul of substrate working solution was added to each well, and the mixture was left to react at 37 ℃ for 15 minutes in the dark. 100ul of stop solution was added to each well and mixed well. And detecting the light absorption value at 450nm by using a microplate reader within 30 minutes.
And (4) taking the concentration of the standard substance as an abscissa and the OD value as an ordinate to prepare a standard curve. And (5) corresponding the OD value of the sample to a standard curve, and calculating the content of the corresponding inflammatory factor.
The results are shown in FIGS. 3C-3F, and compared with the normal group, the expressions of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 in the mustard gas group are all increased, and the expression of the inflammation-inhibiting factor IL-10 is reduced. Compared with the mustard gas group cells, the expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 is reduced, and the expression of the inflammation-inhibiting factor IL-10 is increased by giving the exosome treatment group cells. Compared with an exosome group, the expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 in cells of the miR-146a-5p overexpression engineering exosome group is obviously reduced, and the expression of the inflammation-inhibiting factor IL-10 is obviously increased; the miR-146a-5p knockdown expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 in an exosome group is increased, the expression of an inflammation-inhibiting factor IL-10 is reduced, and the inflammatory reaction is aggravated. The result shows that the miR-146a-5p overexpression engineered exosome can improve the inflammatory response of mustard injury cells.
Example 4 human umbilical cord mesenchymal stem cell exosomes over-expressed by miR-146a-5p improve mustard gas-induced lung injury mouse inflammatory response
Further on the first and third days after mustard gas exposure, mustard gas was administered by tail vein injection to damage the miR-146a-5p overexpression engineered exosomes of ICR mice. On the fifth day after mustard gas contamination, by detecting the expression quantity of miR-146a-5p in mouse lung tissues, observing lung tissue HE stained sections, determining the total protein concentration of alveolar lavage fluid and the lung wet/dry weight ratio, and detecting the expression quantity of inflammatory factors in serum, the improvement effect of miR-146a-5p overexpression engineered exosomes on mustard gas-induced lung injury inflammatory response is evaluated.
(1) Expression level of miR-146a-5p in lung tissue: mouse lung tissue was weighed, 9 times the volume of TRIzol reagent was added thereto, cryogenically ground, centrifuged at 12000rpm at 4 ℃ for 15 minutes, and the supernatant was collected and the subsequent operation was the same as in (1) of example 3.
The result is shown in figure 4A, compared with the exosome group, the gene expression level of miR-146a-5p in the lung tissue of the mouse in the miR-146a-5p overexpression engineering exosome group is remarkably increased, and the gene expression level of miR-146a-5p in the lung tissue of the mouse in the miR-146a-5p knock-down exosome group is remarkably reduced.
(2) Lung tissue HE stained section: the lung tissue was fixed in 4% paraformaldehyde tissue fixative for 24 h. And (4) paraffin embedding after dehydration. And (4) slicing and dewaxing. After hematoxylin-eosin staining. And (5) observing and photographing under a microscope. Two pathology teachers used blind method to score according to the degree of inflammatory cell infiltration of lung tissue, thickening degree of alveolar wall, alveolar hemorrhage and edema. The degree of lung tissue damage is graded from light to heavy into 5 grades. 0 minute: normal; 1 minute: a small amount of inflammatory cells infiltrate into the lung interstitium, and the structure of the alveolus is not obviously changed; and 2, dividing: the lung interstitium has mild to moderate inflammatory change, and the lung structure has no obvious damage; and 3, dividing: the lung interstitium has moderate to severe inflammatory injury, the alveolar space is thickened, and the alveolar structure is obviously damaged; and 4, dividing: severe inflammatory injury, alveolar collapse. Finally, the score is summarized and represents the degree of lung injury.
The results are shown in fig. 4B, the lung tissue of the mustard group mice was severely damaged, the structural destruction was significant, inflammatory cells in the alveolar space were diffused, a large amount of exudates was obtained, and the lung space was significantly thickened. Compared with mustard gas, the exosome group has the advantages of reduced lung injury symptoms, damaged few alveolar structures, partial inflammatory cell infiltration, a small amount of exudates and slight thickening of alveolar walls. Compared with an exosome group, the lung tissue injury symptom of a mouse in the miR-146a-5p overexpression engineering exosome group is obviously relieved. Results show that the miR-146a-5p overexpression engineered exosome can improve the mouse lung tissue injury caused by mustard gas.
(2) Total protein concentration of alveolar lavage fluid: anaesthetizing the mouse, fixing the mouse on an operating table, cutting off the spinal cord after exposing the abdominal cavity, bleeding, carefully and slowly exposing the trachea and the lung portal of the mouse, fixing the exposed trachea by using forceps of the ophthalmology department, transversely cutting off part of the trachea, inserting an lavage needle, and ligating and fixing the trachea and the lavage needle by using an operation suture line. Sucking 800 μ l of precooled 1 × PBS, washing, sucking, pushing again for washing, repeating for three times, recovering 500 μ l of alveolar lavage fluid, centrifuging at 4 deg.C and 3000rpm for 15min, and collecting supernatant as alveolar lavage fluid.
The total protein concentration in alveolar lavage fluid was determined according to the BCA kit instructions. And 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. The protein standard substance is diluted into a protein standard substance solution with the concentration of 0.5mg/mL, and the protein standard substance solution is diluted in a multiple proportion for preparing a standard curve. Mu.l of sample or standard solution and 18. mu.l of diluent were added to the sample wells. 200. mu.l of BCA working solution was added to each well, and the wells were left at 37 ℃ for 30 minutes. And (3) detecting the absorbance of each hole under the wavelength of 562nm by using a microplate reader, and drawing a standard curve by taking the concentration of the protein standard substance as a vertical coordinate and the OD value as a horizontal coordinate. And (5) comparing the OD value of the sample to be detected with a standard curve, and calculating the protein concentration of the sample.
The results are shown in figure 4C, where the concentration of protein in alveolar lavage fluid was significantly increased in the mustard group mice compared to the normal group. Compared with the mustard gas group, the protein concentration in the alveolar lavage fluid of the exosome group is obviously reduced, and the effect of reducing the total protein concentration in the alveolar lavage fluid of the mice injured by the mustard gas by the miR-146a-5p overexpression engineering exosome is more obvious.
(3) Lung wet/dry weight ratio: the lung tissue of the mouse is taken, washed by physiological saline, blotted dry by filter paper, weighed by a balance, and recorded as the wet weight (W) of the lung. And (3) placing the weighed lung tissues in a drying oven at 75 ℃, drying for 72h until the lung tissues are constant in weight, taking out, accurately weighing, and recording as the dry lung weight (D). Lung tissue wet/dry weight ratio (wet/dry weight (W/D)).
The results are shown in fig. 4D, in which the lung tissue wet/dry weight ratio was significantly increased in the mustard gas group mice compared to the normal group. Compared with a mustard gas group, the lung tissue wet/dry weight ratio of the exosome group is obviously reduced, and the effect of reducing the wet/dry weight ratio of the mustard gas-damaged mouse lung tissue by the miR-146a-5p overexpression engineered exosome is more obvious.
(4) The expression level of inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and interleukin-10 (IL-10): the mouse is subjected to eyeball removal and blood collection, is kept stand for 30 minutes, is centrifuged at 3000rpm at 4 ℃ for 20 minutes, and is carefully sucked to obtain a supernatant, namely a detection sample. The subsequent operation was the same as that in (3) of example 3 described above.
The results are shown in FIGS. 4E-4H, and compared with the normal group, the expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 in the serum of the mice in the mustard group is increased, and the expression of the inflammation-inhibiting factor IL-10 is reduced. Compared with the serum of mice in a mustard gas group, the serum of mice in an exosome treatment group has the advantages that the expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 is reduced, and the expression of the inflammation inhibitor IL-10 is increased. Compared with an exosome group, the miR-146a-5p overexpression engineering exosome group has the advantages that the expression of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 in the serum of a mouse is obviously reduced, and the expression of an inflammation-inhibiting factor IL-10 is obviously increased; miR-146a-5p knockdown expressions of proinflammatory factors TNF-alpha, IL-1 beta and IL-6 in an exosome group are all increased, expression of an inflammation inhibitor IL-10 is reduced, and inflammation reaction is aggravated. Results show that miR-146a-5p overexpression engineered exosome can improve the inflammatory response of mice with lung injury caused by mustard.
In conclusion, the invention provides application of miR-146a-5p overexpression engineered human umbilical cord mesenchymal stem cell exosome in preparation of a medicine for treating mustard lung injury caused by qi.
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.
Sequence listing
<110> China people liberation army navy military medical university
Application of miR-146a-5p overexpression engineering stem cell exosome in preparation of medicine for treating mustard air-induced lung injury
<130> specification of claims
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
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<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgagaactga attccatggg tt 22
<210> 3
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctcgcttcgg cagcaca 17
Claims (9)
- Application of miR-146a-5p overexpression engineering stem cell exosomes in preparation of medicines for treating mustard air-induced lung injury.
- 2. Use according to claim 1, characterized in that:wherein, the nucleotide sequence of the gene for coding miR-146a-5p is shown in SEQ ID NO. 1.
- 3. Use according to claim 1, characterized in that:the medicine is a medicine for improving mustard air-induced lung injury inflammatory reaction and cell inflammatory reaction, improving mustard air-induced lung tissue injury, reducing the total protein concentration of mustard air-exposed alveolar lavage fluid or reducing the wet-dry weight ratio of mustard air-induced lung tissue.
- 4. Use according to claim 3, characterized in that:wherein the medicine for improving mustard air-induced lung injury inflammatory reaction and cell inflammatory reaction is a medicine for reducing the expression level of tumor necrosis factor-alpha, interleukin-1 beta and interleukin-6 and improving the expression level of interleukin-10.
- 5. Use according to claim 1, characterized in that:wherein the stem cell exosome is derived from human umbilical cord mesenchymal stem cells.
- 6. The use according to claim 1, wherein the exosomes are prepared as follows:A. human umbilical cord mesenchymal stem cell cultureAdding the mesenchymal stem cell culture medium into the human umbilical cord mesenchymal stem cells, collecting culture supernatant after 48h,the formula of the mesenchymal stem cell culture medium is as follows: adding 5% EliteCell animal free serum cell culture supplement to the mesenchymal stem cell basal medium of Dakewe;B. exosome isolationCollecting culture supernatant of human umbilical cord mesenchymal stem cells, centrifuging for 10min at 300g and 4 ℃, removing precipitate, centrifuging for 10min at 2000g and 4 ℃, removing precipitate, centrifuging for 30min at 10000g and 4 ℃ for supernatant, and collecting supernatant; centrifuging the supernatant at 120000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, then re-suspending by PBS, centrifuging the supernatant at 120000g and 4 ℃ for 70min, then discarding the supernatant, collecting the precipitate, and dissolving the precipitate in 200 mu l of PBS to obtain an exosome solution;C. engineered human umbilical cord mesenchymal stem cell exosomeSequentially mixing an Exo-Fect solution, a miR-146a-5p simulant, a PBS solution and 1 × 10 7 Adding the particle exosome solution into a centrifuge tube according to the volume ratio of 1:2:7:5, turning upside down for three times to mix uniformly, taking notice that vortex oscillation cannot be carried out, vibrating and mixing for 10 minutes at 37 ℃, and immediately transferring the mixture onto ice; adding an ExoQuick-TC solution with the volume being three times that of the Exo-Fect solution, reversing the solution up and down for six times, and uniformly mixing to stop the reaction, wherein the vortex oscillation cannot be carried out; standing at 4 ℃ for 30 minutes, then centrifuging at 13000rpm for 3 minutes at 4 ℃, discarding the supernatant, adding a PBS solution with the volume 300 times that of the Exo-Fect solution into the precipitate, and re-suspending to obtain the miR-146a-5p overexpression engineering exosome suspension.
- 7. An exosome composition derived from human umbilical cord mesenchymal stem cells, which is characterized by comprising miR-146a-5p overexpression engineering stem cell exosomes and pharmaceutically acceptable auxiliary materials.
- 8. Use of the human umbilical cord mesenchymal stem cell-derived exosome composition of claim 7 in the preparation of a medicament for treating mustard gas-induced lung injury.
- 9. A pharmaceutical composition for treating mustard lung injury caused by qi is characterized in that miR-146a-5p overexpression engineering stem cell exosomes are used as the only active components.
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