CN116904394A - Preparation method and application of anti-inflammatory mesenchymal stem cell-derived exosome - Google Patents
Preparation method and application of anti-inflammatory mesenchymal stem cell-derived exosome Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an anti-inflammatory Mesenchymal Stem Cell (MSCs) source exosome, comprising the following steps: 1. MSCs are obtained; 2. MSCs identification; 3. subculturing; 4. three methods pre-treat MSCs: comprises (1) pretreatment of low-concentration inflammatory factors; (2) soft stiffness pretreatment; (3) low concentration inflammatory factors combined with soft stiffness pretreatment; 5. obtaining supernatant of MSCs culture after 24 hours; 6. treating supernatant fluid of MSCs culture by adopting a three-step centrifugation method to obtain the exosomes; 7. a diabetic nephropathy mouse model is constructed, and the tail vein is injected with exosomes and relevant indexes are measured. The invention selects MSCs with wide sources, easy culture, strong activity and better stimulation reactivity by low-concentration inflammatory factors as cells for extracting exosomes, and separates exosomes after stimulation by low-concentration inflammatory factors and soft rigidity, and the obtained exosomes with anti-inflammatory MSCs source enrich PD-L1, has an immunosuppressive effect, and can regulate the conversion of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a preparation method and application of an anti-inflammatory mesenchymal stem cell-derived exosome.
Background
Diabetes Mellitus (DM) is a group of common chronic metabolic diseases characterized by hyperglycemia, which can lead to multiple target organ damage and serious complications. Among them, diabetic Nephropathy (DN) is one of the most important chronic complications of DM patients, and is also a major cause of end-stage renal disease. DM includes type 1 diabetes (T1 DM), type 2 diabetes (T2 DM), and special types of diabetes, with T1DM and T2DM being the most common, both of which can induce DN. DN pathogenesis is closely related to systemic and renal local inflammatory response. It is currently believed that in the pathogenesis of diabetic nephropathy, the inflammatory response in the kidney determines the extent of progression of kidney injury, and macrophages are important innate immune cells that contribute to interstitial proliferation of kidney tissue and glomerular injury. Macrophages can be divided into two main subgroups, the "classical activated" M1 type and the "bypass activated" M2 type, depending on the cytokines and markers secreted. In inflammatory response, M1 and M2 type macrophages can secrete different cytokines to regulate inflammatory processes of the body, M1 type macrophages can secrete pro-inflammatory cytokines such as TNF-alpha, IL-6 and IL-1 beta, and M2 type macrophages can secrete anti-inflammatory cytokines such as IL-10. Thus, macrophages of different phenotypes play a critical role in the development and progression of diabetic nephropathy. Currently, the treatment modalities for diabetic nephropathy mainly include lifestyle modification, oral medication and kidney transplantation, however, these treatment modalities can only temporarily control blood glucose levels to delay kidney damage, and do not mainly play a role from the mechanism of inhibiting kidney inflammatory response. Therefore, how to address the pathogenesis of diabetic nephropathy is a current leading problem.
Mesenchymal stem cells are cells with self-renewal, self-replication and multipotency, with unique immunomodulatory capacity. Research shows that mesenchymal stem cell transplantation can effectively promote macrophage transformation from pro-inflammatory phenotype to anti-inflammatory phenotype, inhibit infiltration of immune cells including macrophages, monocytes and neutrophils to inflammatory regions, reduce local inflammatory infiltration, and relieve tissue injury. Although stem cell transplantation has shown great therapeutic potential in the field of regenerative medicine, it has numerous drawbacks such as ectopic tissue formation, infusion toxicity due to cell retention in pulmonary microvasculature, cell rejection, and the like. Exosomes are vesicle-like vesicles with a bilayer membrane structure secreted by cells and having diameters between 30-100nm, carrying a large number of proteins, mRNA and miRNA associated with the parent cell. There is growing evidence that a new cell-free therapy, i.e. exosomes secreted by mesenchymal stem cells, is an attractive alternative therapy due to its advantages over the parent cells in terms of small volume, easy storage, low immunogenicity, low susceptibility to rejection reactions, the ability to protect the contents from degradation in the circulation and the absence of risk of tumor formation.
Although exosomes have advantages in terms of safety and stability, the required amount of exosomes is far greater than the number of transplanted stem cells under the same curative effect, and the current means for extracting exosomes are still immature and have lower yield. In addition, current means of exosome modification are limited to plasmid, lentiviral transfection, etc., which are too costly and present a biosafety hazard. Therefore, how to safely and effectively improve the content of key proteins in the exosomes with unit concentration while ensuring the cost and to enhance the curative effect of the exosomes with unit concentration are considerable problems.
Disclosure of Invention
The invention aims to provide a preparation method and application of an anti-inflammatory mesenchymal stem cell-derived exosome, so as to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for preparing anti-inflammatory mesenchymal stem cell-derived exosomes, comprising the steps of:
step one, mesenchymal stem cells are obtained: obtaining umbilical cord mesenchymal stem cells in healthy fetal umbilical cord tissue with consent;
step two, mesenchymal stem cell identification: performing phenotype identification, bone formation and lipid formation identification on the mesenchymal stem cells;
Step three, subculturing: subculturing the identified mesenchymal stem cells to a sixth generation, and replacing a serum-free culture medium;
step four, preprocessing mesenchymal stem cells by adopting three methods: comprises (1) pretreatment of mesenchymal stem cells with low concentration of inflammatory factors (IFN-gamma, TNF-alpha, 1-5 ng/ml); (2) Soft stiffness (0.1-10 kPa) pretreatment of mesenchymal stem cells; (3) Low concentration inflammatory factor combined soft rigidity pretreatment mesenchymal stem cells;
step five, obtaining supernatant of mesenchymal stem cell culture: obtaining supernatant of the mesenchymal stem cell culture after continuous culture for 24 hours;
step six, obtaining exosomes prepared by the mesenchymal stem cells: treating the obtained supernatant of the mesenchymal stem cell culture by adopting a three-step centrifugation method, wherein the obtained precipitate is the exosome;
and step seven, constructing a Diabetic Nephropathy (DN) mouse model, injecting exosomes by tail vein and measuring DN related indexes.
As a preferred technical scheme of the invention, the specific effect of the fourth step is to increase the expression of immunosuppressive protein PD-L1 by mesenchymal stem cells and enrich exosomes with PD-L1 protein.
As a preferable technical scheme of the invention, the soft rigidity in the fourth step is prepared by acrylamide with the concentration of 40% and bisacrylamide with the concentration of 2% at the mixing ratio of about 3:1, and the rest is complemented by double distilled water after a proper amount of ammonium persulfate and tetramethyl ethylenediamine are added; dripping the prepared gel on a large glass plate, and immediately reversely buckling a circular slide on the gel; immediately after solidification at room temperature, placing the glass slide in a water tank filled with alcohol, peeling off the glass slide by using a sterile syringe needle, and placing the gel face upwards in a sterile culture dish containing PBS (phosphate buffered saline) with 1% PS; dropwise adding a crosslinking agent Sulfo-SANPAH into PBS in a light-shielding environment; adding the solution into a culture medium dish, placing under a high-intensity UV lamp, irradiating for 30min, discarding the cross-linking agent, adding a proper amount of I-type rat tail collagen solution into PBS, adding the solution into the culture medium dish until the gel is completely soaked, and cooling overnight at 4 ℃; mesenchymal stem cells were cultured at this rigidity for 24h.
As a preferable technical scheme of the invention, the three-step centrifugation method in the step six comprises the specific steps of firstly centrifuging 3000g at 4 ℃ for 20min and discarding the precipitate; transferring the supernatant to a new super-separation tube, and centrifuging at 4 ℃ for 45min with 10000 g; discarding the precipitate, transferring the supernatant to a new super-separation tube, and centrifuging at 120000g for 2h (X2) at 4 ℃; the supernatant was discarded, the bottom pellet was resuspended with an appropriate amount of PBS, and the aspirate was dispensed into sterile EP tubes.
As the application of the anti-inflammatory mesenchymal stem cell-derived exosome, the anti-inflammatory mesenchymal stem cell-derived exosome obtained by separation after pretreatment in a specific mode has a remarkable immunosuppressive effect, can regulate the transformation of macrophage phenotype of a diabetic mouse and reduce the inflammatory infiltration degree of kidney, and is prepared into a medicament for treating diabetic nephropathy.
Compared with the prior art, the invention has the beneficial effects that:
1. the exosome of the mesenchymal stem cells stimulated by the low-concentration inflammatory factors is derived from clinically obtained human umbilical cord tissues, has wide and easily obtained sources and has higher biosafety. .
2. In terms of pretreatment mode, the preparation method obtains exosomes through pretreatment of low-concentration inflammatory factors, soft rigidity pretreatment and combined pretreatment of the low-concentration inflammatory factors and the soft rigidity pretreatment, is rich in PD-L1, has strong immunosuppressive ability, can regulate transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype, lightens the condition of inflammatory infiltration of kidneys of diabetic nephropathy, promotes recovery of injured kidneys, can be prepared into injection for treating the diabetic nephropathy, has higher safety and clinical feasibility, and has great potential in clinical transformation. .
Drawings
FIG. 1 is a flow chart of exosome preparation according to the present invention;
FIG. 2 is a flow chart depicting the identification of mesenchymal stem cell surface markers of the present invention;
FIG. 3 is a graph showing the identification of the adipogenic and osteogenic differentiation potential of mesenchymal stem cells of the present invention;
FIG. 4 is an electron microscope identification of mesenchymal stem cell-derived exosomes treated with low concentrations of inflammatory factors according to the present invention;
FIG. 5 is a Western blot identification of mesenchymal stem cell-derived exosomes treated with low concentration of inflammatory factors according to the present invention, wherein Native is untreated mesenchymal stem cell-derived exosomes, IFN-gamma is 1-5ng/ml IFN-gamma-treated mesenchymal stem cell-derived exosomes, TNF-alpha is 1-5ng/ml TNF-alpha-treated mesenchymal stem cell-derived exosomes, IFN-gamma/TNF-alpha is 1-5ng/ml IFN-gamma and TNF-alpha-combined treated mesenchymal stem cell-derived exosomes;
FIG. 6 is a statistical graph showing the results of PCR of low concentration inflammatory factor treated mesenchymal stem cells and non-induced mesenchymal stem cell PD-L1 expression, wherein Native is untreated mesenchymal stem cells, IFN-gamma is 1-5ng/ml IFN-gamma treated mesenchymal stem cells, TNF-alpha is 1-5ng/ml TNF-alpha treated mesenchymal stem cells, IFN-gamma/TNF-alpha is 1-5ng/ml IFN-gamma and TNF-alpha combined treated mesenchymal stem cells;
FIG. 7 is a graph showing Western blot results of PD-L1 expression of low concentration inflammatory factor treated mesenchymal stem cell-derived exosomes and uninduced mesenchymal stem cell-derived exosomes, wherein Native is untreated mesenchymal stem cells, IFN-gamma is 1-5ng/ml IFN-gamma treated mesenchymal stem cells, TNF-alpha is 1-5ng/ml TNF-alpha treated mesenchymal stem cells, and IFN-gamma/TNF-alpha is 1-5ng/ml IFN-gamma and TNF-alpha treated mesenchymal stem cells in combination;
FIG. 8 is a graph showing the Western blot results of PD-L1 enrichment of untreated and low concentration inflammatory factor treated mesenchymal stem cell-derived exosomes according to the present invention, wherein Native is untreated mesenchymal stem cell-derived exosomes, IFN-gamma is 1-5ng/ml IFN-gamma treated mesenchymal stem cell-derived exosomes, TNF-alpha is 1-5ng/ml TNF-alpha treated mesenchymal stem cell-derived exosomes, and IFN-gamma/TNF-alpha is 1-5ng/ml IFN-gamma and TNF-alpha combined treated mesenchymal stem cell-derived exosomes;
FIG. 9 is a statistical plot of the changes in diabetic nephropathy in mice of each group evaluated in an in vivo model of the mice DN of the present invention, wherein NC is a normal control group, DN is a diabetic nephropathy group, DN+Exo is a post-molding mesenchymal stem cell-derived exosome group injected, DN+TNF- α & IFN- γ -Exo is a post-molding mesenchymal stem cell-derived exosome group pre-treated with low concentrations of inflammatory factors injected. "a" graph shows the weekly fasting blood glucose level of each group of mice, "b" graph shows the weekly weight change of each group of mice, "c" graph shows the glucose tolerance level change of each group of mice, "d" graph shows the spleen factor (mg/g) of each group of mice versus "e" graph shows the kidney factor (mg/g) of each group of mice versus;
FIG. 10 is a statistical plot of the change in urine protein of mice of each group evaluated in an in vivo mouse DN model of the invention, wherein NC is a normal control group, DN is a diabetic nephropathy group, DN+Exo is a post-model injection mesenchymal stem cell-derived exosome group, DN+TNF- α & IFN- γ -Exo is a post-model injection low-concentration inflammatory factor pre-treated mesenchymal stem cell-derived exosome group;
FIG. 11 is a flow cytometry detection graph one of TNF- α & IFN- γ -Exo of the present invention evaluating changes in overall inflammatory response in groups of mice in an in vivo mouse DN model;
FIG. 12 is a graph II of flow cytometry assays for assessing changes in overall inflammatory response of groups of mice of the present invention in an in vivo mouse DN model;
FIG. 13 is a third flow cytometry assay for assessing changes in overall inflammatory response of mice of each group in an in vivo mouse DN model of TNF- α & IFN- γ -Exo of the present invention;
FIG. 14 is a fourth flow cytometry detection graph evaluating changes in overall inflammatory response of mice of each group in an in vivo mouse DN model of TNF- α & IFN- γ -Exo of the present invention;
FIG. 15 is a flow cytometry detection graph five of TNF- α & IFN- γ -Exo of the present invention evaluating changes in overall inflammatory response in groups of mice in an in vivo mouse DN model;
in fig. 11-15, NC is a normal control group, DN is a diabetic nephropathy group, dn+exo is a mesenchymal stem cell-derived exosome group injected after molding, dn+tnf- α & IFN- γ -Exo is a mesenchymal stem cell-derived exosome group pretreated by injection of low concentration inflammatory factors after molding; the upper graph of FIG. 11 shows the ratio of CD4+ T cells in the spleens of mice in each group, the lower graph shows the statistical analysis, the upper graph of FIG. 12 shows the ratio of Th1 cells in the spleens of mice in each group, the lower graph shows the statistical analysis, the upper graph of FIG. 13 shows the ratio of Th17 cells in the spleens of mice in each group, the lower graph shows the statistical analysis, the upper graph of FIG. 14 shows the ratio of Th2 cells in the spleens of mice in each group, the lower graph shows the statistical analysis, the upper graph of FIG. 15 shows the ratio of Treg cells in the spleens of mice in each group.
FIG. 16 is a Western blot results of in vivo mouse DN model evaluation of kidney inflammation change in each group of mice according to the invention, wherein NC is a normal control group, DN is a diabetic nephropathy group, DN+Exo is a mesenchymal stem cell-derived exosome group injected after molding, DN+TNF-alpha & IFN-gamma-Exo is a mesenchymal stem cell-derived exosome group pretreated by injection of low concentration inflammatory factors after molding, IL-6, IL-1 beta, TNF-alpha are M1 type macrophage-related cytokines, IL-10 is M2 type macrophage-related cytokines; the upper graph shows the expression level of inflammatory factors IL-6, IL-1 beta, TNF-alpha and IL-10 in kidney tissues of mice in each group detected by Western blot, and the lower graph shows the statistical analysis;
FIG. 17 is a flow cytometry analysis of the invention in an in vivo mouse DN model for assessing changes in the different phenotypic proportions of macrophages in the kidneys of each group of mice, wherein NC is a normal control group, DN is a diabetic nephropathy group, DN+Exo is a post-model injection of mesenchymal stem cell derived exosomes, DN+TNF- α & IFN- γ -Exo is a post-model injection of low-concentration inflammatory factor pretreated mesenchymal stem cell derived exosomes, CD86+ macrophages are M1 type macrophage markers, CD206+ macrophages are M2 type macrophage markers; the upper panel shows the flow cytometry to detect the changes in the proportion of M1-type and M2-type macrophages in the kidneys of each group of mice, and the lower panel shows the statistical analysis.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-17, the invention provides a preparation method of anti-inflammatory mesenchymal stem cell-derived exosomes, comprising the following steps:
step one, mesenchymal stem cells are obtained: obtaining umbilical cord mesenchymal stem cells in healthy fetal umbilical cord tissue with consent;
step two, mesenchymal stem cell identification: performing phenotype identification, bone formation and lipid formation identification on the mesenchymal stem cells;
step three, subculturing: subculturing the identified mesenchymal stem cells to a sixth generation, and replacing a serum-free culture medium;
step four, preprocessing mesenchymal stem cells by adopting three methods: comprises (1) pretreatment of mesenchymal stem cells with low concentration of inflammatory factors (IFN-gamma, TNF-alpha, 1-5 ng/ml); (2) Soft stiffness (0.1-10 kPa) pretreatment of mesenchymal stem cells; (3) Low concentration inflammatory factor combined soft rigidity pretreatment mesenchymal stem cells;
Step five, obtaining supernatant of mesenchymal stem cell culture: obtaining supernatant of the mesenchymal stem cell culture after continuous culture for 24 hours;
step six, obtaining exosomes prepared by the mesenchymal stem cells: treating the obtained supernatant of the mesenchymal stem cell culture by adopting a three-step centrifugation method, wherein the obtained precipitate is the exosome;
and step seven, constructing a Diabetic Nephropathy (DN) mouse model, injecting exosomes by tail vein and measuring DN related indexes.
Example 1 acquisition of hUC-MSCs
1) Umbilical cord harvesting
The pregnant woman agrees to obtain a healthy neonatal umbilical cord in a first hospital of Jilin university, residual blood is washed by sterile normal saline, two ends of the umbilical cord are ligated, and the umbilical cord is placed in sterile PBS containing 5% penicillin-streptomycin (PS) after high pressure, so that subsequent operation needs to be performed as soon as possible.
2) Human umbilical cord mesenchymal stem cell extraction
After completing each sterile preparation work, the umbilical cord was removed from the superclean bench, placed in a 10cm dish containing PBS, and cut into 2-3cm long sections using high pressure sterile ophthalmic scissors. The arterial vessel and the venous vessel are peeled off from the umbilical cord by using sterile ophthalmic scissors and ophthalmic forceps, and the actions are careful to prevent the vascular rupture. The umbilical cord was then made to about 5mm using a sterile surgical blade 2 And washed clean with PBS. Uniformly sticking the tissue block in a 10cm dish, standing for about 30min, adding appropriate amount of DMEM-F12 complete medium containing 1% PS and 10% FBS after the tissue block sticks to the wall to make the tissue block in wet state, and standing at 37deg.C and 5% CO 2 Culturing in saturated humidity incubator under the condition, and supplementing appropriate amount of DMEM-F12 complete culture medium every 48 h. Care was taken not to add too much medium to detach the tissue mass from the dish bottom. When the cells were climbed out of the tissue mass, 10ml of complete DMEM-F12 medium was supplemented and the medium was changed every 48 hours.
3) Cell passage
The cell growth state is observed well under a microscope, and when the cell density reaches more than about 90%, the passage is carried out. The dish was opened in an ultra clean bench, the medium was aspirated off, 10ml of PBS was added to wash the cells, and the PBS in the dish was discarded. Adding 8ml of pancreatin, taking care of covering a cell layer, shaking lightly for a plurality of times, standing at room temperature for 15-30s, then sucking most of pancreatin, incubating for 15min in a 37 ℃ incubator, stopping digestion when the cell is observed to swell and round under a mirror, adding 10ml of DMEM-F12 complete medium, blowing cell suspension, transferring the suspension cells into a 15ml centrifuge tube, centrifuging at 1000rpm for 5min, sucking the supernatant, re-suspending the cells by the medium, and carrying out subculture according to a proportion.
EXAMPLE 2 phenotypic characterization of mesenchymal Stem cells and osteogenic adipogenic differentiation
1) Phenotypic identification
After digestion of P3 mesenchymal stem cells with pancreatin, 1500rpm/5min, the supernatant was discarded, and the cells were resuspended in 500. Mu.L of staining buffer at 1500rpm/5min. The supernatant was discarded, and the cells were resuspended by adding staining buffer, and the cell number was counted. Will be 5x10 5 100. Mu.L of mesenchymal stem cells were transferred to 1.5ml brown EP tubes for a total of 4 tubes. mu.L of staining blocking solution was added to each tube, and the mixture was gently stirred and mixed by a pipette, and incubated at room temperature for 15min. Adding 10 mu L of staining buffer to the cell suspension of the first tube; adding 10 mu L of mesenchymal stem cells into the cell suspension of the tube 2 to identify an antibody I; add 10 μl of mesenchymal stem cells to tube 3 to identify antibody II; the 4 th tube was added with 10. Mu.L of mesenchymal stem cells to identify isotype control. Gently stirring and mixing, incubating for 15min at room temperature, and mixing once again in the dyeing process. 1ml of staining buffer was added to the 4-tube cells, and the mixture was blown and mixed at 1500rpm/5min. The supernatant was discarded, and cells were resuspended by adding 500. Mu.L of staining buffer per tube and detected on the machine.
2) Will 10 5 And (3) inoculating the mesenchymal stem cells of the well P3 into a 12-well plate, changing the complete culture medium of the DMEM-F12 into fat-forming or osteogenic induction liquid when the cell fusion degree is more than 80%, taking the cells cultured by the complete culture medium of the DMEM-F12 as a control, changing the liquid every two days, performing the induction culture of the fat-forming induction liquid for 3 weeks, and performing the induction culture of the osteogenic induction liquid for 4 weeks.
(1) Bone formation identification:
the culture was discarded, washed 3 times with PBS for 5min each, fixed 15min at 75% ethanol room temperature, and washed 3 times with ddH2O for 5min each. mu.L of 1% alizarin red-Tris-HCl staining solution is added, incubation is carried out at 37 ℃ for 30min, ddH2O is washed 3 times, and a proper amount of PBS is added for observing calcium salt deposition under a microscope.
(2) And (3) lipid formation identification:
the culture was discarded, washed 3 times with PBS, each time with 5min, fixed with 4% paraformaldehyde for 15min at room temperature, and rapidly washed 1 time with 70% isopropanol. Adding 300 mu L of oil red O dye solution filtered by qualitative filter paper, incubating for 20min at room temperature, rapidly washing with 70% isopropanol for 1 time after removing the oil red O dye solution, washing with PBS for 3 times, and observing lipid drops under a proper amount of PBS.
Example 3 preparation and identification of mesenchymal Stem cell-derived exosomes pretreated with Low concentration of inflammatory factors
1) Preparation
(1) P6 mesenchymal stem cells were inoculated into 15cm dishes and cultured in DMEM-F12 complete medium containing 1% PS and 10% fetal bovine serum. After 24 hours, the medium was replaced with serum-free DMEM-F12 and the PS was doubled, and low concentrations of IFN-gamma and TNF-alpha were added to stimulate mesenchymal stem cells, at a dose of 1-5ng/ml, respectively.
(2) The grouping is as follows: a: unstimulated mesenchymal stem cells (Native-MSCs), b: IFN-gamma stimulates mesenchymal stem cells (IFN-gamma-MSCs), c: TNF- α stimulates mesenchymal stem cells (TNF- α -MSCs), d: IFN-gamma and TNF-alpha combine to stimulate mesenchymal stem cells (IFN-gamma/TNF-alpha-MSCs). After 24 hours, cell culture supernatants of each group were obtained. The obtained culture supernatant can be stored at-80deg.C or 4deg.C for 20min by centrifugation at 3000g, and the precipitate is discarded.
(3) Extracting exosomes according to the following steps:
the supernatant was transferred to a fresh super-isolation tube and centrifuged at 10000g for 45min at 4 ℃.
The pellet was discarded and the supernatant was transferred to a fresh super tube and centrifuged at 120000g for 2h (X2) at 4 ℃.
Discarding supernatant, precipitating at bottom to obtain exosome, dissolving with PBS, sucking liquid, packaging into sterile EP tube, and preserving at-80deg.C to avoid repeated freezing and thawing.
The grouping is as follows: a: exosomes of Native-MSCs origin (Native-exos), b: exosomes derived from IFN- γ -MSCs (IFN- γ -exos), c: exosomes of TNF- α -MSCs origin (TNF- α -exos), d: IFN-gamma/TNF-alpha-MSCs derived exosomes (IFN-gamma/TNF-alpha-exos).
2) Electron microscope identification
10 mu L of exosomes derived from mesenchymal stem cells are placed on a copper mesh and settled for 2min. PBS was washed 2 times for 2min each. 3% glutaraldehyde was fixed for 1min.2% (w/v) sodium phosphotungstate for 1min. And (5) observing under an electron microscope.
3) Western blot identification surface marker
(1) Sample preparation:
mu.L of mesenchymal stem cell-derived exosomes were aspirated, 20. Mu.L of pre-chilled protein lysate (RIPA: PMSF=100:1) was added, lysed on ice for 30min, vortexed 1 time every 5min, 12000rpm/20min.
The supernatant was discarded and the protein concentration was measured according to BCA protein kit procedure.
Mixing the protein with SDS protein loading buffer solution and PBS according to a certain proportion, boiling at 100 ℃ for 5min to denature the protein, and directly carrying out the next experiment or preserving at-20 ℃.
(2) And (3) glue preparation:
10% split gum and 5% concentrated gum were prepared according to the following table.
(3) Electrophoresis:
placing the gel plate into an electrophoresis tank, adding an appropriate amount of electrophoresis liquid, adding a sample and a marker, and performing electrophoresis by parameters according to the procedure of separating gel 80V/30min and concentrating gel 120V/90 min. The time of electrophoresis can be adjusted according to the bromophenol blue indicator and the molecular weight of the protein.
(4) Transferring:
cutting gel and PVDF membrane after electrophoresis, and placing the membrane in methanol for activation for 15sec; assembling according to blackboard-sponge-filter paper-gel-PVDF film-filter paper-sponge-red board, placing into a film transfer tank filled with film transfer liquid for film transfer, and setting parameters according to 120V/90 min.
(5) Blocking, incubating the primary antibody and the secondary antibody:
after the film transfer is finished, placing the PVDF film into 5% of skimmed milk powder, and sealing for 1h on a shaking table; adding a primary antibody prepared by TBST, and incubating overnight at 4 ℃; TBST is washed for 3 times, each time for 5min; adding a secondary antibody prepared by TBST, and incubating for 1h on a shaking table; TBST was washed 3 times for 5min each.
(6) Color development:
according to reagent A: ECL color development liquid was prepared at a ratio of reagent b=1:1, images were collected using ECL color development system, and experimental results were analyzed using Image J software.
EXAMPLE 4 identification of PD-L1 expression in mesenchymal Stem cells pretreated with Low concentration of inflammatory factors and mesenchymal Stem cell-derived exosomes
1) qPCR detection of PD-L1 expression in mesenchymal Stem cells
(1) Extraction of RNA
After the mesenchymal stem cells of each group, stimulated or not, were digested with pancreatin, they were centrifuged at 1500rpm for 5min. The supernatant was discarded and resuspended in PBS at 1500rpm/5min (×2). The supernatant was discarded, an appropriate amount of Trizol was added to each tube, cell pellet was repeatedly blown off, and the cell suspension was transferred to a new RNase-free 1.5ml EP tube and allowed to stand on ice for 30min to lyse the cells sufficiently. Chloroform with the volume of 0.2 times of Trizol is added into each tube, and after the chloroform is fully and evenly mixed for 15 seconds, the mixture is kept stand for 3 minutes at the temperature of 4 ℃ and at the speed of 12000rpm/15 minutes. The clear and transparent liquid on the upper layer was transferred to a new EP tube, and an equal volume of isopropanol was added to each tube, and after mixing upside down, the mixture was allowed to stand at room temperature for 10min,4℃and 12000rpm/20min. The supernatant was discarded, 1ml of pre-chilled 75% ethanol (absolute ethanol: DEPC water=3:1) was added, and the pellet was washed upside down, 4 ℃,7500rpm/5min. Removing supernatant, drying at room temperature, dissolving RNA with proper amount of DEPC water when precipitation is transparent (RNA is not easy to dissolve when drying is excessive), detecting RNA concentration, and packaging and storing at-80deg.C.
(2) Reverse transcription (reverse transcription, RT) reaction (20. Mu.L System)
According toII 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit was prepared in liquid form to synthesize cDNA.
Removal of residual genomic DNA
The following mixtures were prepared in an RNase-free EP tube, gently stirred and mixed, and incubated at 42℃for 2min.
Preparation of reverse transcription reaction System
The reaction liquid of the step 1 is reactedII Supermix plus was gently blown and mixed.
Setting up a reverse transcription program
The cDNA can be used for qPCR reaction immediately or stored at-20deg.C after split charging, avoiding repeated freeze thawing to degrade.
(3) Real-time quantitative PCR (Real time quantitive transcriptase polymerase chain Reaction, RT-qPCR)
The cDNA synthesized as described above was diluted 10-fold (cDNA: ddH2O=1:10), according to the followingqPCR SYBR Green Master Mix (High Rox Plus) kit the reaction system was configured.
The required primers were synthesized by Shanghai Bioengineering Co., ltd, and the primers were designed as follows:
reaction procedure
(4) RT-qPCR data analysis
The results of RT-qPCR are expressed as Ct values, Δct=target gene Ct value-reference Ct value, ΔΔct=experimental group Δ -control group Δct, with the final result being 2- ΔΔct, with unstimulated mesenchymal stem cells as control group.
2) Western blot detection of expression of PD-L1 in mesenchymal stem cells and exosomes derived from mesenchymal stem cells
Mesenchymal stem cells were stimulated as in example 3 and culture supernatants were collected to extract mesenchymal stem cell-derived exosomes.
After the mesenchymal stem cells are digested by pancreatin, the cells are resuspended at 1500rpm/5min in PBS and then are lysed by adding an appropriate amount of protein lysate at 1500rpm/5min (x 2); mu.L of mesenchymal stem cell-derived exosomes were taken and lysed by adding 20. Mu.L of pre-chilled protein lysate.
The expression of PD-L1 in mesenchymal stem cells and in mesenchymal stem cell-derived exosomes was examined as in example 3.
The test results of examples 1-4 are shown in fig. 2-8 in sequence, and it can be found from fig. 2-8 that umbilical cord mesenchymal stem cells have a multidirectional differentiation capability under certain induction conditions; the method comprises the steps of carrying out a first treatment on the surface of the The surface markers of the mesenchymal stem cells are detected by a flow cytometer, as shown in fig. 2, and the results show that the expression of CD73 (99.24%), CD90 (98.09%), CD105 (99.33%) is positive, the expression of CD34 (0.07%), CD45 (0.16%), and HLA-DR (0.33%) is negative, and the obtained cells have the phenotype characteristics of the mesenchymal stem cells; as shown in FIG. 5, western Blot demonstrates that both Native-exo and TNF- α IFN- γ -exo express exosome markers CD9, CD63, TSG101.
Example 5 preparation and identification of mesenchymal Stem cell-derived exosomes pretreated with Soft stiffness and Low concentration of inflammatory factors in combination with Soft stiffness
1) Preparation
(1) Uniformly coating a round glass slide with the diameter of about 60mm and a large glass plate with the specification of 240mm multiplied by 120mm which are subjected to high pressure by using affinity silane and stripping silane in an ultra-clean workbench; the gels were arranged as in Table 1, 450. Mu.l of gel was dropped onto a large glass plate, immediately followed by gently reversing the circular slide over the gel (other slides adjusted for gel volume in terms of bottom area ratio); after about 3 minutes at room temperature, the gel is solidified, the gel is immediately placed into a water tank filled with alcohol, a slide glass with the gel is peeled off from a large glass plate by using a sterile syringe needle, the gel is placed upwards, and the slide glass is placed into a sterile culture dish containing Phosphate Buffer Solution (PBS) with 1% PS, and is stored in a refrigerator at 4 ℃; dropwise adding a cross-linking agent Sulfo-SANPAH into PBS in a light-shielding environment until the solution becomes pink; adding the prepared solution into the culture dish, slowly shaking the culture dish to uniformly coat the gel surface, and placing the culture dish under a high-intensity UV lamp (lambda=302-365 nm) for irradiation for 30min; removing cross-linking agent, adding proper amount of I-type rat tail collagen solution into PBS, adding the prepared solution into the culture dish, completely soaking gel, sealing the culture dish with sealing film, and refrigerating overnight at 4deg.C; culturing the mesenchymal stem cells at the rigidity for 24 hours;
TABLE 1 composition ratio of softer stiffness (0.1-10 kPa) Polyacrylamide gel
(2) The grouping is as follows: a: unstimulated mesenchymal stem cells (Native-MSCs), b: mesenchymal stem cells treated with 0.1-10kPa stiffness c: mesenchymal stem cells treated with a combination of IFN-gamma and TNF-alpha at a stiffness of 0.1-10kPa and 1-5 ng/ml. After 24 hours, cell culture supernatants of each group were obtained. The obtained culture supernatant can be stored at-80deg.C or 4deg.C for 20min by centrifugation at 3000g, and the precipitate is discarded.
Culture supernatants were collected and exosomes were extracted and subjected to electron microscopy and Western blot to identify surface markers according to the procedure of example 3, wherein the groupings were as follows:
a: exosomes of Native-MSCs origin (Native-exos), b: exosomes treated with 0.1-10kPa stiffness (Soft-exos), c: exosomes (Soft/IFN-gamma/TNF-alpha-exos) treated with a combination of IFN-gamma and TNF-alpha at a stiffness of 0.1-10kPa and 1-5 ng/ml.
Example 6 identification of PD-L1 expression in mesenchymal Stem cells and mesenchymal Stem cell-derived exosomes pretreated with Soft stiffness and Low concentration of inflammatory factors
The procedure of example 4 was followed to detect the expression of PD-L1 in mesenchymal stem cells by qPCR and to carry out Western blot to detect the expression of PD-L1 in mesenchymal stem cells and in mesenchymal stem cell-derived exosomes, wherein the same procedure as in example 5 was followed.
EXAMPLE 7 construction of a Diabetic Nephropathy (DN) mouse model and tail vein injection of TNF-alpha & IFN-gamma exo
Construction of DN mouse model
SPF-grade male BALB/c mice (6-8 weeks old, 18-22g weight) purchased from Peking Vitolith laboratory animal technology Co., ltd were used, kept in the Jilin university basic medical college animal center with an ambient humidity of 50-60%, an ambient temperature of 22-25℃and light was provided by light and dark cycle (12 h light/12 h dark), and all experimental procedures were approved by the China Jilin university basic medical college laboratory animal ethics committee.
(1) Adaptive feeding for 1 week (all mice were fed normally)
(2) STZ was dissolved in 0.1mol/L, ph=4.5 in citric acid buffer and DN mouse model was prepared by continuous 5d intraperitoneal injection of 60 mg/kg. Note that STZ solution was placed on ice in the dark and used within 30min after dissolution, and mice were fasted and not water-inhibited for 12 hours prior to injection.
(3) Blood glucose level is detected by sampling blood from the tail vein after the intraperitoneal injection of STZ 5d, and the diagnosis standard of diabetes is that the blood glucose level is more than or equal to 16.7mmol/L for three times.
Determination of DN mouse model
At week 12 of the experiment, the mouse was taken to urinate in the morning and the UACR value was determined, and the diagnostic criteria for diabetic nephropathy was UACR value > 30. Mu.g/mg.
Grouping
Mice were randomly divided into 4 groups:
(1) control group (NC group);
(2) diabetic nephropathy group (DN group);
(3) diabetic nephropathy + Exo intervention group (DN + Exo group);
(4) diabetic nephropathy + pretreatment-Exo intervention group (DN + TNF- α & IFN- γ -Exo group).
Tail vein injection of TNF-alpha & IFN-gamma-exo
On week 12 of the experiment, DN+Exo group Native-Exo (50. Mu.g/dose) was given by way of rat tail intravenous injection, DN+TNF-. Alpha. & IFN-. Gamma. -Exo group TNF-. Alpha.IFN-. Gamma. -Exo (50. Mu.g/dose), DN group and NC group were given equal amounts of PBS injection.
5) And after the experiment is finished at the 18 th week, collecting a sample and detecting various indexes.
Example 8 determination of mouse DN-related indicators
1) General physiological index determination
(1) Fasting blood glucose: the fasting blood glucose level was measured every 7 days after the DN molding was successful. The mice were fasted and not watered for 8 hours and were then blood-collected from tail veins, and the fasting blood glucose was measured using a glucometer.
(2) Weight of: body weight levels were measured using an electronic scale every 7 days during exosome treatment.
(3) Sugar tolerance experiments: after 7 days of the last exosome or PBS injection, each group of mice was fasted without water for 16h, 2g/Kg glucose was administered to perfuse the stomach, and blood glucose levels were measured by tail vein blood sampling for each group of mice at 0min, 15min, 30min, 60min, 90min, 120 min.
(4) After the mice were treated at week 18 of the experiment, the weights of the mice and the kidneys were measured using an electronic scale, and spleen factor (mg/g) =spleen mass (mg)/weight (g), and kidney factor (mg/g) =kidney mass (mg)/weight (g) were calculated.
2) Determination of the urine protein/urine creatinine ratio (UACR) of mice
The urine of the mice is reserved before the animals are treated at the 18 th week of the experiment, and the urine is stored on ice, and the urine is sent to a clinical laboratory in Leku-group area of a first hospital of Jilin university after being collected, and the urine protein/urine creatinine ratio (UACR) is measured.
3) Mouse kidney HE staining
(1) Kidney tissue was fixed with conventional 10% folimalin, paraffin embedded and then sectioned (2 μm);
(2) dewaxing: sequentially placing paraffin sections into xylene I (20 min), xylene II (20 min);
(3) hydration: sequentially placing the slices subjected to dewaxing treatment into 100% ethanol I (10 min), 100% ethanol II (10 min), 95% ethanol I (5 min), 95% ethanol II (5 min), and 80% ethanol (5 min) for gradient hydration, and then washing twice with distilled water;
(4) dyeing: immersing the hydrated slice in hematoxylin dye solution (5 min), washing twice with distilled water, differentiating in 1% hydrochloric acid alcohol for 5-10s, washing twice with distilled water, scrubbing with weak ammonia water to turn blue, washing twice with distilled water, observing the cell staining condition under a mirror, then placing in eosin dye solution for 5min, and washing twice with distilled water;
(5) Dehydrating: sequentially adding 80% ethanol (1 min), 95% ethanol I (1 min), 95% ethanol II (1 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), xylene I (10 min) and xylene II (10 min) into the dyed slice;
(6) sealing piece: after slicing and airing, sealing the slices by using neutral resin;
(7) and (3) observation: tissue sections were observed under different magnification light.
4) Mouse kidney immunohistochemistry
(1) Fixing and slicing: kidney tissue was fixed with conventional 10% folimalin, paraffin embedded and then sectioned (2 μm);
(2) dewaxing and hydrating: sequentially adding the slices into xylene I (30 min), xylene II (30 min), 100% alcohol I (10 min), 100% alcohol II (10 min), 95% alcohol I (5 min), 95% alcohol II (5 min) and 80% alcohol (5 min);
(3) washing: washing kidney tissue sections with tap water (1 min);
(4) antigen retrieval: placing kidney tissue slices in a wet box filled with citrate antigen retrieval liquid, heating in an autoclave for 15min, taking out, cooling with running water, cooling to room temperature, washing the slices with PBS for 2min, and repeatedly washing for 3 times;
(5) closing: the sections were soaked in PBS for 10min, then 3% H2O 2-methanol for 10min to eliminate the endogenous catalase effect, and washed 3 times with PBS for 2min each. Dripping the reagent A (10% goat serum sealing liquid) on the tissue to be detected to completely cover, incubating for 30min, and then sucking the liquid by using filter paper;
(6) Incubating primary antibodies: dripping about 100 mu l of corresponding primary antibody on the tissue to be detected to achieve complete coverage, washing the tissue to be detected with PBS for 3 times every 2min in the next day at the temperature of 4 ℃ in a wet box;
(7) incubating a secondary antibody: and (3) dropwise adding the reagent B (reinforcing agent) onto the tissue to be detected, so as to completely cover, incubating for 30 minutes, and washing with PBS for 3 times for 2 minutes each time. Reagent C (HRP polymer) was then added dropwise to the tissue to be tested and incubated for 30 minutes. Washing with PBS for 3 times each for 2min;
(8) color development: adding a drop of color development liquid in the kit into each slice, developing at room temperature for 2-3 minutes, observing the color development condition under a microscope, and immediately stopping the color development by tap water after the color development effect meets the requirement;
(9) counterstaining: counterstaining with hematoxylin dye solution for 5min, and washing with water for 1min;
differentiation of: differentiating kidney tissue slices with 1% hydrochloric acid alcohol solution for 1min, and washing with running water;
returning blue: returning blue with 0.5% ammonia water for 1min, and washing with running water;
dehydrating and drying with gradient alcohol, making xylene transparent, and sealing with neutral gum.
The results of the tests of examples 7-8 are shown in FIGS. 9-10, and from FIG. 9a, the fasting blood glucose levels were significantly elevated in the DN group compared to the NC group (P < 0.0001). After the exosome treatment at week 12, the dn+exo group (P < 0.01) and dn+tnf- α & IFN- γ -Exo group (P < 0.0001) gradually decreased fasting blood glucose compared to DN group. In addition, the degree of fasting blood glucose reduction was greater in the DN+TNF-. Alpha. & IFN-. Gamma. -Exo group than in the DN+Exo group (P < 0.05). As shown in fig. 9b, the body weight gain rate was significantly slowed in DN mice compared to NC group (P < 0.0001). After the start of exosome treatment at week 12, mice in the dn+exo group (P > 0.05) and dn+tnf- α & IFN- γ -Exo group (P < 0.01) began to gain in body weight compared to the DN group. The body weight increase rate of DN+TNF-. Alpha. & IFN-. Gamma. -Exo group was greater than that of DN+Exo group (P > 0.05), but was not statistically significant. As shown in fig. 9c, the increase in blood glucose in NC group mice over time showed a trend of increasing followed by decreasing until the normal blood glucose level was restored, and the increase in blood glucose in DN group mice over time also showed a trend of increasing followed by decreasing, but still higher than the normal blood glucose level after 120min (P < 0.0001). Compared with the DN group, the DN+Exo group (P > 0.05) and DN+TNF-alpha & IFN-gamma-Exo group (P < 0.01) show the trend that the blood sugar of mice increases with time and decreases after increasing, and the blood sugar of mice is lower than that of the DN group after 120min and still higher than that of normal mice. As shown in fig. 9d, DN mice showed reduced spleen factor (P < 0.001), dn+exo group (P < 0.05) and dn+tnf- α & IFN- γ -Exo group (P < 0.01) as compared to NC group mice, and the phenomenon of reduced spleen factor was alleviated. As shown in fig. 9e, DN mice showed an increase in kidney factor (P < 0.05) compared to NC mice, which may be associated with an increase in kidney volume of DN mice, while mice had a reduced body weight gain, whereas dn+exo (P > 0.05) and dn+tnf- α & IFN- γ -Exo (P > 0.05) could slightly reduce the increase in kidney factor of mice, but not statistically significant. As shown in fig. 10, UACR was significantly increased in DN group mice (P < 0.0001) compared to NC group, and UACR was significantly decreased in dn+exo group (P < 0.01) and dn+tnf- α & IFN- γ -Exo group (P < 0.0001) compared to DN group by MSC-Exo treatment.
Example 9 acquisition of splenocytes and detection of CD4+ T cells and their subtype ratios by flow cytometry
1) Acquisition of spleen cells
The spleen was taken under sterile conditions and placed in a plate containing 1% PS in PBS, the cell suspension was prepared by grinding the syringe tail, centrifuging at 1500rpm for 10min, discarding the supernatant, adding 8ml of red cell lysate, and lysing the red cells on ice for 8min. Filtering with 200 mesh sieve, centrifuging at 1500rpm for 5min, discarding supernatant, adding 1ml PBS, and placing on ice for subsequent use.
2) Flow cytometry to detect CD4+ T cell fraction
(1) After spleen cell counting, will contain 1X10 6 The suspension of splenocytes was collected in a 1.5ml EP tube, centrifuged at 4℃for 500g for 8min, the supernatant was aspirated, the cells were resuspended in 100. Mu.l PBS, 5. Mu.l CD3-FITC and 1.25. Mu.l CD4-APC corresponding to the surface staining antibodies were added to the staining tube, finally a tube of negative tube without any antibodies was left, vortexed, incubated for 30 min on ice at 4℃in the absence of light, centrifuged at 500g for 8min, and the supernatant was aspirated.
(2) 200 μl PBS was added to each tube and resuspended, ready for on-machine detection.
3) Flow cytometry to detect the proportion of Th1, th17 and Th2 cells in spleen cells
(1) PMA 50. Mu. Mol/L, monensin 1. Mu.l/1000. Mu.l, 1640 medium with 10% FBS, spleen cells were counted and 1X10 6 Each cell was resuspended in 500. Mu.l of 1640 medium containing 10% FBS and transferred to a 24-well plate, the formulated in vitro stimulator was added according to the standard, incubated at 37℃for 5h, after incubation was completed, the cell suspension in the 24-well plate was transferred to a centrifuge tube, 4℃at 1500rpm,
(2) counting stimulated spleen cells, and containing 1x10 6 The suspension of splenocytes was collected in a 1.5ml EP tube, centrifuged at 4℃for 500g for 8min, the supernatant was aspirated, the cells were resuspended in 100. Mu.l PBS, 1.25. Mu.l of the corresponding surface staining antibody (CD 4-APC) was added to the staining tube, finally a tube of negative without any antibody was left, vortexed and mixed well, incubated on ice for 30 min, centrifuged at 4℃for 500g for 8min, and the supernatant was aspirated.
(3) Fixing: 100 μl of 2% paraformaldehyde was added, and after 60min of fixation, the mixture was centrifuged at 600g for 10min at 4℃to discard the supernatant.
(4) Rupture of membranes: 1ml of 0.1% saponin is added; and (3) centrifuging: 400g,4℃and 5min, the supernatant is discarded and the procedure is repeated twice.
(5) 100 μl of 0.1% saponin was added, and intracellular staining antibodies (Th 1:5 μl IFN-. Gamma. -PE/Cy7, th17:0.5 μl IL17-FITC, th2:1.25 μl IL 4-PE) were added, vortexed, and incubated for 45min on ice in the absence of light. 1ml of 0.1% saponin was added, centrifuged at 600g for 10min at 4℃and the supernatant was discarded.
(6) 200 μl PBS was added to each tube and resuspended, ready for on-machine detection.
4) Flow cytometry detection of Treg cell proportion in spleen cells
(1) Cell counting was performed on spleen single cell suspensions, ensuring 1X 10 per staining tube 6 Centrifuging at 4deg.C for 500g and 8min, sucking supernatant, re-suspending cells with 100 μl PBS, adding 1.25 μl (CD 4-APC) and 3 μl (CD 25-FITC) of corresponding surface staining antibody into the staining tube, leaving a tube of negative tube without any antibody, mixing by vortex, and incubating in ice in dark place for 30minCentrifugation was performed at 4℃for 800g and 8min, and the supernatant was aspirated.
(2) 1ml of a Fixation/membrane-rupture working fluid (Fixation/Permeabilization Concentrate (4X)) was added:
fix/Permeabilization Diluent =1: 3, incubation on ice for 30min,4 ℃, centrifugation at 800g for 10min, and aspiration of supernatant.
(3) 1ml Permeabilization Buffer (1X) was added, incubated on ice for 15min,4 ℃, centrifuged at 800g for 10min, and the supernatant was aspirated.
(4) 100 μ l Permeabilization Buffer (1×) was added and the antibodies were intranucleally stained
Mu.l of Foxp3-PE were incubated on ice for 15min, 1ml Permeabilization Buffer (1X) was added, centrifuged at 4℃at 800g for 8min and the supernatant was aspirated.
(5) 200 μl PBS was added to each tube and resuspended, ready for on-machine detection.
The results of the test of example 9 are shown in FIGS. 11-15 in sequence, as shown in FIG. 11, for CD4 in spleen of DN group mice compared with NC group mice + T cell fraction was significantly increased (P < 0.0001), DN+Exo group (P < 0.0001) and DN+TNF-. Alpha.by MSC-Exo treatment&CD4 of IFN-gamma-Exo group (P < 0.0001) mice + T cell proportion is significantly reduced compared with DN group, wherein DN+TNF-alpha&The IFN-gamma-Exo group had a better reduction effect than the DN+Exo group (P < 0.01).
As shown in FIGS. 12-13, the proportion of Th1, th17 cells in the spleen of the DN group mice was significantly increased (P < 0.0001) compared to the NC group. Through MSC-Exo treatment, the proportion of Th1 cells of mice in DN+Exo group (P < 0.01) and DN+TNF-alpha & IFN-gamma-Exo group (P < 0.0001) is obviously reduced compared with that of the mice in DN group; the Th17 cell ratio of DN+Exo group (P < 0.0001) and DN+TNF-alpha & IFN-gamma-Exo group (P < 0.0001) mice is also obviously reduced compared with DN group. In addition, the reducing effect on Th1 cells is obviously better than that of DN+TNF-alpha & IFN-gamma-Exo group (P < 0.0001); for the reduction of Th17 cells, the DN+TNF-. Alpha. & IFN-. Gamma. -Exo group was slightly better than the DN+Exo group, but was not statistically significant (P > 0.05)
As shown in FIGS. 14-15, the proportion of Th2 and Treg cells in the spleen of mice in group DN was significantly reduced (P < 0.0001) compared to the NC group. Through MSC-Exo treatment, the proportion of Th2 cells of mice in DN+Exo group (P < 0.0001) and DN+TNF-alpha & IFN-gamma-Exo group (P < 0.0001) is obviously increased compared with DN group; the proportion of Treg cells in mice from the DN+ Exo group (P < 0.01) and DN+ TNF-. Alpha. & IFN-. Gamma. -Exo group (P < 0.0001) was also increased compared to the DN group. In addition, the increasing effect on Th2 cells was superior to that of DN+TNF-. Alpha. & IFN-. Gamma. -Exo group (P < 0.05); for the elevation of Treg cells, the dn+tnf- α & IFN- γ -Exo group was slightly better than the dn+exo group, but not statistically significant (P > 0.05).
Example 10 extraction of kidney tissue proteins and Western blot detection of inflammatory factor changes in kidney tissue
1) Extraction of kidney tissue proteins
(1) Pre-chilling the protein lysate on ice (RIPA: pmsf=100:1);
(2) taking out kidney tissue in a refrigerator at-80deg.C, cutting about 50mg of tissue, and shearing with ophthalmic scissors;
(3) to an EP tube (2 ml) was added 500. Mu.l of pre-chilled protein lysate and 50mg of sheared kidney tissue, and the mixture was homogenized using a portable homogenizer;
(4) homogenizing the tissue, placing on ice for 1 hour, and further crushing kidney tissue cells by using an ultrasonic cell disruption instrument;
(5) centrifuge at 4deg.C, 12000g/30min, centrifuging, and collecting supernatant, and placing into new EP tube;
(6) a small sample was taken for protein concentration determination and the remaining sample was stored in a-80 ℃ freezer for subsequent experiments.
2) Protein concentration measurement (BCA method)
(1) Preparing a standard protein solution: adding 800 μl of the preparation solution into protein standard substance, and dissolving thoroughly, and gradient diluting to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/ml;
(2) preparing BCA working solution: uniformly mixing the reagent A and the reagent B according to a ratio of 50:1 by vortex (according to the actual number of samples to be tested) to prepare BCA working solution, and preserving at room temperature for later use;
(3) Adding 20 μl of the mixed solution of the sample to be tested and PBS and the standard protein solution into a 96-well plate respectively, adding 200 μl of BCA working solution into each well, and incubating at 37deg.C in the absence of light for 30min;
(4) and setting the wavelength of the enzyme-labeled instrument to be 450nm, measuring the absorbance of each hole, drawing a standard curve by taking the protein concentration as an abscissa and the OD value as an ordinate, and calculating the protein concentration of each sample according to a standard curve equation.
3) Western blot detection
The same as in 3) of example 3)
As shown in FIG. 16, the results of the test in example 10 are shown in FIG. 16, in which IL-6 (P < 0.01), IL-1β (P < 0.01) and TNF- α (P < 0.05) expression in kidney of DN mice are significantly increased and IL-10 (P < 0.01) expression is significantly decreased as compared with NC mice. Compared with DN group, IL-6 (P < 0.05), IL-1β (P < 0.05), TNF- α (P < 0.01) expression is decreased in kidney of DN+TNF- α & IFN- γ -Exo group mice, and IL-10 (P < 0.001) expression is significantly increased. While the expression of IL-6, IL-1. Beta., TNF-. Alpha.and IL-10 was similarly altered in the kidney of the DN+Exo group mice, but without significant differences (P > 0.05). The effect of DN+TNF-alpha & IFN-gamma-Exo group is superior to DN+Exo group in regulating kidney inflammation injury of mice, has statistical significance on the regulation of IL-1 beta (P < 0.01) and IL-10 (P < 0.05), but has no significant difference (P > 0.05) on the regulation of IL-6 and TNF-alpha.
Example 11 acquisition of kidney cells and flow cytometry detection of changes in the different phenotypical proportions of macrophages
1) Acquisition of kidney cells
Taking kidney under aseptic condition, placing into a plate containing 3ml of 0.2% collagenase IV digestive juice, grinding with the tail of a syringe to prepare cell suspension, capping the aseptic plate, placing into a 37 ℃ incubator for incubation for 60min (shaking every 20 min), transferring the liquid into a new centrifuge tube, centrifuging at 1500rpm for 10min, discarding the supernatant, adding 8ml of erythrocyte lysate, and cracking on ice for 8min. Filtering with 200 mesh sieve, centrifuging at 1500rpm for 5min, discarding supernatant, adding 1ml PBS, and placing on ice for subsequent use.
2) Flow cytometry for detecting different phenotype proportion changes of macrophages
(1) After kidney cell counting, 1X 10 of each staining tube was ensured 6 Individual cells were centrifuged at 4℃for 500g for 8min, the supernatant was aspirated, and the cells were resuspended in 100. Mu.l PBS1.25. Mu.l of the corresponding surface staining antibody (F4/80-APC, CD11 b-PE) was added to the staining tube; 0.4 μl of CD86-FITC, finally a tube of negative tube without any antibody was left, vortexed, incubated on ice for 30 min at 4deg.C in the dark, centrifuged at 500g for 8min, and the supernatant was aspirated.
(2) Adding 100 μl of 2% paraformaldehyde, fixing for 60min, centrifuging at 4deg.C for 600g and 10min, and discarding supernatant;
(3) 1ml of 0.1% saponin is added; and (3) centrifuging: 400g, centrifuging at 4℃for 5min, discarding the supernatant, and repeating the procedure twice;
(4) 100 μl of 0.1% saponin was added for resuspension, 1.25 μl CD206-PE/Cy7 of intracellular staining antibody was added, vortexed and incubated on ice for 45min in the absence of light. 1ml of 0.1% saponin was added, centrifuged at 600g for 10min at 4℃and the supernatant was discarded.
(5) 200 μl PBS was added to each tube and resuspended, ready for on-machine detection.
The results of the test of example 11 are shown in FIG. 17, in which the proportion of monocytes in the spleen of the DN group mice is significantly increased (P < 0.0001) compared to the NC group, and the proportion of monocytes in the DN+Exo group (P < 0.0001) and DN+TNF-. Alpha. -IFN-. Gamma. -Exo group (P < 0.0001) mice are significantly decreased compared to the DN group by MSC-Exo treatment, wherein the decrease effect of DN+TNF-. Alpha. -IFN-. Gamma. -Exo group is slightly better than that of DN+Exo group but has no statistical significance (P > 0.05); compared with the NC group, the kidney of the DN group mice has obviously increased proportion of M1 type macrophages (P < 0.0001), and obviously decreased proportion of M2 type macrophages (P < 0.0001). By MSC-Exo treatment, the proportion of M1 type macrophages in mice of dn+exo group (P < 0.05) and dn+tnf- α & IFN- γ -Exo group (P < 0.05) was significantly reduced compared to DN group, but there was no significant difference between dn+tnf- α & IFN- γ -Exo group and dn+exo group, and no statistical significance (P > 0.05). The proportion of M2 type macrophages in mice of the DN+Exo group is not significantly increased compared with the DN group, and has no statistical significance (P > 0.05), whereas the proportion of M2 type macrophages in mice of the DN+TNF-alpha & IFN-gamma-Exo group (P < 0.0001) is significantly increased compared with the DN group. Compared with the NC group, the proportion of the macrophage M1/M2 in the kidney of the DN group is obviously increased (P is less than 0.0001), the proportion of the macrophage M1/M2 in the kidney of the DN+Exo group (P is less than 0.05) and the DN+TNF-alpha & IFN-gamma-Exo group (P is less than 0.0001) is obviously reduced compared with the DN group, and the effect of reducing the DN+TNF-alpha & IFN-gamma-Exo group is better than that of the DN+Exo group (P is less than 0.001).
In conclusion, a DN mouse model is established by intraperitoneal injection of STZ, and then the DN mouse is treated by tail vein injection of anti-inflammatory mesenchymal stem cell-derived exosome, so that the anti-inflammatory mesenchymal stem cell-derived exosome can effectively reduce blood sugar of the DN mouse model induced by STZ, improve sugar tolerance, improve systemic inflammatory response, simultaneously relieve local inflammatory response of kidneys, lighten infiltration of kidney inflammatory cells and inhibit secretion of partial inflammatory-related cytokines; by detecting the expression level of cytokines in kidney tissue homogenates of each group of mice, the expression level of the M1 type macrophage-related cytokines in the treatment group is reduced, and the expression level of the M2 type macrophage-related cytokines is increased; through flow detection of the kidney tissue single cell suspension, the fact that in the kidney of the mice treated by the anti-inflammatory mesenchymal stem cell-derived exosome, the proportion of M1 type macrophage markers is reduced, the proportion of M2 type macrophage markers is increased indicates that the immunoregulation effect of the anti-inflammatory mesenchymal stem cell-derived exosome on DN can be realized by regulating the phenotypic change of macrophages, and the effect of the anti-inflammatory mesenchymal stem cell-derived exosome pretreated by low-concentration inflammatory factors is more obvious.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (5)
1. A method for preparing an anti-inflammatory mesenchymal stem cell-derived exosome, comprising the steps of:
step one, mesenchymal stem cells are obtained: obtaining umbilical cord mesenchymal stem cells in healthy fetal umbilical cord tissue with consent;
step two, mesenchymal stem cell identification: performing phenotype identification, bone formation and lipid formation identification on the mesenchymal stem cells;
step three, subculturing: subculturing the identified mesenchymal stem cells to a sixth generation, and replacing a serum-free culture medium;
step four, preprocessing mesenchymal stem cells by adopting three methods: comprises (1) pretreatment of mesenchymal stem cells with low concentration of inflammatory factors (IFN-gamma, TNF-alpha, 1-5 ng/ml); (2) Soft stiffness (0.1-10 kPa) pretreatment of mesenchymal stem cells; (3) Low concentration inflammatory factor combined soft rigidity pretreatment mesenchymal stem cells;
Step five, obtaining supernatant of mesenchymal stem cell culture: obtaining supernatant of the mesenchymal stem cell culture after continuous culture for 24 hours;
step six, obtaining exosomes prepared by the mesenchymal stem cells: treating the obtained supernatant of the mesenchymal stem cell culture by adopting a three-step centrifugation method, wherein the obtained precipitate is the exosome;
and step seven, constructing a Diabetic Nephropathy (DN) mouse model, injecting exosomes by tail vein and measuring DN related indexes.
2. The method of preparing an anti-inflammatory mesenchymal stem cell-derived exosome according to claim 1, wherein: the specific effect of the fourth step is to increase the expression of immunosuppressive protein PD-L1 by the mesenchymal stem cells and enrich the exosomes with PD-L1 protein.
3. The method of preparing an anti-inflammatory mesenchymal stem cell-derived exosome according to claim 1, wherein: the soft rigidity in the fourth step is prepared by acrylamide with the concentration of 40% and bisacrylamide with the concentration of 2% at the mixing ratio of about 3:1, and after a proper amount of ammonium persulfate and tetramethyl ethylenediamine are added, the rest is supplemented by double distilled water; dripping the prepared gel on a large glass plate, and immediately reversely buckling a circular slide on the gel; immediately after solidification at room temperature, placing the glass slide in a water tank filled with alcohol, peeling off the glass slide by using a sterile syringe needle, and placing the gel face upwards in a sterile culture dish containing PBS (phosphate buffered saline) with 1% PS; dropwise adding a crosslinking agent Sulfo-SANPAH into PBS in a light-shielding environment; adding the solution into a culture medium dish, placing under a high-intensity UV lamp, irradiating for 30min, discarding the cross-linking agent, adding a proper amount of I-type rat tail collagen solution into PBS, adding the solution into the culture medium dish until the gel is completely soaked, and cooling overnight at 4 ℃; mesenchymal stem cells were cultured at this rigidity for 24h.
4. The method of preparing an anti-inflammatory mesenchymal stem cell-derived exosome according to claim 1, wherein: the three-step centrifugation method in the step six comprises the specific steps of centrifuging 3000g at 4 ℃ for 20min, and discarding the precipitate; transferring the supernatant to a new super-separation tube, and centrifuging at 4 ℃ for 45min with 10000 g; discarding the precipitate, transferring the supernatant to a new super-separation tube, and centrifuging at 120000g for 2h (X2) at 4 ℃; the supernatant was discarded, the bottom pellet was resuspended with an appropriate amount of PBS, and the aspirate was dispensed into sterile EP tubes.
5. The use of a method for the preparation of anti-inflammatory mesenchymal stem cell-derived exosomes according to any one of claims 1-4, characterized in that: the anti-inflammatory mesenchymal stem cell-derived exosomes obtained after pretreatment in a specific mode have remarkable immunosuppressive effect, can regulate the transformation of macrophage phenotype of a diabetic mouse, reduce the inflammatory infiltration degree of kidneys, and can be prepared into medicaments for treating diabetic nephropathy.
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| CN117257975A (en) * | 2023-11-22 | 2023-12-22 | 四川大学华西医院 | Multifunctional extracellular vesicle and preparation method and application thereof |
| CN117695309A (en) * | 2023-12-15 | 2024-03-15 | 东莞市第八人民医院（东莞市儿童医院） | Application of umbilical cord mesenchymal stem cell exosome in preparation of medicines for treating inflammatory diseases related to macrophage inflammatory activation |
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| CN117257975A (en) * | 2023-11-22 | 2023-12-22 | 四川大学华西医院 | Multifunctional extracellular vesicle and preparation method and application thereof |
| CN117257975B (en) * | 2023-11-22 | 2024-03-19 | 四川大学华西医院 | Multifunctional extracellular vesicle and preparation method and application thereof |
| CN117695309A (en) * | 2023-12-15 | 2024-03-15 | 东莞市第八人民医院（东莞市儿童医院） | Application of umbilical cord mesenchymal stem cell exosome in preparation of medicines for treating inflammatory diseases related to macrophage inflammatory activation |
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