CN111643523B

CN111643523B - New application of PD-L1 exosome

Info

Publication number: CN111643523B
Application number: CN201911205272.4A
Authority: CN
Inventors: 程芳; 刘晓燕; 陈红波; 苏丹丹; 蔡湘仪; 徐占雪; 颜福霞; 杨欣蕊; 贺超; 伍颖艺; 肖有梅; 颜海兰; 查华联; 杨敏
Original assignee: Sun Yat Sen University Shenzhen Campus
Current assignee: Sun Yat Sen University Shenzhen Campus
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2023-09-01
Anticipated expiration: 2039-11-29
Also published as: CN111643523A

Abstract

The invention discloses Sub>A new application of PD-L1 exosomes, which is used for researching the new effect of the PD-L1 exosomes on the cellular level and the animal level, and experiments show that the PD-L1 exosomes are applied to Sub>A novel thermosensitive gel for damaging the skin surface, so that the positive T cell activity is reduced, the IL-6, TNF-Sub>A secretion is reduced, the expression of TGF-betSub>A, VEGF-A and other growth factors is increased, the wound contraction and wound re-epithelialization are obviously promoted, the wound healing is accelerated, the PD-L1 exosomes can be used for preparing Sub>A preparation for treating chronic ulcer and/or inflammatory diseases by inhibiting the excessive immune response of T cell activation (including proliferation and growth factor release) to damaged tissues in Peripheral Blood Mononuclear Cells (PBMC), and the preparation has Sub>A great application prospect.

Description

New application of PD-L1 exosome

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of PD-L1 exosomes in preparation of preparations for treating chronic ulcers and inflammatory diseases.

Background

Wound healing is a complex process that requires multiple cells to participate and exert different effects, and can be divided into a blood stage, an inflammatory stage, a proliferation stage and a tissue remodeling stage according to time division. Mainly depends on the natural regeneration process of the epidermal tissue. In many cases, the progress of the regeneration that it involves is insufficient to rescue severely traumatized patients. During the first few days after the wound, inflammatory cells and immune cells accumulate from the vicinity or circulation to the wound site through complement, coagulation molecules and cytokines to clear the wound of cellular debris and bacteria. Excessive and persistent inflammation inhibits healing of damaged tissue, and thus immune cells that inhibit overactivity of damaged tissue are effective methods of treating chronic ulcers and inflammatory diseases.

Chen G et al (2018) (Chen G, huang AC, zhang W, et al Exosomal PD-L1contributes to immunosuppression and is associated with anti-PD-1response.Nature.2018Aug 8.doi:10.1038/s 41586-018-0392-8.) found that melanoma cells secreted PD-L1 positive exosomes into circulating blood, in combination with CD8 ⁺ PD-1 on the surface of T cells can inhibit proliferation and cytotoxic ability of the T cells, weaken anti-tumor immunity of the organism and promote tumor growth, and the PD-1 antibody therapy can inhibit the PD-L1/PD-1 binding mode. Circulating exosome PD-L1 levels are positively correlated with IFN-gamma levels and in patients who respond well to antibodies during PD-1 antibody therapy, there is a significant upregulation of exosome PD-L1 levels, possibly in tumor cells responding to T cell "resuscitationThe feedback mechanism can be used for clinically predicting clinical prognosis and distinguishing patients with good antibody treatment response so as to guide clinical medication.

However, at present, no report is made about the use of exosome PD-L1 in chronic ulcers and inflammatory diseases.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings in the prior art and provide the application of exosome PD-L1 in preparing a preparation for treating chronic ulcer and inflammatory diseases.

Another object of the present invention is to provide a thermosensitive gel with exosome PD-L1 embedded therein.

The above object of the present invention is achieved by the following technical solutions:

the invention discovers at the cellular level and the animal experiment level that the PD-L1 exosome can reduce the activity of the T cells marked by CD4, CD8 and CD3 and inhibit the maturation and proliferation of peripheral blood mononuclear cells, thereby regulating and controlling the immune response of organisms, inhibiting the excessive immune response of organisms to damaged tissues and further playing a good role in treating chronic ulcers and inflammatory diseases.

The invention therefore first provides the following new uses of PD-L1 exosomes:

use of PD-L1 exosomes for the preparation of a formulation for the treatment of chronic ulcers and/or inflammatory diseases.

Use of PD-L1 exosomes for the preparation of a formulation for the treatment of chronic ulcers and/or inflammatory diseases due to hyperimmunization.

Use of PD-L1 exosomes for the preparation of a formulation that promotes tissue repair and regeneration by inhibiting immune and inflammatory responses, increasing growth factor expression.

Use of PD-L1 exosomes in the preparation of a medicament for promoting wound healing.

Use of PD-L1 exosomes in the preparation of immunosuppressants.

Use of PD-L1 exosomes in the preparation of a formulation for inhibiting T cell activity.

Application of PD-L1 exosomes in preparation of preparations for inhibiting proliferation and maturation of peripheral blood mononuclear cells.

Use of PD-L1 exosomes in the preparation of a formulation that promotes expression of a cell growth factor. The cell growth factors are alphSub>A-SMA, TGF-betSub>A, VEGF-Sub>A, etc., to promote tissue repair and regeneration, for example: promoting skin repair and wound healing.

The invention also provides a preparation, which comprises the PD-L1 exosome and pharmaceutically acceptable auxiliary materials thereof.

Preferably, the formulation is a transdermal formulation. Such as solutions, powders, lotions, tinctures, ointments, oils, creams, gels, aerosols, sprays, and the like. Wound healing can be promoted by applying, spraying or spraying the above preparation on the wound.

Preferably, the formulation is a temperature sensitive gel.

The invention provides a thermosensitive gel embedded with PD-L1 exosomes, which is prepared by incubating the PD-L1 exosomes and thermosensitive hydrogel at 35-38 ℃ for 10-20 min and wrapping the exosomes in the hydrogel.

Preferably, the hydrogel is a 20% PF-127 temperature sensitive gel.

Specifically, the invention is to gel and keep PD-L1 exosomes covering the whole wound under the body temperature condition by using a temperature sensitive gel embedded with PD-L1 exosomes in a mouse skin injury model. PD-L1 exosomes were found to decrease T cell activity for CD4, CD8 and CD3 markers. Under the condition that PD-L1 exosomes exist, the expression of the growth factors is increased, and wound surface shrinkage and wound surface re-epithelialization are obviously promoted. The result shows that the PD-L1 exosome can be applied to a novel thermosensitive gel for damaging the skin surface to accelerate wound healing, and a novel visual angle is provided for promoting tissue repair and regeneration by using immunotherapy.

Therefore, the following new uses of the PD-L1 exosome-embedded thermosensitive gel are also within the scope of the invention:

use of a PD-L1-embedded exosome temperature-sensitive gel for the preparation of a formulation for the treatment of chronic ulcers and/or inflammatory diseases.

Use of a PD-L1-embedded exosome temperature-sensitive gel for the preparation of a formulation for the treatment of chronic ulcers and/or inflammatory diseases due to hyperimmunization.

Use of a PD-L1-embedded exosome temperature-sensitive gel in the manufacture of a medicament for promoting skin repair/wound healing.

Preferably, the PD-L1 exosomes of the invention are obtained from melanoma cells stimulated by IFN-gamma or melanoma cells stably expressing PD-L1 after infection, and the PD-L1 protein content in the exosomes obtained by the method is higher.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a new application of PD-L1 exosomes, and the invention researches the new effects of the PD-L1 exosomes on the cellular level and the animal experiment level. The result shows that the PD-L1 exosome is applied to novel temperature-sensitive gel for damaging the skin surface, so that the activity of T cells marked by CD4, CD8 and CD3 is reduced, the expression of growth factors such as TGF-betSub>A, VEGF-A and the like is increased, the wound surface shrinkage and wound surface re-epithelialization are obviously promoted, and the wound healing is accelerated. The thermosensitive gel embedded with the PD-L1 exosome can be used for preparing a preparation for inhibiting proliferation and maturation of peripheral blood mononuclear cells, inhibiting T cell activity or inhibiting excessive immune response to damaged tissues so as to treat chronic ulcers and inflammatory diseases, and has a wide application prospect; a new perspective is also provided for promoting tissue repair and regeneration using immunotherapy.

Drawings

FIG. 1A is a graph showing the detection of PD-L1mRNA levels in SK-MEL cells, IFN-gamma stimulated SK-MEL cells, and SK-MEL cells stably expressing PD-L1 after infection using qPCR techniques.

FIG. 1B is a graph showing the determination of exosome content from SK-MEL cells, IFN-gamma stimulated SK-MEL cells, and SK-MEL cells stably expressing PD-L1 after infection using protein quantification.

FIG. 1C shows the morphology of the exosomes extracted from SK-MEL cells, IFN- γ stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection, as measured using a transmission electron microscope, with a scale of 50nm.

FIG. 1D is a graph showing the particle size distribution of exosomes extracted from SK-MEL cells, IFN- γ stimulated SK-MEL cells, and SK-MEL cells stably expressing PD-L1 after infection.

FIG. 1E is the ZETA potential of exosomes extracted from SK-MEL cells, IFN- γ stimulated SK-MEL cells, and SK-MEL cells stably expressing PD-L1 after infection.

FIG. 1F shows the detection of expression of the exosomes, ALIX, CD63, CD81 and the reference GAPDH, extracted from SK-MEL cells, IFN- γ stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection by Western blot.

FIG. 2A shows, from left to right, PD-L1 exosomes (red) extracted from SK-MEL cells using laser confocal microscopy; SK-MEL cells stably expressing PD-L1 (red light) after cell membrane-infected green light localized infection; 293T (green light) stably expressing PD-1 ingests PD-L1 exosomes (red light); jurkat cells (green light) ingest PD-L1 exosomes (red light) in a scale of 10 μm.

FIG. 2B is a cell streak assay to examine the conditions of exosomes extracted from SK-MEL cells, IFN- γ stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection for promoting healing of HDF cells and HACAT cells, and comparing with negative control (serum-free medium) and positive control (bFGF).

FIG. 2C is a table showing the mobility of each group of cells after 24 hours in the cell scratch test.

FIG. 2D is a graph showing the effect of exosomes on peripheral blood mononuclear cell proliferation capacity. Exosomes, PD-L1 exosomes, IFN- γ exosomes, immunosuppressant tacrolimus FK506 mixed with peripheral blood mononuclear cells for 3 days; the Negative control group (Negative control) was peripheral blood mononuclear cells without exosomes, PD-L1 exosomes, IFN- γ exosomes, immunosuppressant tacrolimus FK506, incubated for 3 days; the Positive control (Positive control) was unincubated peripheral blood mononuclear cells. Detection was performed using a flow cytometer.

FIG. 3A is a feature of temperature sensitive gel PF-127. I is the quasi-solid state of the temperature sensitive gel PF-127 at room temperature, and II is the flowing state of the temperature sensitive gel PF-127 at 4 ℃.

FIG. 3B is a graph showing the rheological viscoelastic characteristics of 20% PF-127 as a function of temperature as measured by a rotational rheometer.

FIG. 3C is an internal pore size profile of a 20% PF-127 temperature sensitive hydrogel detected using a scanning electron microscope.

Fig. 3D shows uptake after detection of HACAT cells by laser confocal microscopy for 1,4,8,24 hours co-incubation with PD-L1 exosomes embedded in temperature sensitive hydrogels. Blue represents HACAT cells and green represents PD-L1 exosomes. The scale bar is 10 μm. FIG. 3E is a graph comparing relative fluorescence intensities of HACAT cells at various time points after uptake of exosomes.

Fig. 4A is a graph of wound healing in mice. Five groups, control group (ctrl), bFGF group, exosome group (EV), IFN-gamma exosome group (EV IFN-gamma), PD-L1 exosome group (EV PD-L1). Representative plots of wounds on day 0, day 3, day 7 and day 10 after molding were selected for the different groups.

Fig. 4B is a graph comparing the rate of change of wound area for different experimental groups compared to day 0.

FIG. 4C is a plot of skin HE after sampling on day 7 of the control group (ctrl), bFGF group, exosome group (EV), IFN-gamma exosome group (EV IFN-gamma), PD-L1 exosome group (EV PD-L1) and an IHC plot examining Ki 67.

FIG. 4D shows the extraction of CD8+ and CD8+ cell content in spleen after sampling on day 7 using a flow analyser for the control group (ctrl), bFGF group, exosome group (EV), IFN-gamma exosome group (EV IFN-gamma), PD-L1 exosome group (EV PD-L1).

FIG. 4E shows mRNA levels of mmp9, α -SMA, TGF- β, IL-6, TNF-a, grazyme B in skin after sampling on day 7 using qPCR for control (ctrl), bFGF, exosome (EV), IFN- γ exosome (EV IFN- γ), PD-L1 exosome (EV PD-L1).

FIG. 5 shows the experimental contents of the present invention.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1 PD-L1 exosome extraction and detection characterization

1. Total RNA in SK-MEL cells, SK-MEL cells stimulated by IFN-gamma and SK-MEL cells stably expressing PD-L1 after infection is extracted, and reverse transcription experiments are carried out by using PrimeScript RT-PCR Kit (TaKaRa) to obtain cDNA. The transcript level of PD-L1, i.e.the change in the mRNA level of PD-L1, was detected by fluorescence quantification. Wherein, the fluorescent quantitative PCR detection uses beta-actin as a reference gene;

the PCR primer sequence for detecting the beta-actin internal reference gene by fluorescent quantitative PCR is as follows:

forward primer F:5'-CTCCATCCTGGCCTCGCTGT-3';

reverse primer R:5'-GCTGTCACCTTCACCGTTCC-3';

the primers for detecting the transcription level of PD-L1 by fluorescent quantitative PCR are as follows:

forward primer F:5'-TCCACTCAATGCCTCAAT-3';

reverse primer R:5'-GAAGACCTCACAGACTCAA-3'.

The results of fluorescent quantitative PCR are shown in FIG. 1A, and the mRNA level of PD-L1 in SK-MEL cells stimulated with IFN-gamma and SK-MEL cells stably expressing PD-L1 after infection is 11.2 times and 54.4 times that of SK-MEL cells. The SK-MEL cells are shown to be capable of elevating the mRNA level of PD-L1 after IFN-gamma stimulation and infection with PD-L1.

2. Exosomes were extracted by ultracentrifugation. After the cells grow to about 70%, changing 0.5% of FBS culture medium without exosomes to culture for 48 hours, collecting the cell culture medium, centrifuging for 500xg,10min;2000xg,20min;10000Xg,40min, removing precipitate, centrifuging supernatant for 100000Xg,70min, collecting exosome precipitate, adding RIPA lysate, and measuring exosome protein concentration by BCA method. The exosome content extracted from SK-MEL cells, IFN-gamma stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection is not significantly different, every 10 ⁶ The total protein content of the exosomes that could be collected by each cell was around 5 μg, as shown in fig. 1B.

Dripping exosomes extracted from SK-MEL cells, SK-MEL cells stimulated by IFN-gamma and SK-MEL cells stably expressing PD-L1 after infection on a copper mesh, performing negative dyeing by uranium acetate, washing the copper mesh twice, airing at room temperature, and detecting morphological characteristics of the exosomes by using a transmission electron microscope; the result is shown in fig. 1C, and the extracted exosomes exhibit a circular bilayer membrane structure.

The particle size distribution and ZETA potential of the exosomes were measured using a dynamic light scattering instrument, and as shown in FIGS. 1D and 1E, the particle size of the exosomes extracted from SK-MEL cells, IFN-gamma stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection was about 100nm, and the ZETA potential was about-27 mV, with no significant difference.

The exosomes were added to 5X loading buffer,100 ℃ and cooked for 20min, then loaded with the same total amount of protein in a 10% sds-PAGE gel, electrophoresed 80mv,2h, blotted with PEVF membrane, 330ma,90min, blocked with blocking solution for 1h, and added with primary antibodies ALIX, CD63, PD-L1, CD81 and GAPDH in dilution ratios according to the description, and gently shaken overnight at 4 ℃. After incubation with the corresponding secondary antibody for 2h, chemiluminescent liquid was added to the membrane and exposed on a chemiluminescent instrument. As a result, as shown in fig. 1F, specific proteins ALIX, CD63, CD81 of exosomes could be detected while the levels of PD-L1 protein in IFN- γ stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection and exosomes were higher compared to SK-MEL cells and exosomes secreted thereby, demonstrating that over-expression of PD-L1 by IFN- γ stimulation and infection could increase the levels of PD-L1 protein in cells and exosomes secreted by cells.

Example 2 functional assay of PD-L1 exosomes at the cellular level

Exosomes suspended in PBS were stained with dye CY5.5, excess dye was removed by centrifugation through a 50KD ultrafiltration tube, and slides were mounted on a cell slide, followed by exosome addition. And (3) carrying out slice throwing by using a slice throwing machine, enabling cells to be attached to a glass slide at 1500Xg for 15min, taking out, sucking redundant liquid by using filter paper, sealing the slice, and carrying out photographing observation under a laser confocal microscope. The SK-MEL cells are infected by the PD-L1 plasmid with OFP, the SK-MEL cells which stably express PD-L1 are screened, the cell membrane is dyed by using a dye WGA488, after 15min, the cells are fixed by 4% paraformaldehyde and then are subjected to sealing, and photographing observation is carried out under a laser confocal microscope, so that the SK-MEL cells which express PD-L1 are proved to be successfully prepared. The 293T cells are infected by PD-1 plasmid with GFP, 293T cells which stably express PD-1 are screened, exosomes which are stained with CY5.5 are added into the cells for co-incubation for 15min, the cells are fixed by 4% paraformaldehyde and then are subjected to sealing, and shooting is carried out under a laser confocal microscope to observe the uptake, so that the PD-L1 exosomes can be combined with 293T cells which express high PD-1 levels. The cell membrane of Jurkat cells was stained with dye WGA488 for 15min, centrifuged, excess dye was washed off with PBS, and after adding the exosomes stained with CY5.5 into the cells for co-incubation for 15min, the cells were spun off with a disk spinning machine for 1500Xg,15min, allowed to adhere to the slide glass, removed, excess liquid was aspirated with filter paper, sealed, and photographed under a laser confocal microscope, confirming that PD-L1 exosomes were able to bind to PD-1 expressing T cells. The above results are shown in FIG. 2A.

Human fibroblast HDF cells and human immortalized epidermal cell HACAT cells are spread in a 24-well plate until the growth is about 60%, a 200 mu L gun head is used for vertically scratching the gun head compared with a ruler, the gun head is washed three times by PBS, the scratched cells are removed, and a serum-free culture medium and a culture medium (with a large amount of immune factors) for culturing PBMC cells are added to simulate an immune environment. Exosomes (100 ug/ml) extracted from SK-MEL cells, IFN-gamma stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection were then added to the experimental group as positive controls, bFGF (2.5 ng/ml) was photographed under a microscope after 24 hours to observe cell healing, as shown in FIG. 2B. Cell migration rate = (0 h scratch area-24 scratch area)/0 h scratch area x 100% was calculated. As shown in fig. 2C, PD-L1 exosomes were able to effectively inhibit the effect of immune factors on cell healing.

To confirm that PD-L1 exosomes were able to inhibit proliferation of immune cells, CFSE proliferation experiments of PBM cells were performed. Placing healthy human blood into an anticoagulation centrifuge tube containing EDTA; the Ficoll separating liquid is centrifuged to extract peripheral blood mononuclear cell PBMC cells, and finally R10 culture medium (RPMI 1640, 10% FBS, L-glutamine, penicillin/streptomycin) is added to break up the cells and count the cell number. CFSE staining solution (5 μm) was added to PBMC cells, incubated at 37 ℃ for 20min in the dark, medium termination marker of 10% fbs was added, and after centrifugation washing, medium was added. The negative control group was not treated at all, and different types of exosomes (100 ug/ml) and the immunosuppressant tacrolimus FK506 (100 nM) were added to the cells as experimental groups, and the negative and experimental groups were incubated for three days. PBMC cells not incubated with exosomes and FK506 after staining with CFSE dye were immediately flow tested as positive control. CFSE fluorescence intensities in PBMC cells were measured using a flow cytometer, and the results are shown in fig. 2D, in which the ordinate represents fluorescence signal intensity, and the abscissa represents the number of cells with CFSE fluorescence signals, and the more the peak is to the right, the more the number of cells with CFSE fluorescence signals, the stronger the inhibition of PBMC cell proliferation. As can be seen from the figure, PD-L1 exosomes were able to inhibit proliferation of PBMC cells.

Example 3 preparation and characterization of temperature-sensitive gels with embedded PD-L1 exosomes

PF-127 (Sigma-Aldrich) powder was weighed, dissolved in water, stirred at 4℃for 1h to prepare a 20% temperature-sensitive hydrogel, which was able to be in a gel state (quasi-solid state) at room temperature and was able to be in a fluid state at 4℃as shown in FIG. 3A, the hydrogel in I was already coagulated and unable to flow at room temperature, and II was the hydrogel just removed from the 4℃refrigerator and still had fluid properties.

The viscosity and elasticity of 20% PF-127 temperature sensitive hydrogel at a temperature ranging from 0 to 40℃were measured using a rotarheometer. The temperature rise rate was 5 ℃/min, the strain amplitude was 1% and the frequency was 0.159Hz. As a result, as shown in FIG. 3B, when the temperature is lower than about 17 ℃, G '(elastic modulus) < G' (viscous modulus), the system is similar to a viscous liquid, and when the temperature is higher than 17 ℃, G '> G', the system is similar to a solid-like colloid.

Pre-cooling 20% PF-127 temperature sensitive hydrogel in a refrigerator at-80 ℃ for 12 hours, placing in a freeze drying box for 15 hours, taking out, placing at normal temperature, and observing the inner aperture of the hydrogel by using a scanning electron microscope, wherein the hydrogel has a porous structure as shown in figure 3C, so that the hydrogel has the function of coating an exosome.

Cell climbing sheets were placed in 12-well plates, HACAT cells were spread, and exosome uptake experiments were performed when the cells grew to 40%. Exosomes were stained with WGA488 dye, after centrifugation of the excess dye with a 50KD ultrafiltration tube, incubated with 20% pf-127 temperature sensitive hydrogel for 15min at 37 ℃, exosomes were wrapped in hydrogel, then the exosomes-embedded hydrogel was added to HACAT with the medium removed, after gelation of the hydrogel at the cell layer for 15min, an equal amount of DMEM medium was added, HACAT cells and exosomes were incubated for 1h,4h,8h,24h, HACAT cells were fixed with 4% paraformaldehyde, nuclei were stained with DAPI (0.5 μg/ml), and then blocked, and observed by photographing with a laser confocal microscope, as shown in fig. 3D. Comparing the relative fluorescence intensities of the cells and exosomes incubated for different times, as shown in fig. 3E, shows that the hydrogel is able to retain the activity of the exosomes and release the exosomes slowly, and that the relative fluorescence intensity is maximal at 24h as time increases, indicating the maximum exosome release.

Example 4 in vivo experiments verify that temperature-sensitive hydrogels embedded in PD-L1 exosomes are capable of promoting skin repair

(1) Establishment of mouse skin incised wound model

Laboratory was kept in a sterile environment with BALB/c mice of 6-8 weeks old purchased from laboratory animal centers at the east school of university in Zhongshan, guangdong province. BALB/c mice were anesthetized with 10% sodium pentobarbital, dehaired with dehairing paste, skin on the back of the mice was sterilized with 75% ethanol, a circle with a diameter of 10mm was drawn on the ridge line in the back of the mice, and skin was cut down along the circle with surgical scissors.

(2) Experimental grouping and administration method

Mice were randomly divided into 5 groups of 6 mice each. The wound is coated with 20% PF-127 temperature-sensitive hydrogel as a control group, the wound is coated with 20% PF-127 temperature-sensitive hydrogel coated with bFGF (0.5 ng/. Mu.L) as a bFGF group, and the wound is coated with 20% PF-127 temperature-sensitive hydrogel coated with an exosome (1. Mu.g/. Mu.L) extracted from SK-MEL cells, IFN-gamma-stimulated SK-MEL cells and SK-MEL cells stably expressing PD-L1 after infection as an EV group, an EV IFN-gamma group and an EV PD-L1 group. The administration was started from day 3 to day 7, 50 μl of hydrogel was applied to the wound each time, and samples were taken on day 7. The body weight of the mice is weighed every day, the wounds of the mice are photographed and observed, as shown in fig. 4A, the change condition of the wound area of each group of mice is compared, as shown in fig. 4B, the EV IFN-gamma group and the EV PD-L1 group can obviously promote wound shrinkage and wound re-epithelialization, and wound healing is accelerated.

(3)CD4 ⁺ And CD8 ⁺ Cell ratio analysis

On the seventh day, the cervical vertebrae sacrificial mice are rapidly removed, and the mice are soaked in 75% ethanol for sterilization (2-3 min). Then opening the abdominal cavity in an ultra-clean workbench by aseptic operation, taking out the spleen, removing the surrounding adipose tissues, washing for 1-2 times by using PBS solution, and transferring into an aseptic culture dish containing precooled PBS solution; placing spleen on 0.75um sieve, shearing with scissors; pouring PBS solution for cleaning to obtain spleen cell suspension; the cell suspension was centrifuged at 1500rpm at 4℃for 5 min; subsequently, the supernatant was discarded, and 2mL of lysis buffer (Acklysis buffer) was added to the ice to lyse the erythrocytes for 3 to 5min, and the mixture was centrifuged at 4℃for 5min at 1500rpm to remove the supernatant. The sample was centrifuged twice with PBS, the supernatant was removed, and 100. Mu.L of the cell stain was added, and 1. Mu.L of PE-CD4 antibody, 1. Mu.L of APC-CD8 antibody, and 1. Mu.L of FITC-CD3 antibody were added to the sample to be tested, respectively. After mixing, placing in ice and incubating for 15min in dark place, centrifuging for 5min at 1500rpm; the supernatant was then discarded, 200. Mu.L of fixative was added, incubated at room temperature for 20min, centrifuged for 5min at 1500rpm, the supernatant was subsequently discarded, and 300. Mu.L of PBS was added for resuspension, and CD4 was subjected to flow cytometry ⁺ And CD8 ⁺ Cell number was measured. As shown in fig. 4D, the numbers of cd4+ and cd8+ cells in the spleen of mice treated with the hydrogel containing PD-L1 exosomes were relatively small after treatment compared to mice in the control group, indicating that PD-L1 exosomes have an inhibitory effect on T cells in the mice.

(4) Histopathological examination

During sampling, the injured skin of the mice is cut off, half of the skin is soaked in 4% paraformaldehyde for fixation, paraffin embedding, slicing, HE staining and IHC is carried out for pathological section observation. The results are shown in FIG. 4C, EV IFN-gamma group, EV PD-L1 group, were effective in reducing inflammatory response at skin, promoting wound repair.

(5) Detecting inflammatory and growth factor levels in skin

RNA was extracted from the other half of the skin and mRNA levels of mmp9, α -SMA, TGF- β, IL-6, TNF-a, grazyme B were measured for each group.

Wherein, the fluorescent quantitative PCR detection uses beta-actin as a reference gene;

forward primer F:5'-AACAGTCCGCCTAGAAGCAC-3';

reverse primer R:5'-CGTTGACATCCGTAAAGACC-3';

the primers for detecting the transcription level of IL-6 by fluorescent quantitative PCR are as follows:

forward primer F:5'-CCTCTGGTCTTCTGGAGTACC-3';

reverse primer R:5'-ACTCCTTCTGTGACTCCAGC-3'.

The primers for detecting the transcription level of TNF-a by fluorescent quantitative PCR are:

forward primer F:5'-CATCCTTGCGAGTGTCAGTGA-3';

reverse primer R:5'-CCCTCACACTCAGATCATCTTCT-3'.

The primers for detecting the transcription level of Granzyme B by fluorescent quantitative PCR are as follows:

forward primer F:5'-TCTCGACCCTACATGGCCTTA-3';

reverse primer R:5'-TCCTGTTCTTTGATGTTGTGGG-3'.

The primers for fluorescent quantitative PCR detection of the transcription level of MMP9 are:

forward primer F:5'-GCAGAGGCATACTTGTACCG-3';

reverse primer R:5'-TGATGTTATGATGGTCCCACTTG-3'.

The primers for detecting the transcription level of TGF-beta by fluorescent quantitative PCR are as follows:

forward primer F:5'-CCACCTGCAAGACCATCGAC-3';

reverse primer R:5'-CTGGCGAGCCTTAGTTTGGAC-3'.

The primers for detecting the transcription level of alpha-SMA by fluorescent quantitative PCR are as follows:

forward primer F:5'-CCCAGACATCAGGGAGTAATGG-3';

reverse primer R:5'-TCTATCGGATACTTCAGCGTCA-3'.

The results of fluorescence quantitative PCR are shown in FIG. 4E, and the EV IFN-gamma group and the EV PD-L1 group can reduce the level of immune factors IL-6, TNF-a and Grazyme B, improve the level of mmp9, alpha-SMA and TGF-beta, control excessive inflammatory reaction of wounds and promote wound repair.

As shown in FIG. 5, the PD-L1 exosomes can inhibit proliferation and maturation of immune cells, so that positive T cell activity is reduced, IL-6, TNF-Sub>A, grazyme B secretion is reduced, TGF-betSub>A, VEGF-A and other growth factors are expressed and wound repair speed can be increased by combining with temperature-sensitive hydrogel. The thermosensitive gel embedded with the PD-L1 exosome can be used for preparing a preparation for inhibiting excessive immune response of T cell activation (including proliferation and immune factor release) in peripheral blood mononuclear cell PBMC to damaged tissues, so as to treat chronic ulcer and inflammatory diseases, and has a wide application prospect.

Claims

Use of pd-L1 exosomes in the preparation of a formulation for promoting wound healing.
2. The application of the thermosensitive gel embedded with the PD-L1 exosome in preparing the medicament for promoting skin repair/wound healing is characterized in that the thermosensitive gel embedded with the PD-L1 exosome is obtained by incubating the PD-L1 exosome and the thermosensitive hydrogel at 35-38 ℃ for 10-20 min and wrapping the exosome into hydrogel.