Inflammation memory activated epidermal stem cell bionic vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses an inflammatory memory activated epidermal stem cell bionic vesicle as well as a preparation method and application thereof. The epidermal stem cell bionic vesicle is obtained by adding lipopolysaccharide into a serum-free culture medium to induce and culture epidermal stem cells, continuously extruding a cell suspension through an extruder of a micron-sized polycarbonate film, and carrying out centrifugal separation. The LEM preparation method provided by the invention is simple, efficient and good in biocompatibility, not only retains the repair characteristic of mother cells, but also can induce neutrophil to transform to N2 phenotype, and has a better inflammation regulation effect, so that wound healing and tissue repair are promoted, and the LEM has a better application prospect and a better application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an inflammatory memory activated epidermal stem cell bionic vesicle and a preparation method and application thereof. Background Art

[0002] Wound repair involves the coordinated action of multiple cells to regulate inflammation, proliferation and remodeling. Changes in the trend of inflammation always affect the overall trend of wound healing. Neutrophils (PMN) are the first immune cells to infiltrate the ulcer surface in the inflammatory phase in the healing process. Fridlender proposed the N1 / N2 functional classification of TANs in 2009, which is similar to the M1 / M2 macrophage classification. N1 is a neutrophil with pro-inflammatory properties and anti-tumor functions, and N2 is a neutrophil with anti-inflammatory and pro-tumor functions. Existing studies have confirmed that neutrophils play an important role in the immune regulation, inflammatory response and tissue repair process, and show that PMN gradually polarizes to the anti-inflammatory N2 phenotype in the wound, promoting the transition of the wound from the inflammatory phase to the proliferation phase, providing a new idea for promoting wound healing.

[0003] N1 and N2 neutrophils can transform into each other, but there are many factors that affect the neutrophil phenotypic polarization: (1) the influence of cytokines; (2) changes in the inflammatory microenvironment; (3) regulation of gene expression and signaling pathways; (4) the influence of the external environment; etc. Therefore, how to induce the transformation of different phenotypes and functions of neutrophils and make wound repair transition from the inflammatory stage to the proliferation stage is a technical problem that needs to be studied urgently.

[0004] Extracellular vesicles (EVs) are small extracellular nano-sized vesicles secreted by most cells, which can be transmitted to other cells as signal molecules and change their functions. EVs contain a variety of bioactive substances, including proteins, nucleic acids, miRNAs and lipids. Marker proteins from mother cells are also often detected in EVs. The composition and function of EVs will change when mother cells are subjected to different stimuli and cell origins. However, the regulatory mechanism and role of EVs secreted by epidermal stem cells stimulated by LPS on the phenotype of neutrophils in the process of wound repair have not been reported. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present application proposes an inflammatory memory activated epidermal stem cell bionic vesicle and its preparation method and application. The present invention uses lipopolysaccharide (LPS) to pretreat epidermal stem cells to prepare epidermal stem cell bionic vesicles (L-EpSCs-MNVs, abbreviated as LEM), and uses Western Blot, ELISA and immunofluorescence techniques to detect the effect of epidermal stem cell bionic vesicles (LEM) on neutrophil polarization. The results showed that compared with EM, LEM bionic vesicles after inflammatory stimulation can induce neutrophil polarization to N2 phenotype, inhibit the expression of pro-inflammatory factors, enhance the expression of anti-inflammatory factors, and accelerate the healing of wounds in diabetic mice.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides an application of an inflammatory memory activated epidermal stem cell bionic vesicle in the preparation of an anti-inflammatory drug, wherein the bionic vesicle is prepared by the following method:

[0008] Lipopolysaccharide (LPS) is added to serum-free culture medium to induce culture of epidermal stem cells (EpSCs), which are resuspended after centrifugation. The cell suspension is sequentially extruded through an extruder with a micrometer-sized polycarbonate membrane, and centrifuged to obtain the biomimetic vesicles L-EpSCs-MNVs (LEM).

[0009] Preferably, the continuous extrusion refers to continuous extrusion through an extruder of polycarbonate films of 10 μm, 5 μm and 1 μm in sequence.

[0010] Preferably, the anti-inflammatory effect refers to excessive inflammatory response in wounds associated with diabetes.

[0011] Preferably, the anti-inflammatory effect refers to up-regulating the expression level of cytokines IL-4 and IL-13, and down-regulating the expression level of pro-inflammatory factor IFN-γ.

[0012] The present invention adopts LEM or EM to incubate neutrophils. Studies have found that the healing-promoting effect of LEM vesicles after inflammatory stimulation depends on infiltrating neutrophils rather than macrophages. Compared with unstimulated vesicles, LEM can induce neutrophils to polarize toward N2 phenotype, improve anti-inflammatory properties, and affect wound healing results.

[0013] Therefore, the present invention also provides the use of the bionic vesicle in promoting the transformation of neutrophils from N1 type to N2 type.

[0014] In addition, the present invention also incubated neutrophils with bionic vesicles, and the immunofluorescence analysis of neutrophil markers showed that compared with the control group and the EM group, the N2 subtype neutrophils in the LEM group stimulated by lipopolysaccharide increased significantly, indicating that LEM can induce neutrophils to polarize to the N2 phenotype (Arg-1), thereby inducing neutrophils to enter another immunometabolic cell fate.

[0015] In the second aspect, the present invention introduces inflammatory memory into injury repair and provides the use of the above-mentioned epidermal stem cell bionic vesicles in the preparation of drugs that promote wound healing.

[0016] Preferably, the wound healing includes skin trauma, burns, chronic wound healing, radiation ulcers, skin cell repair and regeneration, etc.

[0017] Preferably, the wound refers to tissue damage associated with diabetes. The present invention constructs a diabetic mouse model, and then administers drugs by subcutaneous injection to treat mouse skin wounds, verifying that LEM is more effective in promoting wound healing than EM.

[0018] Therefore, the present invention also provides an application of epidermal stem cell bionic vesicles in the preparation of a medicine for promoting wound healing.

[0019] Preferably, the wound surface refers to tissue damage caused by diabetes.

[0020] Preferably, the promoting wound healing refers to: (1) promoting epidermal cell regeneration, and / or; (2) slowing down scar tissue proliferation.

[0021] In a third aspect, the present invention provides an anti-inflammatory composition, comprising inflammatory memory activated epidermal stem cell bionic vesicles prepared by the above method.

[0022] The present invention reveals through experimental studies that LEM regulates inflammation and wound healing by affecting the polarization of neutrophils to N2. Compared with MNVs derived from untreated epidermal stem cells, MNVs separated and cultured from epidermal stem cells pretreated with LPS have enhanced anti-inflammatory effects of LEM, so epidermal stem cell bionic vesicles treated with lipopolysaccharide (LPS) can be used as an effective ingredient of an anti-inflammatory composition.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The LEM preparation method provided by the present invention is simple, efficient, and has good biocompatibility, and retains the repair properties of mother cells. Compared with EM without LPS stimulation, LEM can retain inflammatory memory, promote the transformation of neutrophil polarization to N2 phenotype, and has a better inflammation regulation effect; at the same time, inflammatory memory is introduced into injury repair for the first time, which promotes wound healing and tissue repair, has better therapeutic effect, and has good application prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 NTA result analysis, transmission electron microscopy image, and western detection result of surface markers of L-EpSCs-MNVs prepared in the present invention.

[0026] Figure 2 Analysis of wound treatment results in mouse model experiments, gross observations of mice treated with LEM or EM and quantitative analysis results of wound closure.

[0027] Figure 3 Graphic illustration of wound width in mouse model experimental wound treatment and analysis results of epidermal cell regeneration (length of epidermal cells), HPE (area), wound width (spacing), and wound closure rate.

[0028] Figure 4 Flow cytometric analysis (A) and quantitative analysis (B) of the proportion of N2 phenotype neutrophils (Ly-6G+Arg1+) in diabetic wounds treated with EM / LEM, and qRT-PCR detection of the expression results of chemokines CCL2, CCL3, and CCL5 (C) and cytokines L-4, L-13, and IFN-γ (D).

[0029] Figure 5 In the present invention, neutrophils were incubated with LEM or EM, (A) localization of fluorescently labeled biomimetic vesicles (red), and arrows indicate the merged structure of biomimetic vesicles; (B) representative immunofluorescence images of different groups of cultured neutrophils against N2 (Arg-1) neutrophils. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Lipopolysaccharide was purchased from Merck Sigma-Aldrich (Catalog Number: L1329), and the biological source was Escherichia coli (O127:B8).

[0033] Example 1 Preparation and identification of EpSCs-derived biomimetic vesicles

[0034] EpSCs were extracted and cultured according to conventional methods (refer to the literature "Isolation, culture and identification of epidermal stem cells from newborn mouse skin". DOI: 10.1007 / s11626-009-9245-y), subcultured to the 6th generation, and when the cells grew to 70%-80% confluence, the cell culture medium was discarded, PBS buffer was washed 1-2 times, the culture medium was replaced with serum-free culture medium, lipopolysaccharide (10-1000ng / mL) was added for treatment and cultured for 48 hours, the EpSCs stimulated by LPS were centrifuged and resuspended, the cell suspension was continuously extruded through an extruder with a polycarbonate membrane of 10μm, 5μm, and 1μm in sequence, dispersed in PBS and centrifuged at 100000g for 70 minutes to obtain biomimetic vesicles L-EpSCs-MNVs (abbreviated LEM). EpSCs-MNVs (abbreviated EM) obtained without lipopolysaccharide treatment were used for comparison.

[0035] The size distribution of the prepared L-EpSCs-MNVs was analyzed by NTA, their morphology was characterized by TEM, and the expression of their surface markers was analyzed by Western blotting.

[0036] The results are as follows Figure 1 As shown in the figure, the particle size of L-EpSCs-MNVs is mostly distributed between 100 and 120 nm and is relatively uniform. Transmission electron microscopy images show that it is oval and has a complete double-layer membrane structure. Western blot was used to detect the surface markers of biomimetic vesicles, and the results showed that it expressed TSG101, CD63 and CD81 markers.

[0037] Example 2 Wound healing model establishment and treatment experiment

[0038] The present invention purchases female 6-8 week old type 2 diabetic db / db mice (purchased from Beijing Zhongke Zesheng Biotechnology Co., Ltd.) and prepares for skin wound experiments. All experimental procedures comply with the relevant international regulations outlined in the Guide for the Care and Use of Laboratory Animals. This study was approved by the institutional animal ethics committee for animal experiments. The db / db mouse treatment experimental groups were set as LEM treatment group / EM treatment group / PBS control group (Ctrl group), and each group of diabetic mice was guaranteed to have at least 6 mice, and the incision was prepared with electric scissors and 70% ethanol. The same concentration of biomimetic vesicles (LEM / EM was made into a suspension with PBS, with a concentration of 1 mg / mL) was subcutaneously injected on the day of mouse modeling, and 50 microliters of suspension was injected at each injection point at 5 mm from the wound edge and equidistant from 6 points, and the control group was injected with the same amount of PBS. The wound healing progress was performed every two days, the wound diameter was measured in Photoshop to calculate the closure rate, and the expression of chemokines CCL2, CCL3 and CCL5 was detected by qRT-PCR, and the expression of cytokines L-4, L-13 and IFN-γ was detected at the same time.

[0039] Results Analysis

[0040] like Figure 2 The gross observation images and wound closure curves of mice treated with LEM or EM showed that the wound size in the LEM group was smaller than that in the other two groups at each time point, and the degree of injury was significantly alleviated. Figure 3 The results of the cortical staining, wound width, epidermal cell regeneration (length of long epithelial cells), HPE (new epithelial area), wound width (spacing), and wound closure rate detection and analysis are given. HPE is the new epithelial area, and HF is the distance between two new hair follicles (the longer the distance, the longer the scar distance). It can be clearly seen that the new epithelial area in the LEM group is larger and the scar length is significantly shortened compared with the EM group, indicating that LEM further promotes epidermal cell regeneration, accelerates wound healing, alleviates scar tissue hyperplasia, and has better therapeutic effect and wound healing effect.

[0041] Arg-1 positive cells (Ly-6G+Arg1+) were detected by flow cytometry and quantitative analysis in diabetic wounds treated with PBS / EM or LEM. Figure 4 A, 4B) It can be seen that the LEM group significantly shifted neutrophils toward the Ly-6G+Arg1+ neutrophil population, which is a characteristic of the N2 type. LEM achieved a conversion efficiency of up to about 75.8%.

[0042] At the same time, the present invention detected the expression of chemokines CCL2, CCL3 and CCL5, and the expression levels of cytokines L-4, L-13 and IFN-γ by qRT-PCR. The results showed that the relative mRNA expression of CCL2 / CCL3 / CCL4 was significantly decreased compared with the control group ( Figure 4 C), there was no significant difference between the LEM and EM groups. However, compared with the control group and the EM group, the LEM group significantly upregulated the expression levels of anti-inflammatory cytokines IL-4 and IL-13, and downregulated the expression level of the pro-inflammatory factor IFN-γ ( Figure 4 D), plays a role in regulating excessive inflammatory response in diabetic wounds.

[0043] Example 3 Analysis of the effect of LEM on neutrophil polarization phenotype

[0044] Existing studies have shown that the chronic inflammatory state of diabetic patients hinders the normal healing process, resulting in delayed wound closure and increased risk of infection. This persistent inflammation is often accompanied by an imbalance of pro-inflammatory and anti-inflammatory cytokines, which impairs tissue repair and regeneration. On the one hand, the levels of chemokines CCL2, CCL3, and CCL5 in diabetic wounds are significantly increased, which can induce neutrophils to reach the site of injury and initiate an immune response. On the other hand, L-4 and L-13 in diabetic wounds are significantly decreased, IFN-γ is increased, and changes in the chemokine and cytokine environment may lead to the dysregulated inflammatory response observed in diabetic wound healing. It also explains to a certain extent why there are more neutrophil infiltrations in diabetic wounds, but fewer N2 types. In order to further explore the mechanism of action of LEM, the present invention uses LEM / EM / PBS to incubate neutrophils to explore whether LEM can accelerate wound healing and regulate excessive inflammatory responses in diabetic wounds by affecting the polarization of neutrophils to the N2 phenotype.

[0045] Experimental procedure: To isolate mouse primary neutrophils from mice, femurs and tibiae were extracted from euthanized mice. The bones were flushed using a 25-gauge needle filled with 1× Hanks balanced salt solution (HBSS) 0.38% sodium citrate buffer. The extracted cell suspension was gently disintegrated and filtered through a 40 μm Corning cell strainer to obtain a single cell suspension. Red blood cells were removed using an erythrocyte lysis buffer from Servicebio (Wuhan, China), including a hypotonic lysis step. After washing in HBSS, neutrophils were further purified by a Miltenyi Biotec magnetic associated cell sorting (MACS)-based protocol (kit #130-097-658) and then maintained in RPMI-1640 medium. All cell cultures were stored at 37°C and 5% CO 2 Down.

[0046] By adding 1-2 × 106 Mouse primary neutrophils (with or without 100 μg LEM / EM treatment) were cultured in RPMI medium supplemented with 10% FBS for 12 h, then washed with PBS, and the cells were incubated for another 12 h under serum-free conditions, centrifuged at 300 × g for 10 min to remove cells and debris, and the culture medium was collected and then frozen at -80°C for use.

[0047] Biomimetic vesicles were stained with PKH26 red fluorescent dye for easy visualization, and in vitro studies included co-culture of neutrophils with labeled exosomes and imaging after staining with DAPI. Formalin-fixed, paraffin-embedded tissue sections were dewaxed, rehydrated, and antigen retrieval was performed. To block endogenous peroxidase activity, sections were washed with 3% H 2 O 2 The tissue sections were treated and further blocked with 3% BSA for 1 hour. Tissue sections were modified with anti-mouse antibodies against Arg-1 (Proteintech, 1:400), Ly6G (ThermoFisher, 1:1000) for 12 hours at 4°C, modified with secondary antibodies for 1 hour at room temperature, and DAPI was used to label cell nuclei. The staining results were captured by an inverted fluorescence microscope and measured by ImageJ software.

[0048] Results Analysis

[0049] Arg1 is a marker of N2 status, as shown by immunofluorescence analysis of neutrophil markers ( Figure 5 ), compared with the control group and the EM group, the number of N2 subtype neutrophils in the LEM group stimulated by lipopolysaccharide increased significantly, indicating that LEM can induce neutrophils to polarize toward the N2 phenotype (Arg-1), and this orientation is closely related to regulating excessive inflammatory response in diabetic wounds, enhancing epidermal cell regeneration and accelerating the healing of skin wounds, which is consistent with the in vivo study in Example 2.

[0050] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of an inflammatory memory activated epidermal stem cell bionic vesicle in the preparation of an anti-inflammatory drug, characterized in that: The bionic vesicles are prepared by the following method: adding stimulants to a serum-free culture medium to induce and culture epidermal stem cells, centrifuging and resuspending after culture, and sequentially extruding the cell suspension through an extruder with a micron-sized polycarbonate membrane, and centrifuging and separating.

2. The use according to claim 1, characterized in that: The stimulus is LPS, and the cell suspension is sequentially extruded through 10 μm, 5 μm, and 1 μm polycarbonate membranes.

3. The use according to claim 1, characterized in that: The anti-inflammatory effect refers to the inhibition of excessive inflammatory response in wounds associated with diabetes.

4. The use according to claim 3, characterized in that: The anti-inflammatory effect refers to up-regulating the expression levels of cytokines IL-4 and IL-13, and down-regulating the expression level of the pro-inflammatory factor IFN-γ.

5. Use of the bionic vesicle according to claim 1 in promoting the transformation of neutrophils from N1 type to N2 type.

6. Application of epidermal stem cell bionic vesicles in the preparation of drugs to promote wound healing.

7. The use according to claim 6, characterized in that: The wound surface refers to tissue damage caused by diabetes.

8. The use according to claim 7, characterized in that: The promotion of wound healing refers to: (1) Promote epidermal cell regeneration, and / or; (2) Slow down the proliferation of scar tissue.

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