Composition for recovering epithelial structure of lesion area as well as preparation method and application of composition

Abstract

The invention discloses a composition for recovering an epithelial structure of a lesion area as well as a preparation method and application of the composition. The composition comprises low immunogenic vesicles derived from cells, and the low immunogenic vesicles are positive for CD63, CD9 and TSG101 and negative for Calnexin, and have a cup-shaped double-layer membrane structure. The composition has good biocompatibility, can increase the amount of cells and / or keratin 5 in the epithelium, activate ion transport and metabolism in the epithelium, promote angiogenesis in the epithelium and recover the structural integrity of the epithelium, and is further used for improving or treating lesion areas.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a composition for restoring the epithelial structure of a diseased area, and a preparation method and application thereof. Background Art

[0002] Changes in the epithelial structure of the lesion area can be caused by a variety of reasons, including, for example, trauma, disease, the action of microorganisms and exposure to foreign bodies. The restoration of the epithelial structure of the lesion area is not only important for achieving wound healing, but also plays an important role in restoring tissue function and providing a barrier function to resist infection. Delayed wound healing is an important cause of morbidity in subjects. In some cases, the wound healing process is functionally disordered, which leads to the occurrence of chronic wounds. Chronic wounds are defined as wounds that cannot heal after 3 months. The most common chronic wounds include venous stasis ulcers, diabetic ulcers, pressure ulcers and ischemic ulcers. Chronic wounds have a significant impact on the physical and mental health, morbidity, mortality and care costs of affected individuals.

[0003] The most commonly used conventional methods to assist wound healing include the use of wound dressings and other methods. Many different types of dressings are used to help wounds heal. Some treatments also use the provision of minerals and vitamins to help wounds heal. However, these types of treatments have had little success. In addition, current conventional methods for assisting wound healing also include protecting the wound from mechanical trauma, controlling the surface microbial load through antibiotics, antimicrobial preservatives and other antimicrobial compounds, and using certain types of growth factors. However, current treatments have many shortcomings in terms of anti-inflammatory, biocompatibility and therapeutic efficacy. Summary of the invention

[0004] In order to solve at least some of the problems in the above-mentioned prior art, the present invention prepares a low immunogenic vesicle for restoring the epithelial structure of the lesion area, which can activate ion transport metabolism in the epithelium, promote angiogenesis in the epithelium, and restore the integrity of the epithelial structure. Specifically, the present invention includes the following contents.

[0005] The first aspect of the present invention provides a composition for restoring the epithelial structure of a diseased area, which comprises cell-derived low-immunogenic vesicles, which are CD63, CD9 and TSG101 positive and Calnexin negative, and have a cup-shaped double-layer membrane structure, and a particle size in the range of 30-150 nm.

[0006] In certain embodiments, according to the composition for restoring epithelial structure of a diseased area of ​​the present invention, the cells are selected from at least one of totipotent stem cells, pluripotent stem cells and unipotent stem cells.

[0007] In certain embodiments, according to the composition for restoring epithelial structure in a lesion area of ​​the present invention, the restoration comprises at least one of the following:

[0008] Increase the amount of cells and / or keratin 5 in the epithelial layer;

[0009] Activates ion transport metabolism in the epithelium;

[0010] promotes angiogenesis in the epithelium;

[0011] Restores epithelial structural integrity.

[0012] In certain embodiments, according to the composition for restoring epithelial structure in a diseased area according to the present invention, the low immunogenic vesicles are derived from umbilical cord cells.

[0013] In certain embodiments, according to the composition for restoring epithelial structure in a diseased area according to the present invention, the low immunogenic vesicles are obtained by cell centrifugation.

[0014] The second aspect of the present invention provides a method for preparing the composition according to the present invention.

[0015] The third aspect of the present invention provides use of the composition according to the present invention in the preparation of a drug for restoring the epithelial structure of a diseased area.

[0016] In certain embodiments, according to the use of the present invention, the lesion comprises at least one of a surgical wound, a penetrating wound, a venous ulcer, an arterial ulcer, a laceration, a pressure ulcer, a crush injury, a shear injury, a leg ulcer, a foot ulcer, an abrasion, a puncture, a burn, a diabetic ulcer, an oral ulcer, a contusion, a trauma, a pressure ulcer, an ischemic ulcer, a laceration and a bite wound.

[0017] In a fourth aspect, the present invention provides a method for regulating cell activity in vitro, comprising the step of contacting cells with low immunogenic vesicles in vitro, wherein the cell activity comprises cell proliferation, cell migration and / or changes in gene expression.

[0018] In certain embodiments, according to the method of the present invention, the low immunogenic vesicles are CD63, CD9 and TSG101 positive and Calnexin negative, and have a cup-shaped double-layer membrane structure, and the particle size is in the range of 30-150 nm.

[0019] In certain embodiments, according to the method described herein, the gene expression changes include down-regulation of chemokines CCL3, CXCL1, CXCL2, IL6 and IL-1β and up-regulation of keratins CK5, CK13, KRT13, KRT2, KRT24 and KRT14.

[0020] The present invention demonstrates that the therapy based on low immunogenic vesicles has achieved good results in the restoration and regeneration of epithelial structures in the lesion area, broadens the theory of oral and maxillofacial tissue engineering in the field of soft tissue repair, and shows that low immunogenic vesicles can be used as a promising treatment method to eliminate damaged tissue in the lesion area. In addition, the composition of the present invention has the characteristics of low immunogenicity, good biosafety, stability, easy storage, and easy large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The characterization of low immunogenic vesicles is shown. In the figure, A is a transmission electron microscope (TEM) image of low immunogenic vesicles, scale bar: 100 nm; B is a nanoparticle tracking analysis (NTA) image of low immunogenic vesicles; C is a nano flow cytometry (nFCM) analysis image of low immunogenic vesicles; D is a Western blot detection of marker proteins of low immunogenic vesicles.

[0022] Figure 2 The in vivo toxicity evaluation of low immunogenic vesicles is shown. A is the histopathological images of the heart, liver, spleen, lung and kidney collected from the control group and the low immunogenic vesicle group, scale: 300 μm; B is the blood routine examination of the control group and the low immunogenic vesicle group; C is the performance of various blood biochemical indicators in the two groups, ns (p>0.05).

[0023] Figure 3 The therapeutic effect of low immunogenic vesicles in the rat oral ulcer model is shown. Figure A is a schematic diagram of the treatment of rat oral ulcers with low immunogenic vesicles to evaluate healing; B is an image of the restoration of rat oral mucosal defects using low immunogenic vesicles on days 2 and 4, scale: Figure 4 mm, 6 mm in the lower figure; C is the injury healing rate after 2 or 4 days of treatment with low-immunogenic vesicles; D is the histological observation of oral mucosal defects in rats restored with low-immunogenic vesicles for 2 and 4 days, scale bar: 400 μm; E is multiple immunohistochemistry (mIHC) staining after 2 or 4 days of treatment with low-immunogenic vesicles, scale bar: 500 μm; F is the multiple detection of cytokines and chemokines in the serum of rats with different treatments, ns (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 4The mechanism by which low immunogenic vesicles promote the healing of oral mucosal defects is shown. In the figure, A is a heat map of differentially expressed genes (DEGs) in rat mucosal tissue using the gene expression fold change of the control group and the low immunogenic vesicle group; B is a bubble map showing the inflammation-related KEGG pathways of mucosal tissue in the low immunogenic vesicle treatment group and the control group; C is a volcano map analysis of DEGs between the two groups in mucosal tissue; D is the DEGs of the low immunogenic vesicle group and the control group in GO enrichment analysis; E is a protein blot analysis of IL6 and CK5 in mucosal tissue treated for 4 days; F is the mRNA expression of inflammatory and cytokeratin markers in rat oral mucosa treated for 4 days by RT-qPCR; ns (p>0.05), *p<0.05, **p<0.01.

[0025] Figure 5 The biological effects of low immunogenic vesicles on human oral epithelial cells (HOECs) are shown. Figure A shows the cell viability of HOECs in different groups on day 1, day 2, and day 3. The columns in each group in the figure are control group, low-dose group, and high-dose group from left to right. *p<0.05 compared with the control group on day 1, #p<0.05 compared with the control group on day 2, &p<0.05 compared with the control group on day 3, &&p<0.01; B is a scratch test of HOECs after different treatments, with a scale of 400 μm; C is a quantitative analysis of wound closure rate by measuring the gap size; D is a heat map of DEGs of HOECs using gene expression fold changes between the control group and the low immunogenic vesicle group; E is a genomic enrichment analysis (GSEA) of DEGs after HOECs treated with or without low immunogenic vesicles for 24 hours, NES indicates normalized enrichment score, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] Composition

[0030] In one aspect of the present invention, a composition for restoring the epithelial structure of a diseased area is provided, which comprises cell-derived low immunogenic vesicles, which are CD63, CD9 and TSG101 positive and Calnexin negative, and have a cup-shaped double-layer membrane structure, and a particle size of 30-150nm, preferably 40-150nm, also preferably 50-150nm, and more preferably 60-150nm, for example 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150nm.

[0031] In the present invention, the lesions include but are not limited to at least one of surgical wounds, infiltration wounds, venous ulcers, arterial ulcers, laceration injuries, pressure ulcers, crushing injuries, shearing injuries, lower limb ulcers, foot ulcers, abrasions, punctures, burns, diabetic ulcers, oral ulcers, contusions, trauma, pressure ulcers, ischemic ulcers, lacerations and bites. In a specific embodiment, the composition of the present invention is used to restore or improve the epithelial structure of the ulcer area, especially oral ulcers.

[0032] In the present invention, the recovery includes but is not limited to at least one of the following:

[0033] Increase the amount of cells and / or keratin 5 in the epithelial layer;

[0034] Activates ion transport metabolism in the epithelium;

[0035] promotes angiogenesis in the epithelium;

[0036] Restores epithelial structural integrity.

[0037] In the present invention, the cell is selected from at least one of the following cells: totipotent stem cells (e.g., embryonic stem cells), pluripotent stem cells (e.g., mesenchymal stem cells), and unipotent stem cells (e.g., neural stem cells, muscle stem cells, skin stem cells, etc.). In a preferred embodiment, the cell of the present invention is a pluripotent stem cell, in particular a mesenchymal stem cell, and in a specific embodiment, the cell of the present invention is an umbilical cord mesenchymal stem cell.

[0038] Preparation method

[0039] One aspect of the present invention provides a method for preparing the composition according to the present invention.

[0040] In a preferred embodiment, the preparation method of the present invention comprises:

[0041] (1) Cutting human umbilical cord into pieces and culturing them in culture medium;

[0042] (2) allowing human umbilical cord mesenchymal stem cells to migrate from the tissue for 10-14 days, and after the fusion of the human umbilical cord mesenchymal stem cells reaches 80-95%, centrifugation is performed to collect the cell supernatant;

[0043] (3) Perform gradient centrifugation on the supernatant. Discard the supernatant, resuspend in saline, filter, and freeze for storage.

[0044] First, the human umbilical cord is cut into small pieces and cultured in a culture medium (including but not limited to α-MEM culture medium), and the solution is replaced regularly (for example: every certain time, such as 12-24 hours) to allow human umbilical cord mesenchymal stem cells to migrate from the tissue for 10-14 days (for example, 10, 11, 12, 13, 14 days). After the second generation of human umbilical cord mesenchymal stem cells have reached 80-95% fusion (preferably 81-95%, also preferably 82-95%, further preferably 83-95%, more preferably 84-95%, more preferably 85-95%, such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), the dead cells and debris are removed by centrifugation, and the cell supernatant is collected and subjected to gradient centrifugation. Discard the supernatant, resuspend in saline, filter through a filter (including but not limited to 0.15-0.3 μm filters, such as 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30 μm filters), and store frozen (e.g., -85°C, -84°C, -83°C, -82°C, -81°C, -80°C, -79°C, -78°C, -77°C, -76°C, -75°C).

[0045] In a preferred embodiment, the gradient centrifugation comprises three gradient centrifugation, and the first gradient centrifugation comprises centrifugation at 2500-3500g, preferably 2600-3400g, also preferably 2700-3300g, further preferably at 2800-3200g, for example 2800, 2900, 3000, 3100, 3200g for 20-40min, preferably 21-39min, also preferably 22-38min, further preferably 23-37min, more preferably 24-36min, more preferably 25-35min, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35min. The second gradient centrifugation comprises centrifugation at 9000-11000g, preferably 9200-10800g, further preferably 9400-10600g, more preferably 9600-10400g, for example 9600, 9700, 9800, 9900, 10000g, 10100g, 10200g, 10300g, 10400g for 20-40min, preferably 21-39min, also preferably 22-38min, further preferably 23-37min, more preferably 24-36min, more preferably 25-35min, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35min. The third gradient centrifugation comprises centrifugation at 90000-110000g, preferably 92000-108000g, further preferably 94000-106000g, more preferably 96000-104000g, for example 96000, 97000, 98000, 99000, 100000g, 101000g, 102000g, 103000g, 104000g for 80-100 min, preferably 81-99 min, also preferably 82-98 min, further preferably 83-97 min, more preferably 84-96 min, more preferably 85-95 min, for example 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 min.

[0046] The present invention further provides use of the composition according to the present invention in preparing a drug for improving or restoring the epithelial structure of a diseased area, in particular use of the composition according to the present invention in preparing a drug for improving or treating oral mucosal diseases (such as oral ulcers).

[0047] method

[0048] One aspect of the present invention provides a method for regulating cell activity in vitro, comprising the step of contacting cells with low immunogenic vesicles in vitro, wherein the cell activity comprises cell proliferation and / or cell migration.

[0049] In an embodiment of the method for regulating cell activity in vitro according to the present invention, the low immunogenic vesicles are CD63, CD9 and TSG101 positive and Calnexin negative, and have a cup-shaped double-layer membrane structure with a particle size of 30-150nm, preferably 40-150nm, also preferably 50-150nm, more preferably 60-150nm, for example 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150nm.

[0050] In certain embodiments, the modulation of cellular activity includes gene / expression changes, and the resulting changes in the amount of corresponding protein produced.

[0051] In a preferred embodiment, the gene expression changes include down-regulation of chemokines CCL3, CXCL1, CXCL2, IL6 and IL-1β and up-regulation of keratins CK5, CK13, KRT13, KRT2, KRT24 and KRT14.

[0052] The present invention therefore also provides a method for regulating the amount of cellular genes / proteins in vitro, comprising the step of contacting cells with low immunogenic vesicles in vitro, wherein the regulated amount of genes / proteins includes downregulation of chemokines CCL3, CXCL1, CXCL2, IL6 and IL-1β and upregulation of keratins CK5, CK13, KRT13, KRT2, KRT24 and KRT14.

[0053] Example 1

[0054] The preparation and characterization of low immunogenic vesicles are shown below.

[0055] 1. Preparation method

[0056] The human umbilical cord was cut into small pieces and cultured in α-MEM medium, and the solution was changed every other day. It was observed that human umbilical cord mesenchymal stem cells migrated from the tissue for about 10-14 days. After the fusion of the second generation of human umbilical cord mesenchymal stem cells reached 90%, dead cells and debris were removed by centrifugation, and the cell supernatant was collected for further gradient centrifugation (3000g, 30min; 10000g, 30min, 100000g, 90min). The supernatant was discarded, resuspended in saline, filtered through a 0.22μm filter, and stored at -80°C for later use.

[0057] 2. Characterization

[0058] The morphology of low immunogenic vesicles was observed by TEM. The particle size of low immunogenic vesicles was determined by NTA and nFCM. The surface markers of low immunogenic vesicles CD9, CD63, calnexin and TSG101 were identified by Western blotting.

[0059] Low immunogenic vesicles were derived from umbilical cord and obtained by ultracentrifugation. The double membrane ultrastructure of low immunogenic vesicles was observed using TEM, and the results showed that they had a cup-shaped membrane structure with clear edges and morphology (e.g. Figure 1 NTA showed that the particle size of low immunogenic vesicles was mainly concentrated at 128.8 nm (as shown in A). Figure 1 In addition, the results of nFCM are similar to those of NTA (as shown in B). Figure 1 Finally, the transmembrane proteins CD63 and CD9, the cytoplasmic protein TSG101, and the negative protein Calnexin were detected by Western blotting. The data showed that the positive expression of CD63, CD9, and TSG101 and the negative expression of Calnexin were successfully detected (as shown in FIG. Figure 1 The results show that the low immunogenic vesicles were successfully isolated and prepared.

[0060] Example 2

[0061] The following is an exemplary illustration of the effects of low immunogenic vesicles on wounds and ulcers.

[0062] 1. Experimental Methods

[0063] 1. Rat ulcer model and treatment

[0064] 4-6 week old SD rats were divided into 2 groups, namely a control group (the ulcer was not treated with low immunogenic vesicles) and a low immunogenic vesicle group (the ulcer was treated by spraying a liquid containing low immunogenic vesicles). After anesthesia by intraperitoneal injection of 100 mg / kg sodium pentobarbital, a 4.5 mm diameter filter paper soaked in 50% glacial acetic acid was attached to the buccal mucosa on both sides of the mouth for 30 seconds, and then the residual glacial acetic acid was wiped off with a sterile cotton ball containing saline. 24 hours after surgery (0d), the lesions were treated accordingly every day for 4 consecutive days, during which time the ulcer lesions were evaluated and photographed. The injury healing rate was measured using ImageJ software 2 and 4 days after treatment with or without low immunogenic vesicles. The lesion healing rate was calculated as follows: lesion healing rate = (initial lesion area-lesion area at the evaluation time point) / initial lesion area × 100%.

[0065] 2. Histological Analysis

[0066] Rats were killed 4 days after treatment, and major organs (heart, liver, spleen, lung, kidney) and buccal mucosal tissues around the lesions were collected, fixed in formalin, dehydrated, and embedded in paraffin. Then, the paraffin-embedded samples were sectioned. Finally, the sections were stained with hematoxylin-eosin (H&E) according to the established protocol and analyzed using K-viewer software to evaluate the in vivo toxicity of low immunogenic vesicles and the effects on injury healing.

[0067] 3. Blood test of rats

[0068] For routine blood tests, 1 ml of whole blood was collected and analyzed using a fully automated blood analyzer.

[0069] For blood biochemical examination, whole blood of rats was centrifuged at 3000 rpm for 10 min in a sodium heparin centrifuge tube and plasma was separated and analyzed using a biochemical analyzer.

[0070] 4.mIHC staining

[0071] The sections were incubated with antibodies CD31, IL6, and CK5 in sequence using the mIHC staining kit according to the instructions, and finally scanned using a laser confocal microscope. DAPI was used to mark cell nuclei, CD31 was used to assess angiogenesis, IL-6 was used to detect the degree of inflammation, and CK5 was used to assess epithelial regeneration.

[0072] The following primary antibodies were used: anti-CD31, anti-IL6, anti-CK5. Histological images were analyzed using LAS X.

[0073] 5. Detection of Rat Serum Cytokines

[0074] After 4 days of treatment, blood was collected from the two groups of rats and centrifuged at 3000 rpm for 15 min to obtain serum. Rat serum samples were assayed using the MILLIPLEX kit according to the manufacturer's instructions, and the results were read using Belysa software. The levels of cytokines in serum samples of each group were calculated using a standard curve.

[0075] 6. RNA sequencing (RNA-seq) and validation

[0076] For RNA-seq, rat buccal mucosal tissue was collected after 4 days of treatment, and HOECs were collected after 24 h of treatment. Total RNA was extracted using TRIzol reagent, magnetic beads with Oligo (dT) were selected for enrichment and purification, and then the purified mRNA was fragmented. The fragmented mRNA was then used as a template and reverse transcribed using random primers to synthesize the first and second strands of cDNA. Afterwards, the library was prepared by end repair, base A addition, sequencing adapter addition, magnetic bead screening to recover the target fragments, and PCR amplification. Finally, the quality of the library was checked, and the qualified library was sequenced using the Illumina platform with a PE150 sequencing strategy.

[0077] For western blot validation, the buccal mucosal tissues from the lesions were thoroughly ground in liquid nitrogen, and the relative protein levels of IL6 and CK5 in the ulcer tissues with different treatments were detected. Briefly, protein lysates were added to the sample tissues, the protein concentration was detected, and the proteins were denatured. Then, electrophoresis, membrane transfer, blocking, antibody incubation, and protein detection were performed in sequence. Finally, β-actin was used as the total protein internal reference, and the protein expression of different groups was analyzed by Image Lab software.

[0078] For real-time quantitative PCR (RT-qPCR) validation, buccal mucosa samples were ground into powder in liquid nitrogen, and total RNA was extracted using TRIzol reagent. RNA was then reverse transcribed into cDNA using a reverse transcription kit. RT-qPCR was then performed using the QuantStudio3 system with a SYBR Green Master Mix kit. The primer sequences for CCL3, IL6, IL-1β, CK5, and CK13 genes are shown in Table 1, and GAPDH was used as an internal control gene.

[0079] Table 1 Primer sequences used for RT-qPCR analysis

[0080]

[0081] 7. Cell Proliferation Assay

[0082] The proliferation of HOECs was measured using the Cell Counting Kit-8 reagent. HOECs were seeded (1×10 4 Cells / well) were plated in 96-well plates and cultured overnight at 37°C with different treatments (PBS, 10 μg / mL low immunogenic vesicles, 50 μg / mL low immunogenic vesicles). On days 1, 2, and 3, 10% CCK-8 solution was added to each well and incubated at 37°C for 2 hours. OD values ​​were measured at 450 nm using a microplate reader. All experiments were performed at least three times.

[0083] 8. Scratch Wound Assay

[0084] HOECs were plated in 6-well plates and incubated until fully confluent. The monolayer of HOECs was gently scraped using a 200 μl pipette tip and rinsed three times with PBS to remove detached cells. After 0 and 12 h of incubation with or without different concentrations of low immunogenic vesicles in the culture medium, wound closure was observed using an inverted microscope. The wound closure rate was quantified by ImageJ software and calculated as follows: wound closure rate = (initial wound area-wound area at the assessment time point) / initial wound area × 100%.

[0085] 2. Experimental Results

[0086] 1. In vivo toxicity evaluation of low immunogenic vesicles

[0087] To evaluate the biosafety of low immunogenic vesicles for in vivo application, low immunogenic vesicles were administered to rats by gavage and first subjected to histological testing. No inflammation or histopathological abnormalities were observed in the heart, liver, spleen, lung, and kidney of the rats ( Figure 2 A), indicating that the low immunogenic vesicles did not cause significant organ damage in rats. Routine blood and blood biochemistry tests showed that the low immunogenic vesicle treatment group did not adversely affect hematological parameters compared with the control group ( Figure 2 In addition, no significant side effects were found during the entire low immunogenic vesicle treatment period, which means the safety of low immunogenic vesicles in in vivo treatment.

[0088] 2. Evaluation of the restorative effect of low immunogenic vesicles in a rat oral ulcer model

[0089] This example evaluates the effect of low immunogenic vesicles on the healing of oral ulcers. Rats were randomly divided into three groups, filter paper containing 50% glacial acetic acid was placed on the lower lip mucosa, and the lesions were treated with low immunogenic vesicles 24 hours after injury ( Figure 3 A). By the fourth day of treatment, the oral ulcers in the low-immunogenic vesicle group were basically healed. The oral mucosa of the low-immunogenic vesicle group was bright red, while in the control group, the lesion area healed slowly and the mucosa still showed obvious edema ( Figure 3 B and C). H&E staining showed that in the control group, the lesion area showed little epithelialization and loose disorganized connective tissue with diffuse inflammatory cell infiltration in the submucosal layer. In contrast, the low immunogenic vesicle group showed better mucosal healing, with intact thickened squamous epithelium and significantly less inflammatory cell infiltration compared with the control group ( Figure 3 D).

[0090] Angiogenesis reflects the degree of tissue reconstruction and mucosal function recovery. mIHC staining showed that the endothelial cell marker CD31 had a lower density in the control group, indicating a low level of angiogenesis. In contrast, the low-immunogenic vesicle group showed a significant increase in the vascularized area. In addition, IL6 was used to evaluate the inflammatory response during lesion treatment, and it is worth noting that very few IL6-positive inflammatory cells were detected in the low-immunogenic vesicle group on day 2. At the same time, a large number of IL6-positive cell infiltrations still existed in the control group, indicating that low-immunogenic vesicles have anti-inflammatory activity, thereby contributing to the healing of damaged mucosal tissue. In addition, compared with the control group, the expression of cytokeratin 5 (CK5) present in the epithelial layer was significantly increased in the low-immunogenic vesicle group, confirming that low-immunogenic vesicle treatment restored the original structure of the mucosa ( Figure 3 E).

[0091] As wound healing progresses, the secretion of cytokines at the site of injury changes. The following evaluation of the secretion of inflammatory factors, chemokines, and growth factors in the serum of rats after treatment with low-immunogenic vesicles revealed a significant increase in the secretion of pro-inflammatory factors and chemokines (such as TNFα, IL-1β, IL17, eotaxin, IL12p70, IL5, IP-10, IL-1α, MIP-1α, IL2, MCP-1, and leptin) in the control group. In addition, compared with the control group, the concentrations of secreted anti-inflammatory and growth factors (including IL4, IL10, VEGF, G-CSF, RANTES, and EGF) were significantly increased in the low-immunogenic vesicle group ( Figure 3 F). The results showed that low immunogenic vesicles can promote epithelial tissue regeneration, angiogenesis and anti-inflammatory response, thereby improving the pathological structure of oral ulcers.

[0092] 3. Mechanisms of low immunogenic vesicles promoting healing of oral mucosal defects

[0093] In order to gain a deeper understanding of the intrinsic mechanism and potential downstream signaling pathways of low-immunogenic vesicles in promoting the healing of oral mucosal lesions, RNA sequencing was performed on oral mucosal tissues treated with or without low-immunogenic vesicles. Differentially expressed genes were analyzed in the mucosal tissues of the control group and the low-immunogenic vesicle group, and the results showed that there were significant differences in the expression of genes in the two groups of tissues, indicating that low-immunogenic vesicles significantly affect the physiological function of lesions ( Figure 4 A).

[0094] Inflammation is an important stage in the wound healing cascade, and long-term inflammation is harmful. KEGG analysis showed that the TNF signaling pathway and cytokine-cytokine receptor interactions were significantly enriched after treatment with low-immunogenic vesicles. The TNF signaling pathway affects the development of inflammation by inducing immune cell proliferation and activation and regulating the release of chemokines. Cytokine-cytokine receptor interactions can trigger a variety of downstream signaling pathways that can participate in the regulation of cell growth, immune response, angiogenesis, and tissue repair, restoring internal microenvironment homeostasis. In summary, these data indicate that mucosal tissues treated with low-immunogenic vesicles have a stronger immune activation effect than the control group ( Figure 4 B).

[0095] The volcano plot showed that the expression of chemokines CCL3, CXCL1, CXCL2, and inflammatory factors IL6 and IL-1β in the low-immunogenic vesicle group was downregulated compared with the control group. This further confirmed the anti-inflammatory effect of low-immunogenic vesicles. The low-immunogenic vesicle group had upregulated keratin expression, including KRT13, KRT2, KRT24, and KRT14 ( Figure 4 In addition, GO enrichment analysis showed that biological processes related to ion transport metabolism were activated in the low immunogenic vesicle group ( Figure 4 D), which can explain the excellent effect of low-immunogenic vesicles in restoring the epithelial structure of the lesion area. Compared with the control group, the GO BP items enriched in the low-immunogenic vesicle group were mainly concentrated in the regulation of calcium ion transmembrane transporter activity, which is closely related to inflammation and signal transduction. Based on the in-depth analysis of RNA-seq, we performed RT-qPCR and western blot experiments. The results clearly showed that compared with the control group, treatment of mucosal defect areas with low-immunogenic vesicles reduced the expression levels of inflammatory factors and chemokines, while low-immunogenic vesicles could further increase the expression of cytokeratin ( Figure 4 In conclusion, low immunogenic vesicles modulate immune signaling pathways to exert anti-inflammatory effects by reducing the release of inflammatory factors and chemokines. Notably, low immunogenic vesicles can also restore epithelial structural integrity more quickly by promoting keratin secretion.

[0096] 4. Biological effects of low immunogenic vesicles on HOECs

[0097] In order to study the effect of low immunogenic vesicles in vitro, low immunogenic vesicles at concentrations of 10 μg / mL (low concentration) and 50 μg / mL (high concentration) were selected to treat HOECs. CCK8 assay showed that low immunogenic vesicles promoted the proliferation of HOECs after 1, 2, and 3 days of treatment ( Figure 5 A), indicating that the low immunogenic vesicles are biocompatible for in vitro applications.

[0098] The scratch test showed that 12 hours after the scratch, the migration ability of HOECs treated with low immunogenic vesicles was significantly enhanced compared with that of HOECs treated with PBS in the control group ( Figure 5 B, C). In addition, the ability of low immunogenic vesicles to promote wound closure does not increase with increasing concentration, and effective healing can be achieved by administering only low concentrations of low immunogenic vesicles. The above results demonstrate the ability of low immunogenic vesicles to promote HOECs migration in vitro, further confirming their potential applicability in the treatment of oral mucosal diseases.

[0099] In addition, DEGs analysis of HOECs in the control group and low-immunogenic vesicle group showed significant differences in gene expression between the two groups, indicating that low-immunogenic vesicles significantly affect the physiological functions of HOECs ( Figure 5 D). Using gene set enrichment analysis, it was found that genes related to cell cycle protein binding were enriched in HOECs treated with low immunogenic vesicles, indicating that low immunogenic vesicles induce cell proliferation and metabolism by regulating the cell cycle, thereby further triggering changes in cell biological behavior ( Figure 5 E).

[0100] Conclusion

[0101] The present invention prepares low-immunogenic vesicles, which have good biocompatibility and anti-inflammatory activity in a rat oral ulcer model, and can improve the pathological structure of the lesion area, thereby contributing to the healing of damaged epithelial tissue. In addition, RNA-seq shows that low-immunogenic vesicles regulate immune signal transduction by reducing the release of inflammatory factors and chemokines, thereby accelerating the repair of mucosal damage, and also restore the structural integrity of the epithelium more quickly by promoting keratin secretion. In vitro experiments have shown that low-immunogenic vesicles promote the proliferation and scratch healing of HOECs, once again confirming the healing-promoting ability of low-immunogenic vesicles at the cellular level.

[0102] In summary, the present invention confirms that the therapy based on low-immunogenic vesicles has achieved good results in improving the recovery and regeneration of oral mucosal tissue, broadens the theory of oral and maxillofacial tissue engineering in the field of soft tissue repair, and shows that low-immunogenic vesicles can be used as a promising treatment method to eliminate mucosal damaged tissue.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for restoring the epithelial structure of a lesion area, characterized in that: The invention comprises cell-derived low-immunogenic vesicles, which are positive for CD63, CD9 and TSG101 and negative for Calnexin, and have a cup-shaped double-layer membrane structure, and the particle size thereof is in the range of 30-150 nm.

2. The composition for restoring epithelial structure of a lesion area according to claim 1, characterized in that: The cells are selected from at least one of totipotent stem cells, pluripotent stem cells and unipotent stem cells.

3. The composition for restoring epithelial structure of a lesion area according to claim 1, characterized in that: The recovery includes at least one of the following: Increase the amount of cells and / or keratin 5 in the epithelial layer; Activates ion transport metabolism in the epithelium; promotes angiogenesis in the epithelium; Restores epithelial structural integrity.

4. The composition for restoring epithelial structure of a lesion area according to claim 1, characterized in that: The low immunogenic vesicles are derived from umbilical cord cells.

5. The composition for restoring epithelial structure of a lesion area according to claim 1, characterized in that: The low immunogenic vesicles are obtained by cell centrifugation.

6. A method for preparing the composition according to any one of claims 1 to 5.

7. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicament for restoring the epithelial structure of a lesion area.

8. The use according to claim 7, characterized in that The lesion comprises at least one of a surgical wound, a penetrating wound, a venous ulcer, an arterial ulcer, a laceration, a pressure ulcer, a crush injury, a shear injury, a leg ulcer, a foot ulcer, an abrasion, a puncture, a burn, a diabetic ulcer, an oral ulcer, a contusion, a trauma, a pressure ulcer, an ischemic ulcer, a laceration and a bite wound.

9. A method for regulating cell activity in vitro, characterized in that: The method comprises the steps of contacting cells with low immunogenic vesicles in vitro, wherein the cell activity comprises cell proliferation, cell migration and / or gene expression changes; The low immunogenic vesicles are CD63, CD9 and TSG101 positive and Calnexin negative, and have a cup-shaped double-layer membrane structure with a particle size ranging from 30 to 150 nm.

10. The method for regulating cell activity in vitro according to claim 9, characterized in that: The gene expression changes include down-regulation of chemokines CCL3, CXCL1, CXCL2, IL6 and IL-1β and / or up-regulation of keratins CK5, CK13, KRT13, KRT2, KRT24 and KRT14.

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