Use of plasma-derived exosomes for the preparation of a medicament for improving or treating oral diseases

Abstract

The application discloses application of plasma-derived exosomes in preparation of drugs for improving or treating oral diseases. The plasma-derived exosomes have good biological effects of inhibiting inflammation, promoting angiogenesis and accelerating lesion healing, can accelerate healing of mucosal injury by inhibiting release of inflammatory factors and chemotactic factors, and thus reduce progress of inflammation. In addition, the plasma-derived exosomes can promote proliferation of human oral epithelial cells and healing of scratches, and have good effects in improving oral diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of plasma-derived exosomes in preparation of a drug for improving or treating oral diseases. BACKGROUND

[0002] Oral diseases are a general term for various diseases involving the oral cavity and surrounding tissues, including gingivitis, periodontitis, oral inflammation or oral ulcer, etc. Oral diseases are relatively common diseases, which can cause discomfort to the infected patients, affect the quality of life of the patients, and seriously interfere with the daily life of the patients, such as diet, speech, etc., and further harm the mental health of the patients. There are many causes of oral diseases, such as microorganisms, wounds, low immune function, etc.

[0003] Oral ulcer is a common oral mucosa disease, which is obviously painful when it occurs, and has a burning sensation locally. However, the etiology of oral ulcer is complex, and the clinical treatment is mainly local anti-inflammatory and analgesic, including the use of analgesics, hormones, gargling liquid, and low-intensity laser therapy, etc., but the curative effect is limited. Therefore, it is of great clinical significance to seek an effective therapy to relieve the symptoms of oral ulcer and promote the healing of the lesion. SUMMARY

[0004] To solve at least part of the problems in the prior art, the present application prepares exosomes by plasma separation, which accelerates the healing of damaged tissues in oral diseases by inhibiting the release of inflammatory factors and chemotactic factors, thereby slowing down the progression of the disease. Specifically, the present application includes the following contents.

[0005] In a first aspect of the present application, application of plasma-derived exosomes in preparation of a drug for improving or treating oral diseases is provided.

[0006] In certain embodiments, according to the application, the oral disease refers to a disease associated with up-regulation of pro-inflammatory factors and / or down-regulation of anti-inflammatory factors.

[0007] In certain embodiments, according to the application, the pro-inflammatory factors include at least one of Eotaxin, IL-12p70, IL-5, IP-10, IL-1α, IL-18, TNF-α, MIP-1α, IL-2, Fractalkine, IL-1β and IL-17.

[0008] In certain embodiments, according to the application, the anti-inflammatory factors include IL-4 and / or IL-10.

[0009] In some embodiments, the application according to the present application, wherein the oral disease refers to a disease associated with oral epithelial cell proliferation, expression of keratin CK5 and / or CK13.

[0010] In some embodiments, the application according to the present application, wherein the particle size of the exosome is 50-250 nm.

[0011] In some embodiments, the application according to the present application, wherein the plasma-derived exosome is prepared by plasma extraction and / or ultracentrifugation.

[0012] In some embodiments, the application according to the present application, wherein the plasma-derived exosome is prepared by the following steps:

[0013] (1) collecting plasma;

[0014] (2) isolating plasma-derived exosomes.

[0015] In some embodiments, the application according to the present application, wherein the isolation comprises gradient centrifugation.

[0016] In a second aspect of the present application, a method for in vitro regulating oral-derived cell activity is provided, which comprises the step of contacting plasma-derived exosomes with in vitro cells.

[0017] The plasma-derived exosome of the present application has good biological effects of inhibiting inflammation, promoting angiogenesis and accelerating lesion healing, which accelerates the healing of mucosal injury by inhibiting the release of inflammatory factors and chemotactic factors, thereby reducing the progression of inflammation. In vitro experiments have confirmed that the plasma-derived exosome of the present application can promote human oral epithelial cell proliferation and wound healing. In summary, the plasma-derived exosome of the present application has good effects in improving oral diseases, and can be used as a promising treatment method to regulate, for example, the regenerative repair of mucosal injury. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Characterization of UCBP-Exo is shown. In the figure, A is a TEM image of UCBP-Exo, scale bar: 100 nm; B is a nanoparticle tracking analysis (NTA) image of UCBP-Exo; C is a nanoflow cytometry (nFCM) analysis image of UCBP-Exo; D is a Western blot detection of marker proteins of UCBP-Exo.

[0019] Figure 2The in vivo toxicity evaluation of UCBP-Exo is shown. Figure A shows histopathological images of the heart, liver, spleen, lungs, and kidneys collected from the control group and the UCBP-Exo group, scale bar: 300 μm; B shows the complete blood count of the control group and the UCBP-Exo group; C shows the performance of various blood biochemical indicators in different treatment groups, ns (p>0.05).

[0020] Figure 3 The therapeutic effect of UCBP-Exo in a rat oral ulcer model is shown. Figure A shows the observation of the recovery of oral mucosal defects in rats using UCBP-Exo on days 2 and 4. Scale bar: Top Figure 4 Figure 6mm below; B shows the injury healing rate after 2 or 4 days of UCBP-Exo treatment; C shows the histological observation of rat oral mucosal defects after 2 and 4 days of UCBP-Exo treatment, scale bar: 400μm; D shows the multiplex fluorescence immunohistochemistry (mIHC) staining after 2 or 4 days of treatment with different UCBP-Exo, scale bar: 500μm; E shows the multiplex detection of cytokines and chemokines in rat serum under different treatments, ns (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0021] Figure 4 The mechanism by which UCBP-Exo promotes the healing of oral mucosal defects is illustrated. Figure A is a heatmap of differentially expressed genes (DEGs) in rat mucosal tissues showing fold changes in gene expression between the control and UCBP-Exo groups; B is a bubble diagram showing the inflammation-related KEGG pathway in the mucosal tissues of the UCBP-Exo treatment group and the control group; C is a volcano plot analysis of DEGs between different groups in the mucosal tissues; D is the DEGs in the UCBP-Exo treatment group and the control group in GO enrichment analysis; E is the Western blot analysis of IL6 and CK5 in mucosal tissues after 4 days of different treatments; F is the mRNA expression of inflammation and cytokeratin markers in rat oral mucosa after 4 days of different treatments by RT-qPCR, ns (p>0.05), *p<0.05, **p<0.01.

[0022] Figure 5The biological effects of UCBP-Exo on HOECs are shown in Figure 1. Figure 1A shows the cell viability of the oral epithelial cells (HOECs) in different groups on day 1, day 2 and day 3, and each group of columns from left to right in the figure is the control group, the low-dose group and the high-dose group, *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; Figure 1B shows the scratch test of HOECs after different treatments, the scale bar is 400 μm; Figure 1C shows the quantitative analysis of the wound closure rate by measuring the gap size; Figure 1D shows the heatmap of DEGs of HOECs using the gene expression fold change between the control group and the UCBP-Exo group; Figure 1E shows the gene set enrichment analysis (GSEA) of DEGs after HOECs were treated for 24 hours with or without UCBP-Exo, NES represents the normalized enrichment score, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0023] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The detailed description is intended to be read with the understanding that both the comprehensive and careful presentation of the details as well as the careful presentation of the completely detailed description are not intended to limit the scope of the application but merely to illustrate certain aspects of the application.

[0024] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term “about” in relation to a value or a range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0026] Application

[0027] In one aspect of the present application, the use of plasma-derived exosomes in the preparation of a medicament for improving or treating oral diseases is provided.

[0028] In the present application, examples of the plasma include, but are not limited to, venous blood plasma, such as median cubital venous blood plasma, dorsal hand venous blood plasma; cord blood plasma (CBP) of human umbilical cord blood (HUCB), and the like. In a preferred embodiment, the plasma is cord blood plasma.

[0029] In the present application, the oral disease refers to a disease associated with up-regulation of pro-inflammatory factors and / or down-regulation of anti-inflammatory factors, examples of which include, but are not limited to, gingivitis, periodontitis, stomatitis, or oral ulcer, and the like. In a preferred embodiment, the oral disease is oral mucosa disease. In yet another preferred embodiment, the oral disease is oral ulcer.

[0030] In the present application, the pro-inflammatory factor includes, but is not limited to, one or a combination of Eotaxin, IL-12p70, IL-5, IP-10, IL-1α, IL-18, TNF-α, MIP-1α, IL-2, Fractalkine, IL-1β, and IL-17. In a preferred embodiment, the plasma-derived exosome of the present application is capable of down-regulating the pro-inflammatory factor.

[0031] In the present application, the anti-inflammatory factor includes, but is not limited to, IL-4 and / or IL-10. In a preferred embodiment, the plasma-derived exosome of the present application is capable of up-regulating the anti-inflammatory factor.

[0032] In the present application, the oral disease refers to a disease associated with proliferation of oral epithelial cells and expression of keratin CK5 and / or CK13. In a preferred embodiment, the plasma-derived exosome of the present application is capable of promoting expression of keratin CK5 and / or CK13.

[0033] In the present application, the particle size of the exosome is 50-250 nm, preferably 70-220 nm, more preferably 90-190 nm, further preferably 100-160 nm, more preferably 100-130 nm, such as 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 nm.

[0034] The term "ameliorating or treating" as used herein refers to improving the condition (e.g., promoting wound healing) before or after the occurrence of a disease or disorder in a subject. The degree of such amelioration or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as compared to a reference group under the same conditions but without treatment, as measured by any standard technique.

[0035] In the present application, the plasma-derived exosome is prepared by extraction and / or ultracentrifugation from plasma.

[0036] In a preferred embodiment, the plasma-derived exosomes are prepared by the following steps: (1) collecting plasma; (2) isolating plasma-derived exosomes. In a particular embodiment, the isolation in step (2) comprises ultracentrifugation.

[0037] In one specific embodiment, the plasma-derived exosomes are prepared by the following steps: umbilical cord blood is centrifuged at 800-1500 g, preferably 900-1400 g, for example 900, 1000, 1100, 1200, 1300 g for 10-20 min, preferably 11-19 min, further preferably 12-18 min, for example 12, 13, 14, 15, 16, 17, 18 min to collect the plasma, then the plasma is centrifuged at 2000-4000 g, preferably 2100-3900 g, further preferably 2200-3800 g, further preferably 2300-3700 g, more preferably 2400-3600 g, more preferably 2500-3500 g, for example 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500 g for 20-40 min, preferably 21-39 min, further preferably 22-38 min, further preferably 23-37 min, more preferably 24-36 min, more preferably 25-35 min, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 min. The supernatant is then centrifuged at 9000-11000 g, preferably 9200-10800 g, further preferably 9400-10600 g, more preferably 9600-10400 g, for example 9600, 9700, 9800, 9900, 10000 g, 10100 g, 10200 g, 10300 g, 10400 g for 20-40 min, preferably 21-39 min, further preferably 22-38 min, further preferably 23-37 min, more preferably 24-36 min, more preferably 25-35 min, for example 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 min. This is followed by ultracentrifugation at 90000-110000 g, preferably 92000-108000 g, further preferably 94000-106000 g, more preferably 96000-104000 g, for example 96000, 97000, 98000, 99000, 100000 g, 101000 g, 102000 g, 103000 g, 104000 g for 80-100 min, preferably 81-99 min, further 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.The precipitated exosomes are washed twice with saline and centrifuged 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 60-80min, preferably 61-79min, further preferably 62-78min, further preferably 63-77min, more preferably 64-76min, more preferably 65-75min, for example 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75min, and then resuspended in saline. The exosomes are prepared by filtering the suspension through a filter (including but not limited to a 0.15-0.3μm filter, for example a 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, 0.30μm filter). All operations are carried out at 4℃.

[0038] The present application also provides use of the plasma-derived exosomes in the preparation of a soft tissue repair drug.

[0039] Method

[0040] In one aspect of the present application, a method for in vitro regulation of oral-derived cell activity comprises the step of contacting plasma-derived exosomes with in vitro cells.

[0041] In the present application, examples of in vitro cells include but are not limited to periodontal ligament cells, salivary gland cells, oral mucosa cells, oral epithelial cells, etc. In a specific embodiment, the in vitro cells are human oral epithelial cells.

[0042] In the present application, the plasma-derived exosomes are derived from blood cells, exhibit similar physiological functions as blood cells, mediate cell-to-cell communication by delivering nucleic acids and proteins encapsulated therein to recipient cells, and can more effectively affect recipient cells than transcription factors, thereby having good biological effects of inhibiting inflammation, promoting angiogenesis, and accelerating lesion healing. In a specific embodiment, the plasma-derived exosomes of the present application are derived from umbilical cord blood. Exosomes derived from human umbilical cord blood have good biological safety, are stable and easy to store, and are easy to produce on a large scale.

[0043] In the method for in vitro modulating activity of oral-derived cells according to the present application, the modulating activity further comprises modulating the amount of genes / proteins, preferably, the modulating the amount of genes / proteins comprises down-regulation of chemokines CCL3, CXCL1, IL6 and IL-1β and up-regulation of keratins CK5, CK13.

[0044] The present application therefore also provides a method for in vitro modulating the amount of genes / proteins of a cell, comprising the step of contacting the cell with plasma-derived exosomes in vitro, wherein the modulating the amount of genes / proteins comprises down-regulation of chemokines CCL3, CXCL1, IL6 and IL-1β and up-regulation of keratins CK5, CK13.

[0045] Example 1

[0046] The preparation and characterization of plasma-derived exosomes are shown below.

[0047] 1. Preparation method

[0048] Umbilical cord blood was transferred into a 50 mL centrifuge tube and centrifuged at 1200 g for 15 min to collect the plasma. The plasma was then centrifuged at 3000 g for 30 min. The supernatant was then centrifuged at 10000 g for 30 min, followed by ultracentrifugation at 100000 g for 90 min. The precipitated exosomes were washed twice with saline and centrifuged at 100000 g for 70 min, and then resuspended in saline. The exosomes (UCBP-Exo) were prepared by filtering the suspension through a 0.22 μm filter. All operations were carried out at 4°C.

[0049] 2. Characterization

[0050] The morphology of UCBP-Exo was observed by transmission electron microscopy (TEM). The particle size of UCBP-Exo was determined by nanoparticle tracking analysis (NTA) and nanoflow cytometry (nFCM). The surface markers CD9, CD63, connexin and TSG101 of UCBP-Exo were identified by Western blotting.

[0051] UCBP-Exo was derived from umbilical cord blood and obtained by ultracentrifugation, and the double-membrane ultrastructure of UCBP-Exo was observed by transmission electron microscopy, and the results showed that it had a cup-shaped membrane structure, with clear edges and morphology (Fig. 1A). Figure 1 The nanoparticle tracking analysis showed that the particle size of UCBP-Exo was mainly concentrated at 112.9 nm (Fig. 1B). Figure 1 In addition, the results of nanoflow cytometry were similar to those of NTA (Fig. 1C). Figure 1C). Finally, the transmembrane proteins CD63 and CD9, the cytoplasmic protein TSG101 and the negative protein Calnexin were detected by Western blotting, and the data showed that positive expression of CD63, CD9 and TSG101 and negative expression of Calnexin were successfully detected Figure 1 D). The results showed that the UCBP-Exo was successfully prepared and could be used for further research.

[0052] Example 2

[0053] The following examples show the effect of UCBP-Exo on wounds and ulcers.

[0054] I. Experimental Methods

[0055] 1. Rat ulcer model and treatment

[0056] 4-6 week old SD rats were divided into two groups, namely the control group (no UCBP-Exo treatment at the ulcer site) and the UCBP-Exo group (ulcer treated by spraying liquid containing UCBP-Exo). After anesthesia by intraperitoneal injection of 100 mg / kg sodium pentobarbital, a 4.5 mm diameter filter paper soaked with 50% glacial acetic acid was attached to the buccal mucosa on both sides of the oral cavity for 30 s, and then the residual glacial acetic acid was wiped off with a sterile cotton ball containing saline. Twenty-four hours after the operation (0d), the lesions were treated accordingly every day for 4 consecutive days, during which the ulcer lesions were evaluated and observation photos were taken. Two days and four days after treatment with or without UCBP-Exo, the lesion healing rate was measured using ImageJ software. The lesion healing rate was calculated as follows: Lesion healing rate = (initial lesion area - lesion area at evaluation time point) / initial lesion area x 100%.

[0057] 2. Histological analysis

[0058] Rats were sacrificed after 4 days of treatment, and the main organs (heart, liver, spleen, lung, kidney) and buccal mucosa tissue around the lesions were collected, formalin-fixed, dehydrated, and paraffin-embedded. 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 UCBP-Exo toxicity and the effect on lesion healing.

[0059] 3. Rat blood examination

[0060] For routine blood tests, 1 ml of whole blood was collected and analyzed using a fully automatic blood analyzer.

[0061] For blood biochemical examination, rat whole blood was centrifuged at 3000 rpm for 10 minutes in a heparin sodium centrifuge tube and the plasma was separated. Biochemical analyzer Mindray BS-2000 was used for analysis.

[0062] 4. Multiplexed immunohistochemistry (mIHC) staining

[0063] According to the instructions of the mIHC staining kit, the sections were sequentially incubated with antibodies CD31, IL6 and CK5, and finally scanned with a laser confocal microscope. DAPI was used to label the nuclei, CD31 to assess angiogenesis, IL-6 to detect the degree of inflammation, and CK5 to assess epithelial regeneration.

[0064] The following primary antibodies were used: anti-CD31, anti-IL6, anti-CK5. LAS X was used to analyze the histological images.

[0065] 5. Detection of serum cytokines in rats

[0066] After 4 days of treatment, blood was collected from both 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 the serum samples of each group were calculated using a standard curve.

[0067] 6. RNA sequencing (RNA-seq) and validation

[0068] For RNA-seq, rat buccal mucosa tissues were collected after 4 days of treatment, and HOECs were collected after 24 h of treatment. Total RNA was extracted using TRIzol reagent, and 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 for reverse transcription with random primers to synthesize the first and second strands of cDNA. After that, the library was prepared by end repair, base A addition, sequencing adapter addition, magnetic bead screening recovery of 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 sequencing strategy of PE150.

[0069] To perform Western blot validation, the buccal mucosa tissue at the lesion site was thoroughly ground in liquid nitrogen, and the relative protein levels of IL6 and CK5 in the ulcer tissue of different treatments were detected. Briefly, protein lysate was added to the sample tissue, the protein concentration was detected and the protein was denatured. Then, electrophoresis, membrane transfer, blocking, antibody incubation and protein detection were performed sequentially. Finally, beta-actin was used as a total protein internal reference, and the protein expression of different groups was analyzed by Image Lab software.

[0070] 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. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. RT-qPCR was then performed using the QuantStudio3 system with the SYBR Green Master Mix kit. Primer sequences for the CCL3, IL6, IL-1β, CK5, and CK13 genes are shown in Table 1, with GAPDH used as an internal control gene.

[0071] Table 1 Primer sequences used for RT-qPCR analysis

[0072]

[0073] 7. Cell proliferation assay

[0074] HOEC proliferation was measured using a cell counting kit-8. HOECs were seeded (1 × 10⁻⁸). 4 Cells (100 cells / well) were cultured overnight at 37°C with different treatments (PBS, 10 μg / mL UCBP-Exo, 50 μg / mL UCBP-Exo) in 96-well plates. 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.

[0075] 8. Scratch test

[0076] HOECs were seeded in 6-well plates and incubated until fully confluent. The monolayer of HOECs was gently scraped with the tip of a 200 μl pipette and washed three times with PBS to remove isolated cells. After incubation for 0 and 12 hours with and without different concentrations of UCBP-Exo in the culture medium, wound closure was observed using an inverted microscope. The wound closure rate was quantified using ImageJ software and calculated as follows: Wound closure rate = (Initial wound area - Wound area at assessment time point) / Initial wound area × 100%.

[0077] II. Experimental Results

[0078] 1. In vivo toxicity evaluation of UCBP-Exo

[0079] To assess the biosafety of UCBP-Exo in vivo, rats were administered UCBP-Exo via gavage, and histological tests were performed first. No inflammation or histopathological abnormalities were observed in the heart, liver, spleen, lungs, and kidneys of the rats. Figure 2A), which indicates that UCBP-Exo did not cause significant organ damage in rats. Routine blood and blood biochemistry tests showed that UCBP-Exo treatment did not adversely affect hematological parameters compared to the control group Figure 2 B, C). In addition, no significant side effects were found during the entire UCBP-Exo treatment period, which means that UCBP-Exo is safe in in vivo therapy.

[0080] 2. Evaluation of the therapeutic effect of UCBP-Exo in a rat oral ulcer model

[0081] This example evaluates the effect of UCBP-Exo on oral ulcer healing. Rats were randomly divided into three groups, a filter paper containing 50% glacial acetic acid was placed on their lower lip mucosa, and the lesions were treated with UCBP-Exo 24 hours after injury. By the 4th day of treatment, oral ulcers in the UCBP-Exo group were essentially healed. The oral mucosa of the UCBP-Exo treatment group was bright red, while in the control group, the lesions healed slowly and the mucosa was still significantly edematous Figure 3 A, B). H&E staining showed that in the control group, the lesion area showed a small amount of epithelialization and loose and disorganized connective tissue, with diffuse inflammatory cell infiltration in the submucosal layer. In contrast, the UCBP-Exo group showed better mucosal healing, with complete thickening of the squamous epithelium and significantly reduced inflammatory cells compared to the control group Figure 3 C).

[0082] Angiogenesis reflects the degree of tissue reconstruction and restoration of mucosal function. Multiplex immunohistochemistry (mIHC) staining showed that the endothelial cell marker CD31 was less dense in the control group, indicating a lower level of vascularization. In contrast, the UCBP-Exo-treated group showed a significant increase in vascularized area. IL6 was used to assess the inflammatory response during lesion treatment. There was a large IL6-positive cell infiltration in the control group, indicating that UCBP-Exo has anti-inflammatory activity, thus contributing to the healing of damaged mucosal tissue. In addition, the expression of cytokeratin 5 (CK5) present in the epithelial layer was significantly increased in the UCBP-Exo group compared to the control group, confirming that UCBP-Exo treatment restored the original structure of the mucosa Figure 3 D).

[0083] As the wound healing proceeds, the secretion of cytokines at the injury site changes. The following evaluates the secretion of inflammatory factors, chemokines and growth factors in the serum of rats after UCBP-Exo treatment, and finds that the secretion of pro-inflammatory factors and chemokines such as TNFa, IL-1b, IL17, eosinophil chemotactic factor, IL12p70, IL5, IP-10, IL-1a, MIP-1a, IL2, MCP-1 and leptin in the control group is significantly increased. In addition, the concentration of secreted anti-inflammatory and growth factors, including IL4, IL10, VEGF, G-CSF, RANTES and EGF, is significantly increased in the UCBP-Exo treatment group compared with the control group Figure 3 E). The results show that UCBP-Exo can promote the regeneration of epithelial tissue, angiogenesis and anti-inflammatory response, thereby improving the pathological structure of oral ulcer.

[0084] 3. Mechanism of UCBP-Exo promoting oral mucosa defect healing

[0085] In order to further understand the internal mechanism and potential downstream signaling pathways of UCBP-Exo promoting the healing of oral mucosa lesions, RNA sequencing was performed on the oral mucosa tissue treated with or without UCBP-Exo. Differential gene analysis was performed on the mucosa tissue of the control group and the UCBP-Exo group respectively, and the results showed that there were significant differences in the expression of genes in the two groups of tissues, indicating that UCBP-Exo significantly affected the physiological function of the lesions Figure 4 A).

[0086] Inflammation is an important stage of the wound healing cascade, and long-term inflammation is harmful. KEGG analysis shows that the TNF signaling pathway and cytokine-cytokine receptor interaction are significantly enriched after UCBP-Exo treatment. 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 interaction can trigger multiple downstream signaling pathways, which can be involved in the regulation of cell growth, immune response, angiogenesis and tissue repair, aiming to restore internal microenvironment homeostasis. In summary, these data show that the mucosa tissue treated with UCBP-Exo has stronger immune activation compared with the control group Figure 4 B).

[0087] The volcano plot shows that the expression of chemokines CCL3, CXCL1, CXCL2 and inflammatory factors IL6 and IL-1b in the UCBP-Exo treatment group is down-regulated compared with the control group. Further confirms the anti-inflammatory effect of UCBP-Exo, and as for the UCBP-Exo group, the enriched BP terms are mainly concentrated on the ATP metabolic process Figure 4D), indicating that UCBP-Exo might accelerate tissue healing by regulating aerobic respiration of cells. Based on the in-depth analysis of RNA-seq, we performed RT-qPCR and western blot experiments, and the results clearly showed that the expression levels of inflammatory factors and chemotactic factors were reduced in the mucosal defect area treated with UCBP-Exo compared with the control group Figure 4 E, F). In summary, UCBP-Exo regulates immune signaling pathways to accelerate mucosal injury regeneration by reducing the release of inflammatory factors and chemotactic factors.

[0088] 4. Biological effects of UCBP-Exo on HOECs

[0089] To study the effect of UCBP-Exo in vitro, UCBP-Exo at concentrations of 10 μg / mL (low concentration) and 50 μg / mL (high concentration) were selected to treat HOECs. CCK8 experiment showed that UCBP-Exo promoted the proliferation of HOECs after 1, 2 and 3 days of treatment Figure 5 A), indicating that UCBP-Exo is biocompatible for in vitro application.

[0090] Scratch test showed that the migration ability of HOECs treated with UCBP-Exo was significantly enhanced compared with HOECs treated with PBS in the control group 12 hours after the scratch occurred Figure 5 B, C). In addition, the ability of UCBP-Exo to promote wound closure did not increase with increasing concentration, and effective healing could be achieved by only applying a low concentration of UCBP-Exo. The above results prove the characteristics of UCBP-Exo in promoting the migration of HOECs in vitro, further confirming their potential applicability in the treatment of oral mucosal diseases.

[0091] In addition, DEGs analysis was performed on HOECs in the control group and UCBP-Exo group, respectively, and the results showed that there were significant differences in gene expression between the two groups, indicating that UCBP-Exo significantly affected the physiological functions of HOECs Figure 5 D). Using gene set enrichment analysis, it was found that genes involved in the assembly of mitochondrial respiratory chain complex IV were enriched in HOECs treated with UCBP-Exo, indicating that UCBP-Exo affected aerobic respiration and energy metabolism, thereby regulating the biological effects in HOECs Figure 5 E).

[0092] III. Conclusion

[0093] Oral diseases are highly prevalent and affect the quality of life of patients, however, the traditional treatment has limited effect. The present application prepared UCBP-Exo, which has good biocompatibility and has anti-inflammatory activity in a rat oral ulcer model, thereby helping the healing of damaged epithelial tissue. UCBP-Exo can improve the pathological structure of the lesion area. In addition, RNA-seq shows that UCBP-Exo regulates immune signal transduction by reducing the release of inflammatory factors and chemotactic factors, thereby accelerating the repair of mucosal damage. In vitro experiments show that UCBP-Exo promotes the proliferation and scratch healing of HOECs, again confirming the wound healing ability of UCBP-Exo at the cellular level. In summary, this embodiment demonstrates that UCBP-Exo-based therapy has achieved good results in improving the recovery and regeneration of oral mucosal tissue, broadening the theory of oral and maxillofacial tissue engineering in the field of soft tissue repair, and showing that UCBP-Exo can be used as a promising treatment to eliminate damaged mucosal tissue.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacements for part of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of umbilical cord plasma-derived exosomes for the preparation of a medicament for the treatment of oral inflammatory diseases, characterized in that, The particle size of the exosome is concentrated at 112.9 nm, and CD63, CD9 and TSG101 are positively expressed and Calnexin is negatively expressed, The exosome is prepared by the following steps: (1) collecting plasma; (2) isolating plasma-derived exosomes by ultracentrifugation; Among them, the umbilical cord blood is centrifuged at 1200g for 15min to collect plasma, then the plasma is centrifuged at 3000g for 30min, then the supernatant is centrifuged at 10000g for 30min, followed by ultracentrifugation at 100000g for 90min, the precipitated exosomes are washed twice with saline and centrifuged at 100000g for 70min, then resuspended in saline, and the suspension is filtered through a filter to prepare the exosomes; The oral inflammation disease is oral ulcer inflammation.

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