Application of exosome derived from human dental pulp stem cells in preparation of medicine
Drug preparation through exosomes derived from human pulp stem cells has been solved, and the problem of major side effects in traditional skin inflammation and psoriasis treatment has been achieved, and safe and effective skin inflammation and psoriasis treatment has been achieved.
Patent Information
- Application Number
- CN202510819637.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-08-12
AI Technical Summary
Traditional drugs for treating skin inflammation and psoriasis have problems such as major side effects, liver and kidney toxicity and metabolic difficulties, and long-term use has limited its application.
Exosomes derived from human pulp stem cells are used as drug components, and purified exosomes are extracted and purified through specific centrifugation and mixing steps, and are used to prepare drugs for the treatment of skin inflammation and psoriasis, leveraging their powerful immune regulation and tissue penetration capabilities.
Effectively inhibit excessive inflammatory response of the skin, repair skin damage, promote the recovery of the lesion area, provide safe and effective biological intervention measures, and avoid the side effects of traditional drugs.
Smart Images
Figure CN120459138A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of tissue engineering repair and regeneration technology, and in particular to the use of exosomes derived from human dental pulp stem cells in the preparation of medicines. Background Art
[0002] Inflammatory skin diseases are characterized by chronic inflammation and immune dysfunction. Clinical manifestations include local inflammatory infiltration of skin tissue, disordered tissue structure, and damaged skin barrier, resulting in local tissue function and morphological disorders. In addition, as one of the largest organs in the human body, the skin can serve as an immune protection organ to maintain the homeostasis of the entire body. When the function of skin tissue is severely damaged, it can induce systemic inflammatory diseases. Psoriasis is a common chronic inflammatory skin disease, also known as psoriasis, with a global prevalence of approximately 3%. The cause is relatively complex. It is currently believed that the severity of the cellular immune response is positively correlated with the severity of the disease. In addition to affecting skin tissue, the lesions can often affect other organs and tissues. In particular, psoriasis patients are often accompanied by psoriatic arthritis, and may also be accompanied by hypertension, cardiovascular disease, and other serious threats to the patient's physical and mental health.
[0003] Current psoriasis treatments primarily include hormones, immunosuppressants, and antibiotics. While these medications can modulate the immune system and rapidly alleviate symptoms like inflammation and itching, long-term use can cause numerous side effects, including skin atrophy and thinning, drug dependency, and recurrence upon discontinuation. They are also highly toxic and can cause adverse reactions such as liver and kidney damage, requiring long-term monitoring of liver and kidney function and blood tests, limiting their long-term use. In recent years, stem cells, due to their unique immunomodulatory and tissue repair properties, have been applied in a variety of fields with promising results. To fully leverage the advantages of stem cells while avoiding potential risks such as tumorigenicity, immune responses, and allergic reactions, recent studies have highlighted the potential of stem cell-derived exosomes as novel biotherapeutics.
[0004] Exosomes are tiny vesicles secreted by cells with diameters ranging from 40nm to 160nm. They are the main paracrine components of cells and can act as communication media to deliver bioactive substances from donor cells to the surrounding tissue. Dental pulp stem cells are mesenchymal stem cells with multidirectional differentiation potential derived from dental pulp tissue. They are easily accessible and have good immunomodulatory properties. As donor cells, the exosomes secreted by dental pulp mesenchymal stem cells also have excellent therapeutic effects. Compared with exosomes derived from other mesenchymal stem cells, exosomes derived from dental pulp mesenchymal stem cells have stronger immunomodulatory activity and are more easily absorbed into cells to function. However, their application in the inflammatory skin disease psoriasis has not yet been reported. Summary of the Invention
[0005] The purpose of the present invention is to overcome the defects of traditional drugs for treating skin inflammation or psoriasis, which mainly include hormones, immunosuppressants, antibiotics, etc., which have large side effects, hepatotoxicity and kidney toxicity, and metabolic difficulties. Therefore, the present invention provides an application of exosomes derived from human dental pulp stem cells in the preparation of drugs, especially for the preparation of drugs for treating skin inflammation and the preparation of drugs for treating psoriasis. In addition, a safe and effective biological intervention measure is constructed to replace traditional treatment methods, effectively inhibit excessive inflammatory response of the skin, repair skin damage, and promote recovery of the lesion area, providing a new treatment method for patients with skin inflammation.
[0006] In order to achieve the above-mentioned object, the present invention provides a use of exosomes derived from human dental pulp stem cells in the preparation of a medicine.
[0007] Preferably, the exosomes derived from human dental pulp stem cells are used in the preparation of a drug for treating skin inflammation.
[0008] Preferably, the exosomes derived from human dental pulp stem cells are used in the preparation of a drug for treating psoriasis.
[0009] Preferably, the method for extracting exosomes derived from human dental pulp stem cells comprises: 1) Centrifuge the dental pulp stem cell culture medium for the first time, collect the first supernatant, and centrifuge it for the second time, collect the second supernatant; 2) The second supernatant is mixed with the exosome extraction reagent, allowed to stand, centrifuged for a third time, the third supernatant is discarded, PBS buffer is added to resuspend the exosome particles, centrifuged for a fourth time, and the fourth supernatant is retained to obtain the purified exosomes.
[0010] Preferably, the conditions for the first centrifugation include: temperature of 2°C to 6°C, centrifugal force of 2500×g to 3500×g, and time of 10 min to 15 min.
[0011] Preferably, the conditions for the second centrifugation include: temperature of 2°C to 6°C, centrifugal force of 8000×g to 12000×g, and time of 10 min to 15 min.
[0012] Preferably, the standing conditions include: a temperature of 2°C to 6°C and a time of 12-24 hours.
[0013] Preferably, the conditions for the third centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 8000×g to 12000×g, and time of 40 min to 80 min.
[0014] Preferably, the conditions for the fourth centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 10,000×g to 15,000×g, and time of 1 min to 5 min.
[0015] In the above technical solution, the application of exosomes derived from human dental pulp stem cells in the preparation of drugs, wherein the exosomes derived from human dental pulp stem cells have the advantages of abundant sources and strong immune regulation ability, and at the same time, have good tissue penetration ability and few side effects, which can provide a new treatment for long-term skin inflammation and psoriasis, and have broad application prospects and market value.
[0016] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 This is an experimental flow chart of Example 2 of the present invention for the application of exosomes derived from human dental pulp stem cells in a psoriasis mouse model; Figure 2 These are pictures of normal mice in Example 2 of the present invention, a psoriasis mouse model treated with imiquimod (IMQ) combined with PBS (control group), a psoriasis mouse model treated with imiquimod (IMQ) combined with exosomes (Exo), and a psoriasis mouse model treated with imiquimod (IMQ) combined with 0.1% tacrolimus at different time points; Figure 3 For the normal mice in Example 2 of the present invention, the psoriasis mouse model was treated with imiquimod (IMQ) combined with PBS (control group), the psoriasis mouse model was treated with imiquimod (IMQ) combined with exosomes (Exo), and the psoriasis mouse model was treated with imiquimod (IMQ) combined with 0.1% tacrolimus (tacrolimus). Changes in PASI index and body weight at different time points; Figure 4 This is the angiogenesis of the back skin of mice in different treatment groups on the 8th day in Example 2 of the present invention; Figure 5 HE staining of the back skin sections of mice in different treatment groups on day 8 in Example 2 of the present invention; Figure 6 Quantitative statistical diagram of angiogenesis in the back skin of mice in different treatment groups on day 8 in Example 2 of the present invention, where *P<0.05, **P<0.01; Figure 7 Quantification of the full-thickness skin thickness, dermis thickness, and epidermis thickness of the back skin sections of mice in different treatment groups on day 8 in Example 2 of the present invention, where *P<0.05, **P<0.01; Figure 8 The expression of psoriasis-related cytokines in Example 2 of the present invention, including the expression of Ki67 (brown), a proliferation indicator, in different groups; the expression of KRT-10 (green), a terminal cell differentiation indicator, in different groups; the expression of the pro-inflammatory factor IL-23A (red), in different groups; and the expression of the vascular indicator CD31 (green), in different groups. Figure 9 The expression of psoriasis-related cytokines in Example 2 of the present invention, including the expression of the antimicrobial peptide S100A8 (green) in different groups, the expression of the proinflammatory factor IL-23R (green) in different groups, the expression of the neutrophil marker Ly6G (green) in different groups, and the expression of the proinflammatory factor IL-17A (green) in different groups; Figure 10 This is a statistical graph of the proportion of positively stained cells corresponding to psoriasis-related cytokines in Example 2 of the present invention, where *P<0.05, **P<0.01, ***P<0.001; Figure 11 The results of the expression of psoriasis-related cytokine mRNA levels in Example 2 of the present invention are as follows: the back skin of mice in different treatment groups was collected on the 8th day after treatment, and RNA was isolated and qRT-PCR was performed to detect the mRNA expression of proinflammatory factors (IL-17A, IL-6, IL-23A, IL-1β, TNF-α), chemokine (CXCL-1) and antimicrobial peptides (S100A8, S100A9). Among them, *P<0.05, **P<0.01, ***P<0.001; Figure 12 The results of the expression of psoriasis-related cytokine protein levels in Example 2 of the present invention are as follows: on the 8th day of treatment, the dorsal skin of mice in different treatment groups was collected, tissue proteins were extracted, and the protein expression of pro-inflammatory factors (IL-17A, IL-23R) and antimicrobial peptides (S100A8, S100A9) was detected; Figure 13 This is a statistical analysis chart of the psoriasis-related cytokine protein levels in Example 2 of the present invention, where *P<0.05, **P<0.01, ***P<0.001; Figure 14The expression levels of psoriasis-related cytokines mRNA in Example 3 of the present invention are shown. After 24 hours of treatment with epidermal stem cells in the control group (PBS group), Exo group, Exo+M5 group, and M5 group, RNA was isolated and qRT-PCR was performed to detect the mRNA expression of proinflammatory factors (IL-1α, IL-6, IL-23A, IL-1β, TNF-α), chemokine (CCL-20), and antimicrobial peptides (S100A8, S100A9). *P<0.05, **P<0.01, ***P<0.001. Figure 15 The expression levels of psoriasis-related cytokine proteins in Example 3 of the present invention were analyzed by Western blot analysis of proteins extracted from epidermal stem cells after 24 hours of treatment with the control group (PBS group), Exo group, Exo+M5 group, and M5 group to detect the expression of pro-inflammatory factors (IL-17A, IL-23R), antimicrobial peptides (S100A8, S100A9), differentiation marker KRT-10, and epidermal proliferation marker KRT-5. Figure 16 This is a statistical analysis chart of the expression levels of psoriasis-related cytokine proteins in Example 3 of the present invention, where *P<0.05, **P<0.01, ***P<0.001; Figure 17 This is an experimental flow chart of Example 4 of the present invention; Figure 18 After culturing the foreskin tissue of the control group (PBS group), M5 group, and Exo+M5 group in Example 4 of the present invention for 24 hours, RNA was isolated and the mRNA expression of proinflammatory factors (IL-1α, IL-8, IL-23A, IL-1β, IL-17A) and antimicrobial peptides (S100A8, S100A9) was detected by qRT-PCR experiments. Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION
[0018] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.
[0019] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.
[0020] The present invention provides a use of exosomes derived from human dental pulp stem cells in the preparation of medicines.
[0021] In a preferred embodiment of the present invention, the exosomes derived from human dental pulp stem cells are used in the preparation of a drug for treating skin inflammation.
[0022] In a preferred embodiment of the present invention, the exosomes derived from human dental pulp stem cells are used in the preparation of a drug for treating psoriasis.
[0023] The use of exosomes derived from human dental pulp stem cells in the preparation of drugs of the present invention has the advantages of abundant sources and strong immune regulation ability. At the same time, it has good tissue penetration ability and few side effects. It can provide a new treatment for long-term skin inflammation and psoriasis, and has broad application prospects and market value.
[0024] In a preferred embodiment of the present invention, the method for extracting exosomes derived from human dental pulp stem cells comprises: 1) Centrifuge the dental pulp stem cell culture medium for the first time, collect the first supernatant, and centrifuge it for the second time, collect the second supernatant; 2) The second supernatant is mixed with the exosome extraction reagent, allowed to stand, centrifuged for a third time, the third supernatant is discarded, PBS buffer is added to resuspend the exosome particles, centrifuged for a fourth time, and the fourth supernatant is retained to obtain the purified exosomes.
[0025] In a preferred embodiment of the present invention, the conditions for the first centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 2500×g to 3500×g, and time of 10 min to 15 min.
[0026] In a preferred embodiment of the present invention, the conditions for the second centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 8000×g to 12000×g, and time of 10 min to 15 min.
[0027] In a preferred embodiment of the present invention, the standing conditions include: a temperature of 2° C. to 6° C., and a time of 12-24 hours.
[0028] In a preferred embodiment of the present invention, the conditions for the third centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 8000×g to 12000×g, and time of 40 min to 80 min.
[0029] In a preferred embodiment of the present invention, the conditions for the fourth centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 10,000×g to 15,000×g, and time of 1 min to 5 min.
[0030] The present invention will be described in detail below by way of examples. In the following examples, the drugs and pharmaceuticals are all conventional commercial products. Example 1
[0031] This example is used to illustrate the separation and extraction of exosomes from human dental pulp stem cells: (1) Transfer the collected dental pulp stem cell culture medium to a centrifuge tube and centrifuge it at 3000×g for 10 min at 4°C to remove cell debris. Transfer the centrifugal supernatant to a new centrifuge tube and centrifuge it at 10,000×g for 10 min at 4°C to remove impurities and debris in the sample. (2) The supernatant after removing impurities and debris was transferred to a new centrifuge tube and ECS reagent (Exosome Concentration Solution) was added. The tube was allowed to stand at 4°C for 18 hours, and then centrifuged at 10,000 × g for 60 min at 4°C. The supernatant was discarded. The remaining precipitate was rich in exosome particles. The exosome particles were resuspended in PBS buffer and placed in a 1.5 mL centrifuge tube. The tube was centrifuged at 12,000 × g for 2 min at 4°C. The supernatant was retained to obtain the purified exosomes (Exo). The tube was aliquoted and frozen in a -80°C low-temperature refrigerator for later use. Example 2
[0032] This example illustrates the application of exosomes derived from human dental pulp stem cells in a psoriasis mouse model.
[0033] (1) Eight-week-old male or female BALB / c mice were selected and placed in a deep anesthesia state by inhalation anesthesia. The back skin was then depilated, and 62.5 mg of imiquimod (IMQ) was applied to the back of each mouse daily to establish a psoriasis mouse model. (2) Starting from the fourth day of IMQ application, the exosomes (Exo) extracted in Example 1, 0.1% tacrolimus, and PBS were applied to the back of the mice, respectively. After at least 1 hour, 62.5 mg of IMQ was applied to the same site; (3) Mice were killed on the 8th day after IMQ treatment, and psoriasis skin specimens were collected for subsequent experiments. Changes in skin thickness were detected by HE staining, and the expression of inflammatory response-related factors was detected by tissue section staining; (4) The skin tissues of mice in the back modeling area were collected and RNA and protein were extracted respectively. The expression of inflammatory factors was detected at the mRNA and protein levels respectively.
[0034] The results are as follows Figure 1-13 shown.
[0035] like Figure 5 As shown, the thickness of the skin, epidermis and dermis of the combined treatment group of exosomes and IMQ (Exo+IMQ group) was significantly reduced compared with the control group (IMQ+PBS group), and the effect was similar to that of the positive control group (0.1% tacrolimus+IMQ group), indicating that the exosomes of Example 1 of the present invention have a significant therapeutic effect on psoriasis.
[0036] like Figure 8 As shown, compared with the PBS+IMQ group, the Exo+IMQ group was observed to significantly reduce the number of Ki67-positive cells (brown) in the proliferating epidermal basal layer, and the Exo+IMQ group restored the terminal capacity of epidermal cells, with an increased expression of KRT-10 (green), a differentiation indicator; In addition, Figure 9 As shown, compared with the PBS+IMQ group, the expression of pro-inflammatory factors IL-23A (red), IL-23R (green), IL-17A (green), neutrophil marker Ly6G (green), and antimicrobial peptide S100A8 (green) related to immune response was significantly reduced in the Exo+IMQ group. The expression of CD31 (green), an indicator of vascular induction due to inflammation, was significantly reduced in the Exo+IMQ group. like Figure 11 and Figure 13 As shown in the data, compared with the control group (PBS+IMQ group), the mRNA and protein expression levels of inflammatory factors and antimicrobial peptides in the Exo+IMQ group were significantly reduced, and the effect was similar to that of the positive control group (0.1% tacrolimus+IMQ group). Example 3
[0037] This example is used to illustrate the application of exosomes derived from human dental pulp stem cells in an in vitro psoriasis-like cell model; Epidermal stem cells were induced with 10 ng / ml-M5 (IL-1α, IL-17A, IL-22, OSM and TNF-α) to establish a psoriasis-like in vitro cell model. The cells were divided into the following groups: Control group (PBS group): epidermal stem cells were treated with PBS buffer; Exo group: epidermal stem cells were treated with exosomes extracted from Example 1; Exo+M5 group: The exosomes extracted in Example 1 were used to treat the M5-induced psoriasis-like in vitro cell model; M5 group: epidermal stem cells treated with M5 were used to establish the psoriasis-like in vitro cell model; The above cell RNA and protein were collected, and the expression of mRNA and protein levels of cytokines related to inflammatory response were detected by qRT-PCR and Western Blot technology. The results are as follows Figure 14-16 shown.
[0038] Depend on Figure 14-16 The results show that the exosomes of the present invention can effectively inhibit the mRNA and protein expressions of various inflammatory factors and antimicrobial peptides induced by M5, and the mRNA and protein expressions of various inflammatory factors and antimicrobial peptides in the exosomes alone treatment group did not show significant changes compared with the blank group. Example 4
[0039] This example is used to illustrate the application of exosomes derived from human dental pulp stem cells in an in vitro psoriasis-like skin model.
[0040] The psoriasis-like in vitro skin model was established by inducing the foreskin tissue from the foreskin tissue by 10 ng / ml-M5 (IL-1a, IL-17A, IL-22, OSM or TNF-α). Control group (PBS group): PBS buffer was used to treat the in vitro cultured foreskin tissue; Psoriasis-like in vitro skin model group (M5 group): The in vitro cultured foreskin tissue was treated with M5 to establish the psoriasis-like in vitro skin model; Exo+M5 group: The exosomes extracted in Example 1 were used to treat the M5-induced psoriasis-like in vitro skin model; The expression of mRNA levels of cytokines related to inflammatory response was detected by qRT-PCR technology. Figure 18 shown.
[0041] Depend on Figure 18 It can be seen from the content that the exosomes of the present invention can effectively inhibit the expression of pro-inflammatory factors (IL-1α, IL-8, IL-23A, IL-1β, IL-17A) and antimicrobial peptides (S100A8, S100A9) mRNA induced by M5.
[0042] In summary, the exosomes derived from human dental pulp stem cells of the present invention can be used to prepare drugs for treating skin inflammation, especially for treating psoriasis. The prepared drugs have significant therapeutic effects on psoriasis and do not have the side effects of hormonal drugs, providing a better choice for patients with dermatitis and psoriasis.
[0043] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.
[0044] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.
[0045] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.
Claims
1. Application of exosomes derived from human dental pulp stem cells in the preparation of drugs.
2. The use according to claim 1, characterized in that Use of the exosomes derived from human dental pulp stem cells in the preparation of a drug for treating skin inflammation.
3. The use according to claim 1, characterized in that Use of the exosomes derived from human dental pulp stem cells in the preparation of a drug for treating psoriasis.
4. The use according to any one of claims 1 to 3, characterized in that The method for extracting exosomes derived from human dental pulp stem cells comprises: Centrifuge the dental pulp stem cell culture medium for the first time to obtain the first supernatant, centrifuge it for the second time to obtain the second supernatant; The second supernatant was mixed with the exosome extraction reagent, allowed to stand, centrifuged for a third time, the third supernatant was discarded, PBS buffer was added to resuspend the exosome particles, centrifuged for a fourth time, and the fourth supernatant was retained to obtain the purified exosomes.
5. The use according to claim 4, characterized in that The conditions for the first centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 2500×g to 3500×g, and time of 10 min to 15 min.
6. The use according to claim 4, characterized in that The conditions for the second centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 8000×g to 12000×g, and time of 10 min to 15 min.
7. The use according to claim 4, characterized in that The standing conditions include: a temperature of 2° C. to 6° C. and a time of 12-24 hours.
8. The use according to claim 4, characterized in that The conditions for the third centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 8000×g to 12000×g, and time of 40 min to 80 min.
9. The use according to claim 4, characterized in that The conditions for the fourth centrifugation include: temperature of 2° C. to 6° C., centrifugal force of 10,000×g to 15,000×g, and time of 1 min to 5 min.