CN112691186B

CN112691186B - Composition comprising mesenchymal stem cells and exosomes and application thereof in tissue repair

Info

Publication number: CN112691186B
Application number: CN202110001358.6A
Authority: CN
Inventors: 韩卫星; 曹燕; 黄玉燕; 张涛; 陈博
Original assignee: Guangdong Peptide Normal Stem Cell Biotechnology Co ltd
Current assignee: Guangdong Peptide Normal Stem Cell Biotechnology Co.,Ltd.
Priority date: 2021-01-04
Filing date: 2021-01-04
Publication date: 2021-09-10
Anticipated expiration: 2041-01-04
Also published as: CN112691186A

Abstract

The present invention relates to compositions comprising mesenchymal stem cells and exosomes and uses thereof in tissue repair. The repairing peptide provided by the invention is combined with adipose-derived stem cells and epidermal stem cell exosomes for use, so that the repairing peptide can effectively repair an ulcer model of a diabetic mouse, has remarkable effect of promoting the repair of diabetes loss, and has good application prospect and application value.

Description

Composition comprising mesenchymal stem cells and exosomes and application thereof in tissue repair

Technical Field

The invention relates to the field of biology, in particular to a composition containing mesenchymal stem cells and exosomes and application thereof in tissue repair.

Background

The chronic skin ulcer caused by the trauma is one of the causes of hospitalization of the diabetic, the condition of the chronic skin ulcer is complex and easy to repeat, serious complications can be caused, the treatment is relatively troublesome, the treatment method is limited, the morbidity rises year by year, and the chronic skin ulcer is a problem which is urgently overcome in the clinical practice at present. Wound healing is known to be a complex and delicate process, involving a variety of cells and molecules. The current research shows that over hundreds of physiological factors are related to poor healing of diabetic wounds, such as reduction of growth factors, reduction of angiogenesis, small number of macrophages, functional damage, abnormal collagen deposition, reduction of keratinocyte and fibroblast proliferation and the like.

Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, originating from the mesoderm and ectoderm in early developmental stages. MSCs were first found in bone marrow and clinically applied to the treatment of various hematological diseases, cardiovascular diseases, autoimmune diseases, etc., and have been receiving increasing attention because of their multipotential, hematopoietic support, immune regulation, and self-replication characteristics. Kuo and the like research the effect of mesenchymal stem cells from bone marrow in the wound healing of streptozotocin-induced enhanced diabetes rat models, and the results show that compared with a control group, the wound area of mice in a mesenchymal stem cell treatment group is obviously reduced, the complete healing time of the wound is shorter than that of the control group, and the comparison difference has statistical significance. Histological analysis shows that compared with a control group, the local proinflammatory reaction of the mesenchymal stem cell group is weakened, and the expression of CD45 is obviously inhibited. Immunohistochemical analysis indicated that the expression of epidermal growth factor, vascular endothelial growth factor, prolyl 4-hydroxylase was significantly increased in the treated group compared to the control group. Therefore, the mesenchymal stem cells can obviously enhance the healing of the diabetic wound, which provides a new idea for clinically searching an effective treatment method. Human umbilical cord blood-derived Endothelial Progenitor Cells (EPCs) are often used for the treatment of various ischemic diseases because they promote neovascularization. However, the positive role and the related mechanism of the compound in diabetic wound healing are rarely reported. Related researches are carried out by Kim and the like by taking nude mice with diabetes induced by streptozotocin as a model, and the proliferation of keratinocytes and fibroblasts in the diabetic wound surface of the EPCs transplantation group is found to be 3 days earlier than that of a PBS control group, so that the wound surface closure is remarkably accelerated.

In addition, the epithelization of the wound surface is very important for the healing of the wound surface, and related researches show that the epidermal stem cells have an important effect on the epithelization of the wound surface, so that the epidermal stem cells have positive significance for the healing of the wound surface. The abnormally distributed epidermal stem cells in the diabetic wound surface have lower activity and less quantity than normal skin, and can not effectively heal the diabetic wound surface within a common time, so that the existing epidermal stem cells in the diabetes cannot play a due role in the process of repairing the wound surface and healing the wound surface. The epidermal stem cells are used as seed cells to culture tissue engineering skin, promote wound healing, and avoid a series of problems of skin source shortage, skin supply area scar and the like during skin sheet transplantation. The compound skin constructed by the sunflower and the like by utilizing the epidermal stem cells is used for repairing the wound surface of a nude mouse, the proliferation capacity of the new skin is strong, and the growth speed and the skin quality are both ideal. If the composite skin or the stem cell membrane containing the epidermal stem cells is used for repairing the diabetic wound and enhancing the healing capacity of the diabetic wound, an ideal mode is possibly provided for treating the diabetic wound.

Recent studies have shown that the tissue repair and regeneration functions exerted by stem cells are largely achieved not only by proliferation and differentiation at the site of injury, but also by their paracrine action. RNA or protein in the exosome (MSC-exo) derived from the mesenchymal stem cell can play a role in cell homing, regulate the proliferation and differentiation of cells, limit injury, regulate immune response and promote self-repair and tissue regeneration after cell injury. Baglio et al isolate exosomes secreted by human bone marrow and adipose-derived mesenchymal stem cells, and confirm that the exosomes secreted by the human bone marrow and adipose-derived mesenchymal stem cells are rich in specific miRNA and tRNA by methods such as electron microscope labeling, protein analysis, RNA sequencing and the like. Quesemberry et al suggested that MSC-exo plays a critical role in stem cell biology, and speculated that exosomes play an important role in promoting cell neogenesis and tissue injury repair. But at present, the exosome is not applied to the diabetic tissue repair.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides an application of promoting the repair of the diabetic damage by using adipose-derived stem cells and epidermal stem cell exosomes and repair peptides together.

In addition, a method for screening a polypeptide capable of promoting repair of diabetic lesions and the polypeptide obtained by screening are also provided.

In particular, the repair peptide can specifically promote the proliferation of human keratinocytes and human normal fibroblasts, particularly the proliferation in a high-sugar environment.

Further, the repair peptide is GTC-2 polypeptide, and the sequence of the repair peptide is shown as SEQ ID NO: 1 is shown.

The repair peptide can be obtained by adopting a chemical synthesis method.

The invention aims to provide application of repair peptide in preparing products in the aspects of skin wound, burn and scald, chronic wound healing, radiation ulcer, skin cell repair, regeneration and the like.

Further, the repair is in the context of a diabetic patient.

Further, the product is preferably a medical product, a skin care product or a cosmetic product.

Further to the present invention, the formulation type of the product is preferably, but not limited to, tablet, granule, solution, emulsion, cream, gel, mask or dressing, etc.

The invention further provides an application of the adipose-derived stem cells and the epidermal stem cell exosomes in combination with the GTC-2 polypeptide in treating the tissue loss repair of a diabetic patient.

The invention further provides application of the adipose-derived stem cells and the epidermal stem cell exosomes in preparing a pharmaceutical composition for treating tissue loss repair of a diabetic patient together with the GTC-2 polypeptide.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In another aspect, the invention provides a pharmaceutical composition comprising an adipose stem cell and an epidermal stem cell exosome in synergistic with a GTC-2 polypeptide. The pharmaceutical composition comprises an effective amount of the polypeptide compound with the structure and a pharmaceutically acceptable carrier, and the composition can be prepared according to the well-known technology in the field, for example, the composition is mixed with the conventional pharmaceutical excipients to prepare injection, or the preparation type of local administration such as emulsion, ointment, cream or nasal administration dosage forms, or the like, or the preparation with sustained release effect such as microspheres, liposomes, and the like.

The "pharmaceutically acceptable carrier" of the present invention includes any pharmaceutical excipients, such as fillers, diluents, excipients, and the like. Examples include, but are not limited to, lactose, sucrose, glucose, starch, celluloses (e.g., carboxymethyl cellulose, light propyl methyl cellulose, etc.), ethylene glycol, soybean oil, sesame oil, ethanol, sterile physiological saline, sterile water, ethanol, and the like. Typically, the pharmaceutically acceptable carrier is a water-soluble pH buffer, such as citrate, phosphate, and the like; and optionally antioxidant, stabilizer (such as amino acid), bacteriostatic, etc.

In another aspect, the invention provides the use of the above composition in the preparation of a medicament for treating a disease associated with the repair of tissue loss in diabetes, including type I diabetes and type II diabetes.

The invention provides a stem cell preparation, which comprises the following components in concentration:

the concentration of adipose-derived stem cells is 1-9 × 10⁶Per mL;

the concentration of the exosome is 1-5 mg/mL;

the concentration of the GTC-2 polypeptide reaches 1-5 mg/mL;

dissolving with compound electrolyte injection, glucose injection or normal saline.

In embodiments provided herein, the solvent is physiological saline.

Subcutaneous infusion of the Stem cell preparation the stem cell preparation of the present invention is injected subcutaneously into patients in an amount of 0.01 to 0.1ml/kg per body weight or 50ul per time.

Compared with the prior art, the invention has the following advantages and effects:

the repair peptide provided by the invention can specifically promote the proliferation of human keratinocytes and human normal fibroblasts, particularly the proliferation under a high-sugar environment. Moreover, the repair peptide can be prepared by a chemical synthesis method or a genetic engineering method, and the production cost and the use cost are lower. The repairing peptide provided by the invention is combined with adipose-derived stem cells and epidermal stem cell exosomes for use, so that the repairing peptide can effectively repair an ulcer model of a diabetic mouse, has remarkable effect of promoting the repair of diabetes loss, and has good application prospect and application value.

Drawings

FIG. 1 Effect of the polypeptides on the proliferation of two cells

FIG. 2 is a diagram showing the results of PCR detection of corresponding genes in cells by GTC-2 polypeptide

FIG. 3 diameter distribution diagram of exosomes

FIG. 4 result chart of wound healing rate

FIG. 5 is a graph showing the effect of various treatment regimens on the expression level of mRNA for endothelial cell growth factor

Detailed Description

The invention discloses a composition for preventing and treating the tissue loss of diabetes, application thereof and a stem cell preparation, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Example 1 preparation of a polypeptide that promotes cell proliferation in a high sugar Environment

Hacat human immortalized keratinocyte (cat number YCL-0090, Yipu organism) and NHDF (normal human skin fibroblast) (cat number: HTX2167, Shenzhen luxury Tuo biotechnology Limited) are used as target cells, polypeptide specificity screening for promoting cell proliferation is carried out by adopting a polypeptide library constructed by the inventor at 37 ℃, 3 polypeptides capable of promoting two kinds of cell proliferation under a high sugar environment are obtained and are respectively named as GTC-2, GTC-15 and GTC-29, the dodecapeptide is synthesized and labeled with FITC by Beijing Zhongkoku matt biology technology Limited, and the purity of the polypeptide is more than 95%.

Example 2GTC-2 polypeptide Activity assay

HaCat human immortalized keratinocyte culture solution: the cells were cultured in RPMI1640 medium containing 10% fetal calf serum and 50mmol/L glucose, and 1% of a solution of 10000U/ml-10000. mu.g/ml penicillin-streptomycin was added.

Normal human skin fibroblast culture solution: culturing cells with MEM culture medium containing 10% fetal calf serum and 50mmol/L, adding 1% volume of 10000U/ml-10000 μ g/ml penicillin-streptomycin, 1% volume of sodium pyruvate, and 5ml glutamine.

Taking out the cryopreservation tube storing HaCat human keratinocytes and human normal fibroblasts from liquid nitrogen, rapidly placing the tube in a 37 ℃ water bath kettle to rapidly melt the cells, placing the sucked cell sap and 4ml of complete culture solution in a centrifuge tube after the liquid in the tube is completely melted, centrifuging at 1000rpm for 5 minutes, then discarding the supernatant by using a suction tube of an autoclave, uniformly mixing the precipitated cells by using 5ml of respective corresponding culture solution, inoculating the cells in a sterile culture dish for culture, replacing the fresh culture solution the next day, and continuing cell culture. Liquid changing: as described above, HaCat human keratinocytes were cultured in RPMI1640 medium, while human normal fibroblasts were cultured in MEM medium. Note that the growth of the cells was observed under an inverted microscope, and the fresh medium was changed every 2 to 3 days as needed. Passage: when the cell association rate reaches about 80%, digesting the cells to a suspension state by using 1-2 ml of 0.25% pancreatin, then adding equal volume of complete culture solution, centrifuging at the speed of 1000rpm, taking out a centrifuge tube after 10 minutes, removing the supernatant, blowing the uniformly-mixed precipitated cells by using fresh culture solution, and performing petri inoculation and passage by using a proper proportion. The cells were placed in NEST-Dish honeycomb cell-slide dishes (46 plates/Dish) and the cells were plated at 2.0X 10⁴The density of each ml was inoculated in a petri dish and when cell growth was observed to be stable, all the slides were collected. The collected cell slide was divided into 3 groups according to the slide dish from which it originated, and the detection was repeated 3 times. Each time, the concentration of each group is divided into 3 concentrations of 1 blank control group and 1 polypeptide treatment group for detection, and the specific process is as follows: the clips are grouped as follows: blank control group (culture solution) the experimental group (polypeptide) was divided into 3 groups of high (100. mu.g/ml), medium (50. mu.g/ml) and low (10. mu.g/ml) by concentration. The treatment time was 48 hours per polypeptide. The drug-treated slide was applied to MTT assay. Such a detection process was repeated 3 times, and a reliable experimental result was obtained. The results are shown in FIG. 1.

The detection result of MTT (figure 1) shows that the polypeptide can effectively promote the proliferation of two kinds of cells under three concentrations, namely high concentration, low concentration, medium concentration and medium concentration, particularly the proliferation effect on keratinocytes is more obvious, and within 48h, the OD of the keratinocytes can reach 0.37 at the highest concentration of GTC-2 polypeptide and is obviously improved compared with 0.13 of a control group.

EXAMPLE 3 PCR assay of the Effect of GTC-2 Polypeptides on the corresponding genes of cells

Respectively extracting RNA from two cells of cells treated by the HaCat human immortalized keratinocyte control group and the low-concentration group in the embodiment 2 to perform reverse transcription polymerase chain reaction, wherein the reverse transcription system is 20 mu l and contains template RNA; the operation is carried out according to the instruction provided by a TaKaRa RNALAPCRMkit, and the reaction conditions are as follows: 30 minutes at 55 ℃, 5 minutes at 99 ℃ and 5 minutes at 5 ℃; the PCR reaction system was 15. mu.l containing 3. mu.l of reverse transcription product, 0.9. mu.l MgCl₂(25mM), 1.2. mu.l PCR reaction buffer, 0.075. mu.l Taq polymerase, 0.3. mu.l upstream primer, 0.3. mu.l downstream primer, and H₂O (1% DEPC) complement system. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 1min, annealing at 58 ℃ for 55 sec, extension at 72 ℃ for 40 sec, circulation for 35 times, extension at 72 ℃ for 10 min, and finally cooling to 4 ℃ to finish the reaction. The specific primer sequences are as follows:

β-actin：F:5′-GCATGGAGTCCTGTGGCAT-3′，R:5′ -CTAGAAGCATTTGCGGTGG-3′

Wnt2：F:5'-GCCACACGCTGCACCTAAAGC-3'，R: 5'-CAATTACCCTAAGGGTGGTAGC-3'

Smad3：F:5′-CATAGGTGCTTTGGGCGTAT-3′，R:5′ -GCTGCAAGGTGAAGATGT CA-3′

survivin: the results are shown in FIG. 2 for F: 5'-GGCATGGGTGCCCCGACGTTG-3' and R:5'-CAGAGGCCTCAATCCATG GCA-3', PCR.

As can be seen from fig. 2, Smad3, Wnt2 and survivin were significantly up-regulated on the transcriptional level. Because of Smad3, Wnt2 and survivin genes play a role in promoting cell proliferation during injury repair. Therefore, it was preliminarily determined that the polypeptide promoted cell proliferation by activating the repair pathway. Particularly, the relative expression level of Wnt2 can be increased from 70 to about 110, and the promotion effect is obvious.

Example 4 preparation of adipose-derived Stem cells

Taking adipose tissue, washing with PBS for 3 times, and shearing the adipose tissue into pieces with tissue scissors1mm³Adding 0.1% collagenase I into the small blocks, digesting in water bath at 37 ℃ for 30min, placing in a centrifuge, centrifuging at 1200r/min for 5min, leaving cell precipitate, resuspending with a proper amount of PBS, centrifuging again under the above conditions, discarding supernatant, adding DMEM/F12 culture medium containing 10% fetal calf serum by volume fraction, inoculating in a 10cm culture dish, and culturing at cell concentration of 1 × 10⁹L-¹Placing the mixture at 37 ℃ and 5 percent of CO by volume fraction₂And (3) changing the liquid after 48 hours in a saturated humidity incubator, adding 0.25% of pancreatin for digestion when the cell growth is observed to reach 80%, subculturing according to the ratio of 1: 3, and culturing to the 3 rd generation for subsequent experiments. Detecting the surface molecular markers of the adipose-derived stem cells by using a flow cytometer, taking 3 rd generation human adipose-derived stem cells, adding 0.25% of pancreatin for digestion to prepare 500 mu L of single cell suspension, respectively adding 2 mu L of antihuman CD90-FITC, CD44-PE, CD105-PerCP, CD73-APC, negative antibodies and positive antibodies according to the requirements of a flow identification kit, incubating for 30min at 37 ℃, washing for 3 times by using PBS, and detecting by using the flow cytometer. Flow cytometry results showed that more than 95% of cells positively expressed CD73 (100%), CD44 (100%), CD90 (99.6%), CD105 (98.7%), while the molecular markers CD34, CD45, CD19 negatively expressed cells accounted for 99.6%. Indicating that the adipose-derived stem cells are successfully separated.

Example 5 preparation of epidermal stem cell exosomes

Taking foreskin skin, separating out keratin cells and fibroblasts by using a two-step method of DispaseiI enzyme and trypsin, and enriching and separating out epidermal stem cells by using a quick IV type collagen adhesion method. Collecting supernatant of second-generation fibroblast in exponential growth phase, filtering, mixing with keratin cell serum-free culture medium in equal proportion, adding fetal bovine serum, epidermal growth factor, bovine pituitary extract, etc. to obtain epidermal stem cell culture solution, and performing in-vitro amplification culture of human epidermal stem cell with keratin cell as control. And (3) passing to the 2 nd generation, detecting the expression of B1 integrin and keratin 19 by using immunohistochemical staining, and finding that the staining is positive, thereby indicating that the epidermal stem cells are prepared.

Taking the 4 th generation human epidermal stem cells, firstly culturing the cells in a DMEM/F12 culture medium containing 10% fetal bovine serum by volume fraction until the cells grow to 80% toThe culture is continued for 24h by using a serum-free basic culture medium, the culture process is at 37 ℃ and the volume fraction is 5 percent CO₂The constant temperature oven is used for carrying out the process; collecting the basic culture medium into a centrifuge tube after 24h, filtering by a 0.22 mu m filter, transferring into a 100ku ultrafiltration concentration centrifuge tube, and centrifuging at 5000 Xg for 30min at 4 ℃ to obtain a concentrated solution containing exosome. The quantity and size distribution of the human epidermal stem cell exosomes are measured by preparing the exosomes into 1mL0.5g/L solution, injecting a sample into a sample tank until the sample tank is full, detecting the particle size and the concentration by using a NanosightNS300 particle size analyzer, and detecting the size of the human adipose stem cell exosomes by using the particle size analyzer as shown in figure 3, wherein the abscissa represents the diameter range, and the ordinate represents the concentration/intensity of the human adipose stem cell exosomes, so that most of the exosomes are in the diameter range of 30-110nm and the concentration reaches 5 mg/ml.

Example 6 establishment of mouse model

Preparation of diabetic foot ulcer rat model: taking a Wistar rat of 4 months old, injecting STZ solution into the fasting abdominal cavity of the rat according to 50mg/kg, wherein the STZ solution is dissolved by citric acid-trisodium citrate buffer solution with the pH value of 4.4 and the concentration of 0.1mol/L, the final concentration is 10mg/mL, injecting the same amount of citric acid-trisodium citrate buffer solution into the fasting abdominal cavity of a normal control group, and measuring the peripheral fasting blood glucose to be more than or equal to 16.7mmoI/L after 1 week, namely the model building success of the rat diabetes is obtained. A3 mm by 6mm full-thickness rectangular skin defect was made on the backs of both hind legs of a diabetic rat, which was regarded as successful foot ulcer molding and was recorded as day 0. Foot ulcers were disinfected with 0.5% iodophor 1 time per day.

Example 7 mouse model treatment experiment

Adipose-derived stem cell transplantation group: preparing single cell suspension from adipose-derived stem cells with PBS, wherein the cell concentration is 5.0 × 10⁶one/mL. On the day after molding (day 0), the adipose stem cell transplantation group was injected with 50uL of cell suspension at 5mm from the wound margin at 6 points equally spaced from each injection point subcutaneously on the dorsum of the foot on both sides.

Epidermal stem cell exosome + adipose-derived stem cell transplantation group: preparing adipose-derived stem cells into single cell suspension with PBS, adding epidermal stem cell exosome, and mixing the above twoAfter mixing, the concentration of the adipose-derived stem cells is 5.0 multiplied by 10⁶Per mL, the concentration of exosome reaches 1 mg/mL. On the day after molding (day 0), the epidermal stem cell exosome + adipose stem cell transplantation group was injected with 50uL of cell suspension per injection point at 5mm from the wound margin and 6 points equidistant under the dorsal surface of both sides.

An epidermal stem cell exosome + adipose-derived stem cells + GTC-2 polypeptide transplantation group: preparing adipose-derived stem cells into single cell suspension with PBS, adding epidermal stem cell exosome and GTC-2 polypeptide, mixing, and making the adipose-derived stem cells with the concentration of 5.0 × 10⁶The concentration of exosome reaches 3mg/mL, and the concentration of GTC-2 polypeptide reaches 1 mg/mL. On the day after modeling (day 0), the epidermal stem cell exosome + adipose-derived stem cell + polypeptide transplantation group was injected with 50uL of cell suspension per injection point at 5mm from the wound margin and 6 points equidistant under the dorsal surface of bilateral hind feet.

The normal foot ulcer control group and the diabetic foot ulcer control group were injected with PBS in equal amounts.

Collecting a sample: digital photographs were taken of the foot wound on day 6 after cell transplantation, and dorsal foot wound tissue specimens were collected. The tissue specimen was used for RT-PCR detection in the middle of the wound. Healing rate of wound surface: photographing the instep wound surface at each time point, calculating the area of the instep wound surface by using Image-Pro Plus 6.0 software, and calculating the wound surface healing rate according to a formula. The healing rate is (original wound area-current measurement area)/original wound area is 100%. RT-PCR detection of the expression of mRNA of vascular endothelial cell growth factor in wound tissue: total RNA was extracted from day 6 tissues, and 1ug of total RNA was extracted by RNAiSoPlus, and then reverse-transcribed into cDNA, followed by PCR using 2OuL reaction system. Primer sequence of rat vascular endothelial growth factor: upstream 5 '-gtcctcacttggatccgaca.3', downstream 5 '. cctggcagcaaacagctcc.3'; primer sequence for rat GAPDH: upstream 5'-GGCACAGTCAAGGCTGAGAATG-3', downstream 5'-ATGGTGGTGAAGACGCCAGTA-3'. Specific PCR parameters: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 10s, amplification at 60 ℃ for 35s, extension at 72 ℃ for 1min for 35 cycles, and final extension at 72 ℃ for 5 min. By using 2^-△△CTThe expression level of mRNA was calculated.

The results of the healing rate are shown in figure 4. The result of fig. 4 shows that the combination of adipose-derived stem cells, exosomes and polypeptides adopted by the invention can synergistically and remarkably promote the wound healing rate of minor diabetic ulcers to reach 92%, and the effect is more remarkable.

The change in the expression level of mRNA is shown in FIG. 5. Because the healing of the diabetic decimal wound surface is slow and related to the damage of angiogenesis, the vascular endothelial cell growth factor is the strongest vascular growth promoting factor and can promote the division and proliferation of vascular endothelial cells and induce angiogenesis. The results in fig. 5 show that the combination of adipose-derived stem cells, exosomes and polypeptides adopted by the invention can synergistically and significantly promote the expression of endothelial cell growth factor mRNA, and the expression is up to 4, which is significantly improved compared with 1.4 of the control group of diabetic foot ulcer.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

Sequence listing

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Claims

1. The application of the fat stem cell and the epidermal stem cell exosome in preparing the medicine for treating the tissue loss repair of the diabetic patient by the cooperation of the GTC-2 polypeptide is characterized in that: the sequence of the GTC-2 polypeptide is shown as SEQ ID NO: 1 is shown.

2. Use according to claim 1, characterized in that: the medicine or the medicine composition further comprises a pharmaceutically acceptable carrier.

3. Use according to claim 2, characterized in that: wherein the pharmaceutically acceptable carrier comprises any pharmaceutically suitable excipient.

4. Use according to claim 3, characterized in that: wherein the pharmaceutically acceptable carrier is a water-soluble pH buffer.

5. A stem cell preparation is characterized by comprising the following components in concentration:

the concentration of adipose-derived stem cells is 1-9 × 10⁶Per mL;

the concentration of the epidermal stem cell exosome is 1-5 mg/mL;

the concentration of the GTC-2 polypeptide reaches 1-5 mg/mL;

dissolving by using normal saline;

the sequence of the GTC-2 polypeptide is shown as SEQ ID NO: 1 is shown.

6. The formulation according to claim 5, characterized in that the stem cell preparation is administered by subcutaneous infusion into the patient subcutaneously according to the body weight of the patient, in a standard of 0.01-0.1ml/kg or in an amount of 50ul per time.