CN113274410A - Application of exosome hydrogel complex in preparation of medicine for repairing skin scar - Google Patents
Application of exosome hydrogel complex in preparation of medicine for repairing skin scar Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention discloses an application of an exosome hydrogel compound in preparation of a medicine for repairing skin scars. The exosome hydrogel compound is preferably composed of natural polypeptide and human umbilical cord mesenchymal stem cell exosomes, has the characteristics of antibiosis, self-repairing, injectability and wound surface adhesion, and intelligently responds for a long time to control release of exosomes and maintain the bioactivity of the exosomes. The hydrogel is used as a carrier, and the exosome can continuously improve and repair the skin scar.
Description
Technical Field
The invention relates to the field of stem cell processing and application, in particular to application of an exosome-hydrogel complex in preparation of a medicine for repairing skin scars.
Background
The skin is the outermost tissue of the human body and is the largest organ of the human body. Its main function is to protect the body from the invasion of external pathogens, and at the same time, it also has the functions of perspiration and feeling. However, skin tissue is often damaged, such as burns, scalds, high temperature injuries, diabetic foot ulcers, and the like. The acute and chronic skin wounds not only affect the physiological and functional mental health of patients, but also bring huge social and economic burden. Therefore, more and more researches are devoted to accelerating the healing of the skin wound and promoting the repair and regeneration of skin tissues.
In the field of skin repair and regeneration, stem cell-centered related therapies have achieved a variety of surprising therapeutic effects. However, more and more researches at present prove that the stem cells mainly rely on a paracrine mode to promote the repair of skin wounds in the process of wound repair, and the stem cells have slightly low transplanting and differentiating rates to the injured parts and short action. In addition, the use of stem cells has limitations such as short shelf life. Therefore, it is necessary to find a therapeutic method which has the effect of stem cell therapy and can overcome the defects of stem cells.
Exosomes are one of the extracellular vesicles secreted by cells, and are secreted by stem cells. The major components are the phospholipid bilayer and carry the associated content. Exosomes are about 30-100nm in diameter and are similar in size to viruses. Exosomes contain a variety of substances, and their contents may vary depending on the type of secreting cell. Its main components are protein, miRNA, mRNA, DNA and lipid. In addition, there are other kinds of RNA molecules, such as piRNA, snRNA, etc. The exosome can transfer the content and the like carried by the exosome to a receptor cell, influence the microenvironment of the cell and regulate the expression of related proteins, and the defect of stem cell treatment is overcome.
The healing and regeneration of the wound surface of the skin are complex physiological processes, and various tissues and cells are required to perform synergistic action to replace, repair and reconstruct the missing cell structure and tissue hierarchy. The physiology generally divides the healing of wounds into several phases: bleeding stage, inflammation stage, proliferation stage, and tissue reconstruction stage (scar formation stage).
Scarring after skin damage is one of the difficulties in wound healing. The scar exceeding a certain limit can cause various complications, such as disfigurement of the appearance, dysfunction of the function and the like, and bring great physical and mental pain to patients, especially the scar left after burn and scald and serious trauma. However, the current research finds that the exosome also has a certain effect on inhibiting the scar formation. Hu et al extracted exosomes of human adipose-derived mesenchymal stem cells and applied them to the skin defect repair of mice. By injecting the exosome into the animal body through veins, the exosome is traced and found to be recruited to the periphery of the wound of the skin, thereby playing the function and greatly improving the speed of wound healing. And the exosome is found by histological analysis to promote the synthesis of collagen in the early stage of wound healing so as to improve the repair speed, and inhibit the synthesis of collagen in the later stage so as to inhibit the formation of scar tissue. But not of a permanent nature.
Disclosure of Invention
In view of the above, in order to solve the above technical problems, the present invention aims to provide an application of an exosome hydrogel complex in preparing a drug for repairing a skin scar, wherein the drug can permanently improve and repair the skin scar.
The adopted technical scheme is as follows:
an application of an exosome-hydrogel complex in preparing a medicament for repairing skin scars.
Further, the exosome hydrogel complex is a complex of human umbilical cord mesenchymal stem cell exosomes and hydrogel.
Further, the hydrogel is a natural polypeptide.
Further, the native polypeptide is epsilon-polylysine.
Further, the human umbilical cord mesenchymal stem cell exosome is prepared by the following method:
s1, preparation of mesenchymal stem cells: selecting umbilical cord-derived mesenchymal stem cells for in-vitro subculturing;
s2, separating and identifying the exosome derived from the mesenchymal stem cells.
In the above-mentioned technical solution,
the exosome is used as an important component in a paracrine way of stem cells, and has a good application prospect in promoting the repair and regeneration of skin wounds. Compared with the method for directly utilizing cells to repair tissues, the exosome has the advantages that the exosome has incomparable advantages with various stem cells:
1. the safety is good. The exosome is used for tissue repair, so that the risks of immunological rejection, vessel occlusion and mutation and tumor formation can be avoided, and the safety is greatly improved.
2. Easy to store and transport. The exosome obtained by separation can be stored for a long time at the temperature of minus 80 ℃, and the outer membrane of the exosome can protect the content of the exosome. Especially for RNA molecules, degradation thereof by external RNAses is avoided.
3. The effect is fast and the efficiency is high. The exosome can be directly taken in by cells, the function of target cells is influenced, and the action efficiency is improved.
4 has no ethical limitation. Exosomes serve as a subcellular structure secreted by cells, which have no associated ethical limitations when applied.
5. The source is wide. The exosome can be extracted from culture supernatants of various cells, various body fluids and blood, has wide sources and is convenient to use.
6. Exosomes are mainly present in bone and lung 24 hours after exosome injection. Other studies have shown that most exosomes are distributed in blood-rich organs such as the kidney, spleen and lung. Unlike systemic administration, local administration can maintain high concentrations of exosomes at the target.
A hydrogel is a hydrophilic polymer that can absorb several times its weight in water, a property that allows cells to attach, proliferate, and differentiate.
The exosome hydrogel compound can prolong the release speed of exosome and has a slow release effect. Traditional stem cell therapies include systemic application and local injection. Systemic application stem cells are injected into animals, relying on the stem cells to home to the lesion automatically; systemic exosome injections into animals were found to accumulate major exosomes in the spleen and liver, and most exosomes were cleared 6 hours after injection. Topical application of exosomes to damaged tissues has targeting efficacy, however these vesicles have a short lifetime and are not associated with the influence of proteases or changes in pH. Local treatment is the direct implantation of stem cells into the wound. While exosomes may be anchored to biomaterials/scaffolds such as fibronectin, type I collagen, hydrogels, tricalcium phosphate, polylactic-glycolic acid (PLGA), and hydrogel gels to support their delivery and facilitate controlled release of exosomes.
The invention has the beneficial effects that:
the exosome hydrogel compound is preferably composed of natural polypeptide and human umbilical cord mesenchymal stem cell exosomes, has the characteristics of antibiosis, self-repairing, injectability and wound surface adhesion, and intelligently responds for a long time to control release of exosomes and maintain the bioactivity of the exosomes. The hydrogel is used as a carrier, and the exosome can continuously improve and repair the skin scar.
Drawings
The drawings are briefly described as follows:
FIGS. 1-2 are electron micrographs of human umbilical cord mesenchymal stem cell exosomes.
Figure 3 is a photograph of the indicated cell transplant wound at day 14 after initial treatment.
Figure 4 is a photograph of scarring in mice of different treatment groups 25 days after transplantation.
Fig. 5 is a graph of wound diameters for different treatment groups.
Fig. 6 is a quantification plot of scar length for different treatment groups.
FIG. 7 is a representative image of immunohistochemistry. It showed vascular smooth muscle actin (α -SMA) expression in the indicated treatment groups. Scale bar 500 mm.
Figure 8 is a representative image of purified exosome particles.
FIG. 9 is a graph of particle size distribution in purified uMSC-Exos. As determined by NanoSight. Red arrows indicate exonucleases. Scale bar 1 mm.
FIG. 10 is a graph of the exact particle size distribution of purified uMSC-Exos. The dashed line indicates the peak particle size of the purified uMSC-Exos, as determined by laser light scattering evaluation.
FIG. 11 is a Westernblot analysis identification purification diagram. uMSC-Exos using CD81 and CD63 antibodies.
Figure 12 is a representative immunohistochemistry map showing α -SMA expression in the indicated exosome-treated group. Phosphate buffered saline was used as a control. Scale bar 500 mm.
Abbreviations: medium, umbilical cord mesenchymal stem cell culture Medium; mock, phosphate buffered saline group; n, normal region; α -SMA, smooth muscle actin; UEFS, supernatant of umbilical cord-derived mesenchymal stem cells without exosomes; uMSC-Exos, umbilical cord mesenchymal stem cell-derived exosomes; w, wound site.
Detailed Description
The invention is described in further detail below for a better understanding of the invention, without limiting the scope of the invention thereto.
Example 1
Preparing a human umbilical cord mesenchymal stem cell exosome:
preparation of mesenchymal stem cells
Collecting umbilical cord of parted fetus of healthy parturient, mincing, filtering with screen, and mixing with Phosphate Buffer Solution (PBS). The umbilical cord tissue was centrifuged at 1500r/min for 5min at a radius of 10 cm. Tissues were suspended in Dulbecco's Modified Eagle Medium (DMEM)/F12 containing 10% Fetal Bovine Serum (FBS) and transferred to culture flasks. And changing the culture solution after 4 days of culture, changing the culture solution once after 3 days, and carrying out subculture when the fusion rate reaches about 90%. And observing the adherent growth and morphology of the stem cells under a light microscope.
Separation and identification of exosome derived from mesenchymal stem cells
After stem cells reached 80% confluency, fresh α -MEM was used instead in FBS-free medium, culture was continued for 3 weeks, and the culture was transferred to a conical tube and centrifuged at room temperature (16500g, 4 ℃, 20 minutes) to remove dead cells and cell debris. Then, the supernatant was transferred to a new tube by filtration using a 0.22m filter, and centrifuged at 120000g for 70min at 4 ℃ to granulate the exosomes. The supernatant was discarded. The particles formed on the walls of the conical tube are dialyzed (soaked in dialysis bag for three days) with distilled or deionized water to remove small molecule protein fragments and ions. The dialysate was then lyophilized to give white flocculent exosome powder. Dissolved in PBS at a concentration of 1000g/mL and stored at-80 ℃ for subsequent use. Exosomes were analyzed by Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA), and their characteristic molecules, such as CD9, CD63, and CD81, were identified by Western blotting. After identification, human umbilical cord mesenchymal stem cell exosome (uMSC-Exo) is obtained, and is shown in figure 1 and figure 2.
Example 2
The hydrogel complex of the human umbilical cord mesenchymal stem cell exosome of the embodiment is formed by compounding the human umbilical cord mesenchymal stem cell exosome (uMSC-Exo) of the embodiment 1 and epsilon-polylysine. Epsilon-polylysine is a natural polypeptide that can be used as a hydrogel.
Example 3
The human umbilical cord mesenchymal stem cell exosome hydrogel compound has the following repairing effect on skin scars:
the hydrogel complex of human umbilical cord mesenchymal stem cell exosome of example 2 can promote the generation of type I and type III collagens in the early stage of wound healing, and accelerate wound healing. However, at a later stage, the expression of collagen is inhibited, and scar formation can be reduced. The human umbilical cord mesenchymal stem cell exosome hydrogel complex can inhibit scar formation.
Mice were housed in a specific pathogen-free environment from the Shanghai laboratory animal research center with a 12 hour light cycle and were freely available with standard food and water. Adult male ICR mice (Hauschka mice, switzerland) and nude mice (BALB/c-n) were used for this study. Mice were anesthetized with 10% chloral hydrate (0.3ml/100g), and after hair was removed from the dorsal surface, 1.5cm of skin of uniform diameter was removed from the dorsal surface of the mice to form a full-thickness skin defect. The wound was maintained with Tegaderm (3M, St. Paul, Minn., http:// www.3m.com) for 1 day post-surgery followed by open dressings. Skin contractures were continuously assessed on days 10, 14 and 25 by measuring passive extension and skin collection on day 25.
For the uMSC-Exo treatment of the model, the hydrogel served as a scaffold and the uMSC-Exo was dissolved into the hydrogel according to the manufacturer's instructions. Briefly, 100mg/ml of uMSC exons dissolved in Phosphate Buffered Saline (PBS) were first prepared in one tube and a 1% (10mg/ml) hydrogel was prepared in another tube using sterile water. The two components were immediately mixed in a 1: 1 ratio and injected around the wound 48 hours after wound healing. PBS, HEK-293-exon (100mg/ml) and uMSC-exon free supernatant (UEFS; concentrated medium left after exon removal) were used as controls.
In order to clarify the regulation and control effect of uMSCs on scar formation in the wound healing process, a whole-layer skin defect nude mouse model is established, hydrogel-coated uMSCs are compared with HEK-293T cells or PBS as a control, and the influence of uMSCs on wound healing is researched. On day 14 post-treatment, we found that the cut edges of the uMSC groups were on average smoother than the control groups. On day 25 post-treatment, the skin defects of the uMSC groups were closed with less scarring than the other groups (see FIG. 3, FIG. 4, FIG. 5 and FIG. 6). We assessed a-SMA expression by Immunohistochemical (IHC) staining and found that a-SMA expression was significantly reduced in the uMSC treated group compared to the HEK293T and PBS treated group (see figure 7).
The paracrine capacity of exosomes may play a key role in performing their function in promoting wound repair. In view of the important role of exons as secretory factors, we investigated the role of the uMSC exons in wound repair. We collected and purified exons from culture supernatants of uMSCs and HEK293 cells and analyzed using NanoSight, Laser Vertriebsesellschaft (ALV Laser Vertriebsesellschaft mbH, Langen, Germany, http:// www.alvgmb.de) and Western-blot (see FIG. 8, FIG. 9, FIG. 10 and FIG. 11). Next, we tried to elucidate the function of uMSC-Exos in vivo. We injected equal amounts of hydrogel-coated uMSC exon, HEK-293T cell derived exon (HEK293 exon), PBS or UEFS (concentrated medium left after exon removal) around the wound.
The results show that: on day 14 post-treatment, the mean area of wound surface was minimal in the uMSC-Exo group and the incision edges were smooth. After 25 days, the defect was closed and scar formation was significantly reduced in the uMSC-Exo group compared to the control group. IHC staining showed that in the uMSC-Exo treated group, a-SMA expression was also significantly reduced and the healed tissue was more well-aligned (see FIG. 12). These results indicate that uMSC-Exos can promote wound healing, reduce scarring and myofibroblast formation in situ.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (5)
1. An application of an exosome-hydrogel complex in preparing a medicament for repairing skin scars.
2. The use according to claim 1, wherein the exosome hydrogel complex is a complex of human umbilical cord mesenchymal stem cell exosomes and hydrogel.
3. The use of claim 2, wherein the hydrogel is a native polypeptidic.
4. The use according to claim 3, wherein the native polypeptide is epsilon-polylysine.
5. The use according to claim 2, wherein the human umbilical cord mesenchymal stem cell exosome is prepared by the following method:
s1, preparation of mesenchymal stem cells: selecting umbilical cord-derived mesenchymal stem cells for in-vitro subculturing;
s2, separating and identifying the exosome derived from the mesenchymal stem cells.
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