CN111888379A - Application of stem cell-derived microvesicles in preparation of scar repairing preparation - Google Patents
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Abstract
The invention provides application of stem cell-derived microvesicles in preparation of a preparation for repairing scars, and relates to the technical field of scar skin repair. In the invention, the extracellular vesicles from mesenchymal stem cells obviously inhibit the expression of collagen III, the collagen III in a high-concentration microvesicle and low-concentration microvesicle treatment group is obviously lower than that in a PBS group, and the expression of collagen I also shows a descending trend; the high-concentration micro vesicles and the low-concentration micro vesicles can promote reasonable distribution of skin structures, and are particularly characterized in that the collagen structure is more reasonable, and auxiliary structures are remarkably increased; therefore, the microvesicles derived from the stem cells have the effect of repairing scars, the low-concentration microvesicles and the high-concentration microvesicles can promote the repairing effect of scar skin, but the scar area after the high-concentration microvesicles are repaired is smaller than that of the low-concentration microvesicles, the treatment effect is obvious, and the microvesicles can be used for preparing a preparation for repairing scars.
Description
Technical Field
The invention belongs to the technical field of skin scar repair, and particularly relates to application of stem cell-derived microvesicles in preparation of a scar repair preparation.
Background
Scar (scar) is generally the change in the appearance, morphology and histopathology of normal skin tissue caused by various traumas, and is an inevitable product in the process of repairing human wounds. When the growth of the scar exceeds a certain limit, various complications can occur, such as disfigurement, dysfunction of functional activities and the like, which bring great physical and mental pains to patients, in particular to the scar left after burns, scalds and serious trauma. Collagen is the main component of extracellular matrix participating in wound healing, and in normal human skin, collagen I and collagen III are mainly contained, the content of the collagen I is large, the collagen I plays a role of a bracket, the ratio of the collagen I to the collagen III is 10:1, in the scar healing process, fibroblasts are activated, collagen is synthesized and secreted, the collagen III is mainly used, and a small amount of the collagen I is also contained. In recent years, research on skin regeneration medicine based on stem cells, particularly Mesenchymal Stem Cells (MSCs), has attracted much attention, and research shows that MSCs can promote repair of skin wounds by differentiation into skin cells, sweat gland cells and paracrine, and have clinical application prospects in repair of scars. However, the ethical, safety, difficulty in storage and transportation, and the like of stem cells limit the clinical application of the stem cells.
The current therapeutic means for scars only comprise: for the depressed scars, atrophic scars and hypertrophic scars with relatively serious degrees, surgical excision is a main means, but the scars cannot be completely removed in any surgical mode, only the harm caused by the scars can be improved or corrected to the maximum extent, and new scars are preferably formed after the surgery; the most common medicaments for treating scars clinically comprise triamcinolone, triamcinolone acetonide, dexamethasone and the like, but the long-term application of a large amount of medicaments can cause cushing's syndrome and certain side effects; freon and other low-temperature cold sources are adopted to treat freckles, senile plaques and the like, the freckles, senile plaques and the like are directly contacted with a diseased part, the diseased tissue is necrotized and shed after 7-10 days, and the epidermis is healed, but the defects of slow change of skin pigment after operation, slow healing of wounds, incomplete scar removal and the like are also overcome; chemical medicament carbolic acid is adopted and smeared on the surface of scar skin, and fresh and tender pink skin can appear after the skin of the scar falls off, so that the purpose of removing the scar is realized, but the yellow people are not suitable for the scar removing method.
Therefore, a safe and effective treatment is needed for the treatment of scars and new scars that are formed during the treatment of scars.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of stem cell-derived microvesicles in the preparation of a preparation for repairing scars, wherein the stem cell-derived microvesicles directly interfere with scars, and the dosage and safety are controllable.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of stem cell-derived microvesicles in preparing a preparation for repairing scars.
Preferably, the stem cells comprise human umbilical mesenchymal stem cells.
Preferably, the preparation comprises an injection, and the number of the microvesicles in the injection is 4.5 multiplied by 108～4.5×1011/200μl。
Preferably, the injection solution takes phosphate buffer solution as a solvent.
The invention also provides a preparation for repairing scars, which takes microvesicles derived from human umbilical mesenchymal stem cells as active ingredients, and the quantity of the microvesicles is 4.5 multiplied by 108～4.5×1011/200μl。
Preferably, the preparation also comprises a phosphate buffer.
Preferably, the preparation formulation comprises an injection.
Preferably, the formulation comprises a cosmetic or skin care product.
The embodiment proves that extracellular vesicles from mesenchymal stem cells obviously inhibit the expression of collagen III, the collagen III in a high-concentration microvesicle and low-concentration microvesicle treatment group is obviously lower than that in a PBS group, and the expression of collagen I also shows a descending trend; the high-concentration micro vesicles and the low-concentration micro vesicles can promote reasonable distribution of skin structures, and are particularly characterized in that the collagen structure is more reasonable, and auxiliary structures are remarkably increased; therefore, the microvesicles derived from the stem cells have the effect of repairing scars, the low-concentration microvesicles and the high-concentration microvesicles can promote the repairing effect of scar skin, but the scar area after the high-concentration microvesicles are treated is smaller than that after the high-concentration microvesicles are treated, and the repairing effect is obvious.
Drawings
FIG. 1 is an identification of MSCs and their microvesicles, wherein A represents umbilical cord mesenchymal stem cells; b is transmission electron microscope picture of umbilical cord mesenchymal stem cells; c is a surface labeling diagram of the microvesicles of the umbilical cord mesenchymal stem cells;
FIG. 2 is a Nanosight technique for measuring the particle size of microvesicles, wherein A represents the peak value of the microvesicles; b represents a Nanosight technology microvesicle tracer diagram;
figure 3 is a graph comparing the results of a microvesicle-repaired rat scar model, wherein figure 3A is a graph comparing the first, fourth and seventh days of treatment; FIG. 3B is a graph comparing the tenth, thirteenth and sixteenth days of treatment;
FIG. 4 is a graph showing the results of staining a pathological section HE of skin tissue;
FIG. 5 is a graph showing the results of Masson collagen staining of skin tissue;
FIG. 6 is a histochemical staining diagram of collagen, wherein FIG. 6A is a chemical staining diagram of collagen I, and FIG. 6B is a chemical staining diagram of collagen III;
FIG. 7 is a diagram of RT-PCR detection of collagen I and collagen III expression in skin tissues;
FIG. 8 is a graph showing the results of microvesicle treatment in human scar experiments.
Detailed Description
The invention provides application of stem cell-derived microvesicles in preparing a preparation for repairing scars.
The stem cells of the present invention preferably include human umbilical cord mesenchymal stem cells, and the source of the human umbilical cord mesenchymal stem cells is not particularly limited in the present invention, and it is preferable to culture and expand MSCs in vitro according to the HucMSC isolation culture and identification method (QiaoChun et al, human sensory stem cells isolated from the mesenchymal cardiac cord. CellBiol int.2008 Jan; 32(1): 8-15). The invention extracts microvesicles from human umbilical mesenchymal stem cells, and the specific steps preferably comprise: culturing with serum-free medium for 48h after the growth area of the MSC is 80%, collecting culture supernatant, centrifuging at 2000g for 10min, and removing cell debris to obtain supernatant (MSC-CM) secreted by umbilical mesenchymal stem cells; centrifuging at differential speed, removing cell debris and organelles, transferring to 100000 Da MWCO ultrafiltration centrifugal tube with specification of 15ml, concentrating, transferring the concentrated solution to 5ml sucrose/D with concentration of 30%2Centrifuging at 100000 g for 3h on an O density pad at 4 ℃, collecting a buffer pad (containing microvesicles) with the bottom of 5ml, diluting and washing the buffer pad with PBS, putting the buffer pad into a 100000 Da MWCO ultrafiltration centrifugal tube for washing, quantifying the collected microvesicle concentrated solution, filtering and sterilizing the microvesicle concentrated solution by a 0.22 mu m filter membrane, measuring the number of microparticles of the extracellular microvesicles by a Nansigt method, subpackaging and refrigerating at-70 ℃ for later use. By using the extraction method, the micro-bubbles obtained by extraction have a bubble-shaped structure with the diameter of about 100nm, are uniform, and have the original concentration of the number of particles of 2.2-2.5 multiplied by 108/μl。
The preparation formulation of the preparation of the invention preferably comprises injection, and the number of the micro-vesicles in the injection is preferably 4.5 x 108～4.5×1011/200. mu.l. In the invention, the injection solution preferably takes phosphate buffer solution as a solvent.
The invention also provides a preparation for repairing scars, which takes microvesicles derived from human umbilical mesenchymal stem cells as active ingredients, and the quantity of the microvesicles is 4.5 multiplied by 108～4.5×1011Mu.l/200. mu.l. The preparation of the invention preferably also comprises a phosphate buffer; the preparation formulation preferably comprises an injection. The formulations of the present invention preferably comprise a cosmetic or skin care product.
The invention also provides a method for repairing scar by using the preparation, preferably injecting the preparation into the subcutaneous tissue of the scar part at four points, wherein the injection amount is preferably 4.5 x 108～4.5×1011200 mul, the injection amount is determined according to the optimal effect of the preliminary experiment; or uniformly applying the preparation on cleaned scar, and gently beating and massaging until completely absorbed; the amount of the coating is preferably 4.5 × 108～4.5×1011200 mul, the smearing amount is determined according to the effect of animal experiments.
The use of the microvesicles derived from stem cells provided by the present invention for preparing a preparation for repairing scars will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
The main materials and sources used in the examples are respectively as follows:
MSC culture reagent: low-sugar DMEM, fetal bovine serum (Gibco), trypsin (Sigma), carbon dioxide incubator (Forma), serum-free medium (shanghai cockiosel;
inverted microscope, fluorescence microscope, biological microscope, electron microscope, super clean bench, bench centrifuge.
Microvesicle extraction reagent: heavy water (D)2O, shanghai chukaso), analytically pure sucrose (guangzhou chemical reagent factory), rabbit anti-human CD9 antibody (Bioworld Technology, usa), rabbit anti-human CD63 antibody (Epitomics, usa), BCA protein quantification kit, horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG secondary antibody (beijing kang, century), HRP chemiluminescenceSubstrate, 100-kDa MWCO ultrafiltration centrifuge tube, 0.22 μm sterile filter membrane (Millipore, USA); transmission electron microscope (FEI Tecnai 12, Philips); ultracentrifuge (beckman, usa).
Example 1
Obtaining microvesicles derived from umbilical cord mesenchymal stem cells (hucMSC)
(1) Isolated culture of umbilical cord MSCs: in vitro culture of expanded MSC (Qiao Chun et al, human mesenchyme stem cells isolated from the said umbellicalcord. cell Biol int.2008 Jan; 32(1):8-15) according to the established HucMSC isolation culture and identification method, healthy human MSC (A in figure 1) with strong proliferation capacity and good state is selected, culture supernatant is collected by serum-free culture medium for 48h after the growth area covers 80%, MSC-CM is obtained after centrifugation for 2000g and 10min to remove cell debris, and then the MSC-CM is refrigerated at-70 ℃ for standby.
(2) And (3) separating and purifying microvesicles in supernatant secreted by umbilical cord mesenchymal stem cells: centrifuging at differential speed, removing cell debris and organelles, transferring to 100000 Da MWCO ultrafiltration centrifugal tube with specification of 15ml, concentrating, transferring the concentrated solution to 5ml sucrose/D with concentration of 30%2Centrifuging at 100000 g for 3h on O density pad at 4 deg.C, collecting buffer pad (containing microvesicle) with bottom 5ml, diluting and washing with PBS, washing in 100000 Da MWCO ultrafiltration centrifuge tube, quantifying the collected microvesicle concentrate, filtering with 0.22 μm filter membrane for sterilization, determining number of microparticles of extracellular microvesicle by Nansigt method, packaging, and refrigerating at-70 deg.C for use
(3) The basic morphology of the microvesicles was observed by transmission electron microscopy: 20 mu L of MSC micro-capsules are fully and uniformly mixed and then are dripped on a sample-carrying copper net with the diameter of 2mm, after the mixture is kept stand at room temperature for 5min, residual liquid at the edge of the copper net is slightly absorbed by filter paper, then the copper net is reversely buckled on 30g/L phosphotungstic acid (pH6.8) liquid drops, negative dyeing is carried out at room temperature for 5min, finally the copper net is dried under an incandescent lamp and is placed under a transmission electron microscope for observation and photographing, and the micro-capsules with the diameter of about 100nm have vesicle structures as shown in B in figure 1;
(4) detecting the micro vesicle surface marker protein by western blot: preparing 12% SDS-PAGE electrophoresis gel, fully cracking the extracted microvesicles, adding 1/4 volumes of 5 xSDS loading buffer, boiling for 5min, loading according to 200 mug protein total amount, transferring the proteins onto PVDF membrane by electrotransfer (350mA, 120min), sealing for 1h at room temperature with TBS/T containing 50g/L skimmed milk, reacting with rabbit anti-human CD9 antibody and rabbit anti-human CD81 antibody (1:500) at 4 ℃ overnight, washing the membrane for 3 times with TBS/0.5% Tween20 for the next day, incubating with HRP-labeled goat anti-rabbit IgG secondary antibody at 37 ℃ for 1h, washing the membrane for 3 times with TBS/0.5% Tween20, adding a premixed chemiluminescent substrate, and detecting by a chemiluminescent gel imaging system, wherein the labeling of the microvesicles derived from umbilical cords is shown as C in figure 1.
(5) The particle size of the microvesicle is about 100nm and is relatively uniform by the Nanosight technology. The number of particles at an initial concentration of 2.2 to 2.5X 108μ l, as shown in FIG. 2.
Example 2
Construction of scar rat model and repair effect of hucMSC-microvesicle on scar rat
(1) Construction of SD rat skin scar model: 8-week-old female SD rats weighing 200g were anesthetized with 10% chloral hydrate and applied 8% Na on the backs2S, performing unhairing treatment, preparing a circular full-layer skin defect wound surface with the diameter of 2cm by using a sterile surgical instrument, scabbing for 2-3 days, and forming a skin scar model about 7 days;
(2) intervention treatment of HucMSC-microvesicles: three experimental groups, PBS control group, high concentration group, low concentration group (high concentration: 4.5X 10)11Experimental animals only; and (3) low concentration: 4.5X 108Experimental animals only); three of each group, each rat was treated by injecting 200 μ l Phosphate Buffered Saline (PBS) into the subcutaneous tissue around the scar to dissolve high-concentration and low-concentration microvesicles, and PBS was used as a blank control; the time for observing the repairing effect is 1d, 4d, 7d, 10d, 13d and 16d respectively, and photographing is carried out.
(3) As shown in fig. 3A and 3B, compared with control PBS, both low-concentration microvesicles and high-concentration microvesicles can promote the repair effect of scar skin, but the scar area of the high-concentration microvesicle treatment group is smaller than that of the low-concentration microvesicle treatment group, and the repair effect is significant.
(4) The results of hematoxylin and eosin staining (HE staining) of the skin histopathological section are shown in fig. 4, and compared with the PBS group control, the high-concentration microvesicles and the low-concentration microvesicles can promote the reasonable distribution of the skin structure, which is particularly shown in that the collagen structure is more reasonable and the accessory structures are significantly increased.
(5) The results of Masson collagen staining of skin tissues of PBS group and microvesicle group are shown in fig. 5, the expression of collagen iii was significantly inhibited by extracellular vesicles derived from mesenchymal stem cells, the expression of collagen iii was significantly lower in the high-concentration microvesicle and low-concentration microvesicle treatment groups than in the PBS group, and the expression of collagen i also showed a decreasing trend.
(6) The histochemical collagen staining results for skin tissues of PBS and microvesicle groups are shown in FIG. 6, in which the expression of collagen III is significantly inhibited by extracellular microvesicles derived from mesenchymal stem cells (FIG. 6B), the expression of collagen III is significantly lower in the high-concentration microvesicle and low-concentration microvesicle treatment groups than in the PBS group, and the expression of collagen I is also reduced (FIG. 6A).
(7) The expression of collagen I and collagen III in mouse skin tissues detected by RT-PCR is shown in FIG. 7, the expression content of collagen I and collagen III in PBS group is obviously increased, and the expression content of collagen I and collagen III in high-concentration microvesicle and low-concentration microvesicle treatment groups is obviously reduced. (the primer sequences are shown in Table 1)
TABLE 1 RT-PCR detection of expression primer sequences for collagen I and collagen III in mouse skin tissue
Example 3
Repairing effect of human scar microvesicle
(1) Basic case and repair scenario: the first caesarean section operation is carried out on a female 31 years old in 11 months in 2017, and the second caesarean section operation is carried out on a female 2 months in 2020, and scar tissues are generated after the first caesarean section operation;
the repair scheme comprises the following steps: divided into 6 times of treatment each4.5X 10 times11Microparticles/volume 200 μ l. The nano-micro-capsules are stored in a freezing layer of a refrigerator, and one nano-micro-capsule is taken out before use and naturally dissolved at room temperature. After cleaning scars, taking pictures of the scars, then lying flatly to uniformly smear the completely melted nano micro-capsules on the scars, gently patting and massaging until the nano micro-capsules are completely absorbed, treating once every 3 days, and primarily treating 6 times.
(2) Observation of the repairing effect (as shown in fig. 8):
1) gross evaluation of scars
Includes the scar protrusion degree: the convex portions of the scar gradually flatten;
② color of scar: the color of the scar gradually becomes lighter from purple red, and the lighter red gradually approaches the skin color;
width of scar: the width of the scar gradually narrows.
2) Evaluation of scar hardness: when lying flat and relaxed, the scar is lightly pressed, the scar gradually becomes soft, the hardness is gradually narrowed, and the depth is shallow.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (8)
1. Application of stem cell-derived microvesicles in preparing a preparation for repairing scars.
2. The use of claim 1, wherein the stem cells comprise human umbilical mesenchymal stem cells.
3. The use of claim 1, wherein the formulation comprises an injectable solution, and the number of microvesicles in the injectable solution is 4.5 x 108～4.5×1011/200μl。
4. The use according to claim 3, wherein the injectable solution comprises a phosphate buffer as a solvent.
5. A preparation for repairing scars is characterized in that the preparation takes microvesicles derived from human umbilical mesenchymal stem cells as an active ingredient, and the number of the microvesicles is 4.5 multiplied by 108～4.5×1011/200μl。
6. The formulation of claim 5, further comprising a phosphate buffer.
7. The formulation of claim 5 or 6, wherein the formulation comprises an injectable solution.
8. The formulation of claim 5, wherein the formulation comprises a cosmetic or skin care product.
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