CN115025288A - Exosome-hydrogel mixed system and preparation method thereof - Google Patents
Exosome-hydrogel mixed system and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biomedical materials, and particularly relates to an exosome-hydrogel mixed system and a preparation method thereof. The components of the exosome hydrogel mixed system comprise exosomes derived from mesenchymal stem cell subsets and methacryloylated gelatin, wherein the mesenchymal stem cell subsets are CD 146-bearing + And CD271 + A labeled subpopulation of cells; the exosome expresses human angiopoietin-like protein 4. Can quickly promote the proliferation, tube formation and migration of vascular endothelial cells, can play the beneficial effect brought by stem cell treatment to a great extent, and simultaneously avoidsThe adverse reaction related to stem cell transplantation, such as tumor formation and the like.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to an exosome-hydrogel mixed system and a preparation method thereof.
Background
Spinal Cord Injury (SCI) is a serious traumatic disease of the central nervous system, and more than 200 million of the existing traumatic Spinal Cord Injury patients in china and 10-14 million new patients are increased every year. After the spinal cord injury occurs, blood vessels at the spinal cord are obstructed, blood circulation can not be performed, and even death can be caused. SCI not only consumes enormous medical resources, but also places a heavy economic burden on individuals, homes, and society. However, there is still no effective treatment for SCI patients with severe neurological dysfunction. At present, the treatment such as spinal fixation, intramedullary decompression, hormone application and the like is mainly adopted clinically to reduce secondary injury; many therapeutic means such as cell therapy, nerve factor, gene therapy, etc. are in experimental stage. Therefore, effective strategies for treating spinal cord injuries and promoting functional recovery are urgently sought.
Multiple studies indicate that mesenchymal stem cell transplantation can promote the functional recovery of spinal cord injury under the conditions of aging, brain injury, neurodegenerative diseases and the like. At present, the main sources of clinical mesenchymal stem cells are bone marrow, umbilical cord and the like. However, in conventional clinical treatment, there are many problems to be solved, including low survival rate of transplanted cells, cell de-differentiation, tumor formation, etc. There is therefore an urgent need to find a therapeutic means and method that can exert a positive therapeutic effect on stem cells while reducing the associated risks.
Exosomes (EXOs) are nanoscale vesicles secreted by various cells, and carry in vivo various bioactive components such as maternal cell nucleic acids and mirnas, proteins, and the like. The exosome implantation can also avoid the risks of tumors and the like caused by the direct transplantation of stem cells and a plurality of medical ethical problems related to autologous neural stem cell transplantation. The exosome derived from the stem cells has the similar function as the stem cells, can effectively promote tissue repair and regeneration, shows strong protective capability in the field of central nervous system injury repair, and opens a new way of cell-free treatment.
There is an urgent need for a material that can promote revascularization to repair and regenerate blood vessels in spinal cord injuries.
Disclosure of Invention
The application provides an exosome hydrogel mixed system and a preparation method thereof, and aims to solve the technical problem of how to promote angiogenesis.
In a first aspect, the present application provides an exosome-hydrogel mixed system, the components of which comprise exosomes derived from mesenchymal stem cell subpopulations with CD146 as carrier and methacrylated gelatin + And CD271 + A labeled subpopulation of cells; the exosome expresses human angiopoietin-like protein 4.
Optionally, the mass concentration of the exosome in the exosome-hydrogel mixed system is 100-200ug/mL, preferably 100 ug/mL.
Optionally, the mesenchymal stem cell subpopulation is derived from human umbilical cord Wharton jelly;
and/or the exosome hydrogel mixed system has photosensitive curing performance;
and/or the diameter of the exosome is less than or equal to 150 nm.
The exosome hydrogel mixed system has photosensitive curing performance; the diameter of the exosome is less than or equal to 150nm, and the positive effect of removing impurities can be achieved by controlling the diameter of the exosome to be less than or equal to 150 nm.
Optionally, the exosome-hydrogel mixed system is applied to the local part of the injured spinal cord in a dressing form, and the dosage of the exosome-hydrogel mixed system is 80-100ul/cm 2 。
Optionally, the number of exosomes in the damaged spinal cord local is 1.5 × 10 7 ～2.5×10 7 Per cm 2 。
Optionally, the exosome-hydrogel mixed system further contains a vascular endothelial growth factor.
The exosome hydrogel mixed system also contains Vascular Endothelial Growth Factor (VEGF), and the positive effect of promoting angiogenesis can be achieved by adding VEGF and activating a PI3K/AKT signal channel related to angiogenesis.
In a second aspect, the present application provides a method for preparing an exosome-hydrogel hybrid system, the method comprising the steps of:
obtaining a human umbilical cord sample after parent separation;
sorting the cells in the human umbilical cord Wharton jelly sample to obtain a marker CD146 + And CD271 + The mesenchymal stem cell subpopulation of (a);
culturing the mesenchymal stem cell subset, and harvesting cell supernatant;
filtering, separating and resuspending the cell supernatant to obtain exosomes;
detecting the protein content in the exosome to obtain the exosome containing the target protein amount;
and mixing the exosome with the methacrylated gelatin, and performing co-incubation to obtain an exosome hydrogel mixing system.
Optionally, the target protein is human angiopoietin-like protein 4.
The positive effects of activating a PI3K/AKT signal channel related to angiogenesis and promoting angiogenesis can be achieved through the human angiopoietin-like protein 4.
Optionally, the sorting is by sorting the mesenchymal stem cell subpopulations by flow cytometry with anti-CD 146 antibodies and anti-CD 271 antibodies.
Specifically, the CD146 can be obtained by sorting the human umbilical cord Wharton's jelly mesenchymal stem cells by stepwise utilization of anti-CD 31, anti-CD 45, anti-CD 235a, anti-CD 73, anti-CD 90, anti-CD 146 and anti-CD 271 + And CD271 + Cell subsets and exosomes thereof.
Optionally, culturing the mesenchymal stem cell subpopulation, and collecting cell supernatant, specifically including: culturing the mesenchymal stem cell subset, adding an exosome-free culture medium when the confluency of the mesenchymal stem cell subset is 60% -70%, and harvesting supernatant after 48-72 h.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the exosome hydrogel mixing system provided by the embodiment of the application comprises the components of exosomes derived from mesenchymal stem cell subsets and methacrylated gelatin, and the mesenchymal stem cell subsets are CD 146-bearing + And CD271 + The marked cell subset is extracted from exosomes, the exosomes express a plurality of types of proteins for promoting angiogenesis, the exosomes have high activity and human angiopoietin-like protein 4, and can quickly promote the proliferation, tube formation and migration of vascular cells by activating a PI3K/AKT signal path related to angiogenesis, so that the therapeutic effect brought by stem cell therapy can be exerted to a great extent, and meanwhile, related adverse reactions brought by stem cell transplantation, such as tumor formation and the like, are avoided; when in use, the photoinitiator is added to achieve the curing effect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.
FIG. 1 is a diagram of the human umbilical Wharton's jelly mesenchymal stem cell subpopulation (CD146+ CD271+ HUCWJMSCs) provided by the present application;
FIG. 2 is a diagram illustrating the extraction and identification of exosomes of mesenchymal stem cell subpopulation of human umbilical Wharton's jelly provided in the embodiments of the present application;
FIG. 3 is a CD146 provided in the embodiments of the present application + CD271 + HUCJMSC-EXOs can promote the function recovery of spinal cord injury mice;
FIG. 4 shows an embodiment of the present applicationProvided CD146 + CD271 + The effect of HUCWJMSC-EXOs on the biological behavior of vascular endothelial cells;
FIG. 5 is a CD146 provided by an embodiment of the present application + CD271 + Function recovery effect of HUCWJMSC-EXOs-hydrogel mixed system spinal cord injury mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Umbilical cord samples used in the examples of this application were obtained from healthy mothers after delivery, approved by the ethical review committee of xiangya hospital, south China university, and given informed consent from all participants before sample collection; DMEM/F-12 medium, fetal bovine serum, penicillin and streptomycin, collagenase type II, 0.25% trypsin was purchased from Gibco; CD31 antibody, CD45 antibody, CD235a antibody, CD90 antibody, CD73 antibody, CD146 antibody and CD271 antibody were purchased from Biolegend corporation.
Example 1
Human umbilical cord Wharton jelly mesenchymal stem cell CD146 + CD271 + Subgroup (CD 146) + CD271 + HUCWJMSC).
(1) The study was approved by the ethical review committee of xiangya hospital, central university, and informed consent was obtained from all participants prior to sample collection. Human umbilical cord samples were obtained from healthy mothers after delivery and were processed within 6 hours after collection. The umbilical cord was first washed twice with Phosphate Buffered Saline (PBS) containing 100U/mL penicillin and 100. mu.g/mL streptomycin to remove the blood that was expected to remain. The washed umbilical cord was cut into 3-4 cm long pieces in a petri dish, and the amniotic membrane was separated from the umbilical vein and the umbilical artery to obtain Wharton jelly. Then theCutting HUATONG glue into 1mm pieces 3 Adding the fragment into 1g/L type II collagenase, digesting for 30min with 37 deg.C constant temperature oscillator, and then digesting for 30min with 0.25% pancreatin at 37 deg.C constant temperature oscillator. The digest was filtered through a 70um cell sieve, the filtrate was centrifuged at 400G for 5min, and the supernatant was discarded and washed 2 times with PBS.
(2) Separation of CD146 by flow cytometry + CD271 + HUCWJMSC firstly separates some impurity cells by using a general technology, and then carries out flow cytometry method sorting on human umbilical cord Wharton jelly mesenchymal stem cells by using CD73 antibody and CD90 antibody. CD146 + CD271 + HUCWJMSC represents a cell subset highly enriched in CD146 and CD271 human umbilical cord Wharton jelly mesenchymal stem cells, wherein HUCWJMSC represents human umbilical cord Wharton jelly mesenchymal stem cells.
(3) And (3) carrying out flow cytometry to sort the human umbilical cord Wharton jelly mesenchymal stem cell subsets by using anti-CD 146 antibodies and anti-CD 271 antibodies as required. The human umbilical cord Wharton jelly mesenchymal stem cell subset marker is CD146 + And CD271 + 。
(4) Human umbilical cord Wharton's jelly mesenchymal stem cell subpopulation was transferred to DMEM/F-12 medium and supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin.
As shown in fig. 1, the 6 drawings in fig. 1 are sequentially marked by arrows: removing fragments to obtain a cell process diagram (the abscissa is relative intensity of a signal of an area under a forward scattering light-curve and reflects the volume of cells, the ordinate is relative intensity of a signal of a lateral scattering light and reflects granularity of cells), obtaining a single cell diagram (the ratio of single cells obtained in a frame is higher, the abscissa is relative intensity of a signal of an area under a forward scattering light-curve and the ordinate is relative intensity of a signal of a forward scattering light-width), obtaining a live cell diagram (the live cells are obtained in a frame, dead cells are obtained outside the frame; DAPI is 4', 6-diamidino-2-phenylindole, the abscissa is relative intensity of a DAPI fluorescent signal, the ordinate is relative intensity of a signal of a lateral scattering light and reflects granularity of cells), and obtaining CD45/235a/31 - Cytogram (relative intensity of fluorescence signal with CD45/235a/31 on abscissa and lateral scattered light signal on ordinate, which reflects the granularity of cellsEndothelial cells, immune cells and blood cells are removed), and a HUCWJMSC cytogram (human umbilical cord Wharton jelly mesenchymal stem cells in frame with the abscissa of CD 90) is obtained + Relative intensity of fluorescence signal, ordinate CD73 + Relative intensity of fluorescence signal, CD90 + And CD73 + Marker for HUCWJMSC), obtaining CD146 + CD271 + Map of HUCWJMSC (with CD146 in frame) + CD271 + Labeled cell subsets with CD146 abscissa + Relative intensity of fluorescence signal, ordinate CD271 + Fluorescence signal relative intensity), wherein the part circled in the figure is the target cell, and the 6 figures are the obtaining process of the target subcellular population; the positive rate of markers CD73 and CD90 of the stem cells after sorting is 5.96 percent (the positive rate can be directly measured from the graph); the positive rate of CD146 and CD271 of the sorted cells was 14.7%, and the two positive rates indicated that CD146 was obtained + And CD271 + HUCWJMSC subgroup.
However, in the prior art, only unsorted human umbilical cord Wharton jelly mesenchymal stem cells are often obtained, and the human umbilical cord Wharton jelly mesenchymal stem cell subset with a specific marker is not obtained, but the human umbilical cord Wharton jelly mesenchymal stem cell subset with CD146 provided by the application + And CD271 + Compared with other unsorted human umbilical cord Wharton's jelly mesenchymal stem cells, the marked cell subgroup and exosomes extracted from the marked cell subgroup have obviously stronger capacities of promoting proliferation, tube formation and migration of vascular cells through research.
Example 2
CD146 in human umbilical cord Wharton jelly mesenchymal stem cells + And CD271 + Preparation of exosomes of cell subsets
Waiting for CD146 + CD271 + When the cell subset grows to 60-% 70% confluence, collecting culture supernatant, filtering with 0.22 μm filter membrane, and centrifuging at 4 deg.C and 500G for 10 min; centrifuging at 2000g for 30min at 4 ℃; centrifuging at 4 deg.C at 10000g for 60 min; centrifuging 110000g for 90min, discarding the supernatant, and resuspending the precipitate with phosphate buffer; centrifuging again at 110000g for 90min, discarding the supernatant, resuspending the precipitate with a small amount of phosphate buffer solution to obtain exosomes, storing at-80 deg.C, FIG. 2, A. CD146 under light microscope + And CD271 + Pictures of labeled cell subsets cultured for 7 days with microscope magnification of 100 ×, illustrating the sorted CD146 + And CD271 + The marked cell subset has the characteristic of long spindle shape of typical mesenchymal stem cells. B. For transmission electron microscope observation of CD146 + CD271 + Form of HUCWJMSC-EXOs. C. CD146 acquisition using Nanoparticle Tracking Analysis (NTA) + CD271 + HUCWJMSC-EXOs particle size distribution and image, with the abscissa as the exosome particle size and the ordinate as the relative concentration. D Western Blot (WB) analysis of specific markers for exosomes showed significant presence of CD63 and TSG 101.
Example 3
Subgroup exosome promoting functional repair capability of mouse spinal cord injury model
All animal operations were authorized by the animal ethics committee of the university of south and middle. General anesthesia, skin preparation, disinfection and sheet laying. The skin, subcutaneous, muscle and vertebral plates were sequentially incised centered on the T10 spinous process to reveal the spinal cord. An improved Allen's beating method is adopted, and a spinal cord beating model is established with the beating force of 10g multiplied by 25 mm. The wound is irrigated, the incision is closed layer by layer, and the dressing covers the wound and is fixed. All mice are raised in an animal laboratory, the environmental temperature is 22-24 ℃, and the relative humidity is 60% -80%. After operation, the feed is fed in cages, and the feed is fed by the standard feed and drinking water. After operation, 3d is subjected to intramuscular injection of penicillin 2WU/Bid to resist infection. The bladder is squeezed 2-4 times a day to help urinate until the micturition reflex is restored.
Evaluating motor function and motor coordination and stabilization function of hind limbs of the mice after spinal cord injury by using BMS (Basso Mouse Scale) primary score and secondary score system respectively 1, 3, 7, 14, 21, 28,42 and 56 days after spinal cord injury, wherein the BMS primary score is 9 scores in total, and from 0 score to 9 scores, the higher the score is, the better the motor function is recovered; the BMS score was 11 in total, with higher scores being better for motor stability and coordination. Each mouse was double-blinded for 5 minutes in the experimental design by two researchers familiar with BMS scoring. The average score for each mouse was then recorded.
In fig. 3, the abscissa of the graph a is Pre-sur (time point before spinal cord injury), SCI 1D, SCI 3D, SCI 7D, SCI 14D, SCI 21D, SCI28D, SCI42D and 56D (time points of days 1, 3, 7, 14, 21, 28,42 and 56 after spinal cord injury in turn), and the ordinate represents the BMS main score, and from 0 to 9, the score is higher, indicating that the hindlimb motor function recovery of the mouse is better; the abscissa of the graph B is Pre-sur (time point before spinal cord injury), SCI 1D, SCI 3D, SCI 7D, SCI 14D, SCI 21D, SCI28D, SCI42D and 56D (time points of 1, 3, 7, 14, 21, 28,42 and 56 days after spinal cord injury in turn), the ordinate represents BMS major and minor scores, and from 0 to 11 points, the higher the score is, the better the movement stability and coordination recovery of hind limbs of the mouse is indicated; 0/50/100/150/200 in the figure represent different concentrations of exosomes (in ug/mL) respectively.
As can be seen from FIG. 3, 100- + CD271 + The HUCWJMSC-EXOs treatment can obviously improve the motor function and the motor stability and coordination of the double lower limbs after the spinal cord injury of the mice.
Example 4
Subgroup exosomes improve vascular endothelial cell biological behavior
(1) The subgroup exosomes promote vascular endothelial cell proliferation. Vascular endothelial cells were seeded in 96-well plates and treated with the subpopulations of exosomes prepared in example 2 (concentration 100-. Add 10. mu.l of CCK-8 reagent to the medium in each well. After incubation at 37 ℃ for 4 hours, the absorbance was measured at 450nm using a microplate reader. As can be seen from FIG. 5, CD146 is expressed + And CD271 + Compared with the exosome derived from unsorted cells, the exosome extracted after the cell subpopulation has stronger effect of promoting vascular endothelial cell proliferation, and is caused by high expression of human angiopoietin-like protein 4.
(2) The subpopulation of exosomes promotes the lateral migration of vascular endothelial cells. Spinal cord microvascular endothelial cells were seeded in 6-well plates, and when the confluency of the cells became 90%, the vascular endothelial cells were treated with the subpopulation exosomes (concentration 100-. The distance between both sides of the scratch was measured by microscopic observation at 0 hour and 12 hours, respectively. As can be seen from fig. 5, the subgroup exosomes of the present application have a stronger ability to promote the lateral migration of vascular endothelial cells.
(3) The subpopulation of exosomes promotes the longitudinal migration of vascular endothelial cell proliferation. Mixing spinal cord microvascular endothelial cells at a ratio of 1-2 × 10 4 The cells/ml were cultured in 24-well transwell upper chambers without serum in the cell culture medium. Add 500. mu.l of medium containing 10% FBS to the lower chamber and then separately add CD146 + CD271 + HUCJMSC-EXOs (concentration 100-200. mu.g/ml), HUCJMSC-EXOs (concentration 100-200. mu.g/ml) and PBS (control group) were added into the lower chamber for intervention, after 12h of culture, the non-migrated cells in the upper chamber were wiped off, the cells migrated to the lower chamber were fixed with 4% paraformaldehyde for 20min, and then stained with 0.1% crystal violet for 15min and counted. As can be seen from FIG. 5, the subgroup exosomes of the present application have stronger ability to promote the longitudinal migration of vascular endothelial cells.
(4) The subgroup exosomes promote vascular endothelial cell tube formation. Mixing spinal cord microvascular endothelial cells at a ratio of 8-10 × 10 3 The density of individual cells/well was transferred to a 48-well plate previously plated with 120. mu.l of Matrigel (Corning), and the subpopulations of exosomes prepared in example 2 (concentration 100-. Cultured in a medium with 5% CO 2 The number of branch nodes formed by each group of cells and the length of the segment are calculated and compared by observing under a microscope in an incubator at 37 ℃ for 12 h. As can be seen from fig. 5, the subgroup exosomes of the present application have a stronger ability to promote vascular endothelial cell tube formation.
As can be seen from FIG. 4, A is a vascular endothelial cell proliferation test, and the higher absorbance represents the stronger proliferation capacity of vascular endothelial cells, and compared with unsorted HUCWJMSC-EXOs, CD146 + CD271 + HUCJMSC-EXOs promoted vascular endothelial cells more strongly at 4 h. B is a transverse migration experiment of vascular endothelial cells, and the phenomenon that the vascular endothelial cells migrate to the middle in the figure shows that compared with unsorted HUCWJMSC-EXOs, the CD146 gene is + CD271 + HUCWJMSC-EXOs have stronger effect of promoting the transverse migration of vascular endothelial cells at 12 h. C is a longitudinal migration experiment of vascular endothelial cells, and the cell number in the figure shows that the CD146 is compared with unsorted HUCWJMSC-EXOs + CD271 + HUCJMSC-EXOs has stronger effect of promoting the longitudinal migration of vascular endothelial cells in 12 h; d is the vascular endothelial cell tube formation experiment, and the vascular endothelial cell tube formation characteristic in the figure shows that compared with unsorted HUCWJMSC-EXOs, the CD146 + CD271 + HUCJMSC-EXOs has stronger effect of promoting vascular endothelial cell tube formation in 12 h.
Example 5
Construction and application of subgroup exosome hydrogel mixed system
Constructing a mouse spinal cord striking model, and taking CD146 as a model + CD271 + HUCJMSC-EXOs, VEGF and GelMA light-cured hydrogel (i.e. methacrylated gelatin) mixed in CD146 + CD271 + The concentration of HUCWJMSC-EXOs is 100-. Heating the mixed system of the subgroup exosome hydrogel to 37-40 ℃ to make the mixed system in a water phase, and locally applying the mixed system to the injured spinal cord in a dressing form according to 100ul/cm 2 The subpopulation exosomes-gel sustained release formulation was overlaid, in addition, PBS-gel formulation, HUCWJMSC-EXOs-gel formulation and VEGF-gel formulation were overlaid respectively as controls. At 365- 2 Irradiating with light for at least 30s under the condition to change the hydrogel from water phase to solid phase, and suturing the wound. CD146 + CD271 + HUCJMSC-EXOs represents CD146 + CD271 + Human umbilical cord Wharton jelly mesenchymal stem cell exosome.
Example 6
Influence of subgroup exosome hydrogel mixed system on recovery of spinal cord injury function after application
The therapeutic effect of the exosome-hydrogel hybrid system on locomotor function of the hind limbs of mice after spinal cord injury was evaluated on days 1, 3, 7, 14, 21, 28,42 and 56 after spinal cord injury using bms (basso Mouse scale) primary and secondary scoring system on the basis of example 3.
As can be seen from FIG. 5, the abscissa and ordinate have the same meaning as those in FIG. 3, in which A.CD146 is shown + CD271 + The HUCWJMSC-EXOs hydrogel mixed system treatment can obviously improve the movement function of the two lower limbs of the mouse after spinal cord injury. B.CD146 + CD271 + The HUCWJMSC-EXOs hydrogel mixed system treatment can obviously improve the movement stability and coordination of the two lower limbs after the spinal cord injury of the mice.
BMS main score and side score system experiments prove that when the mass concentration is 100-. Proved by vascular endothelial cell proliferation, tube formation and migration experiments, the subgroup exosome has better effect of promoting the recovery of spinal cord injury function compared with an unsorted cell exosome, and the effect is that the capacity of promoting angiogenesis is stronger.
BMS main scoring and auxiliary scoring system experiments prove that the exosome hydrogel mixed system is applied to the local part of the injured spinal cord in a dressing mode, and the dosage of the exosome hydrogel mixed system is 80-100ul/cm 2 . The number of exosomes active in the damaged spinal cord section at this time is about 1.5 × 10 7 ～2.5×10 7 Per cm 2 The effect of promoting the recovery of spinal cord injury function can be achieved by promoting the vascular proliferation to about 80% of the level before injury.
It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. An exosome hydrogel mixed system, which is characterized in that the components of the exosome hydrogel mixed system comprise exosomes derived from mesenchymal stem cell subsets and methacrylated gelatin, wherein the mesenchymal stem cell subsets are CD 146-carrying + And CD271 + A labeled subpopulation of cells; the exosome expresses human angiopoietin-like protein 4.
2. An exosome-hydrogel hybrid system according to claim 1, wherein the mass concentration of the exosome in the exosome-hydrogel hybrid system is 100-200ug/mL, preferably 100 ug/mL.
3. An exosome hydrogel mixing system according to claim 1, wherein the mesenchymal stem cell subpopulation is derived from human umbilical cord Wharton's jelly;
and/or the exosome hydrogel mixed system has photosensitive curing performance;
and/or the diameter of the exosome is less than or equal to 150 nm.
4. The exosome-hydrogel hybrid system according to claim 1, wherein the exosome-hydrogel hybrid system is applied to an injured spinal cord local in the form of a dressing, and the amount of the exosome-hydrogel hybrid system used is 80-100ul/cm 2 。
5. The exosome-hydrogel hybrid system according to claim 1, wherein the number of exosomes in the local injured spinal cord is 1.5 x 10 7 ～2.5×10 7 Per cm 2 。
6. An exosome-hydrogel hybrid system according to claim 1, further comprising a vascular endothelial growth factor.
7. A preparation method of an exosome-hydrogel mixed system is characterized by comprising the following steps of:
obtaining a human umbilical cord sample after parent separation;
sorting cells in the human umbilical cord Wharton jelly sample to obtain a marker CD146 + And CD271 + The mesenchymal stem cell subpopulation of (a);
culturing the mesenchymal stem cell subset, and harvesting cell supernatant;
filtering, separating and resuspending the cell supernatant to obtain exosomes;
detecting the protein content in the exosome to obtain the exosome containing the target protein amount;
and mixing the exosome with methacryloylated gelatin, and performing co-incubation to obtain an exosome hydrogel mixing system.
8. The method of claim 7, wherein the protein of interest is human angiopoietin-like protein 4.
9. The method of claim 7, wherein the sorting is by sorting the subpopulations of mesenchymal stem cells by flow cytometry with an anti-CD 146 antibody and an anti-CD 271 antibody.
10. The method according to claim 7, wherein the culturing the mesenchymal stem cell subpopulation and the supernatant harvesting comprise: culturing the mesenchymal stem cell subset, adding an exosome-free culture medium when the confluency of the mesenchymal stem cell subset is 60% -70%, and harvesting supernatant after 48-72 h.
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