CN112870228B - Multifunctional microenvironment protection exosome hydrogel and preparation method and application thereof - Google Patents

Multifunctional microenvironment protection exosome hydrogel and preparation method and application thereof Download PDF

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CN112870228B
CN112870228B CN202110077305.2A CN202110077305A CN112870228B CN 112870228 B CN112870228 B CN 112870228B CN 202110077305 A CN202110077305 A CN 202110077305A CN 112870228 B CN112870228 B CN 112870228B
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崔文国
林嘉盈
王臻
俞小华
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Hangzhou Xianshi Biotechnology Co ltd
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Abstract

The invention provides a multifunctional microenvironment protection exosome hydrogel and a preparation method and application thereof. The preparation method comprises the following steps: (1) Obtaining adipose-derived mesenchymal stem cells, digesting the mesenchymal stem cells to obtain primary cells, performing subculture, and taking the adipose-derived mesenchymal stem cells for later use; (2) Taking mesenchymal stem cells from fat sources, placing the mesenchymal stem cells into a DMEM medium containing 10% fetal calf serum for culture, and extracting exosomes released by the mesenchymal stem cells by an ultracentrifugation method; (3) Dissolving sulfhydryl-polyethylene glycol in adipose-derived stem cell exosome, and then AgNO 3 The water solution is mixed with the diluted water solution to form transparent multifunctional microenvironment protective exosome hydrogel. Compared with the existing reports, the multifunctional microenvironment protection exosome hydrogel has better repairing effect, can promote the formation of new blood vessels and the regeneration of endometrial tissues, better helps to recover fertility and improve pregnancy and birth rate.

Description

Multifunctional microenvironment protection exosome hydrogel and preparation method and application thereof
Technical Field
The invention belongs to the technical field of thin endometrium repair, and particularly relates to a multifunctional microenvironment protection exosome hydrogel and a preparation method and application thereof.
Background
Successful pregnancy requires a good quality embryo, a well-tolerated endometrium, and a synchronized, coordinated interaction of the two. The thickness of the endometrium has some effect on endometrial receptivity and affects the successful implantation of the embryo. Due to low estrogen level, infection, inflammation, uterine cavity operation history in pregnancy (such as uterine cleaning after drug abortion, induced abortion and postpartum uterine cleaning) and uterine cavity adhesion, endometrium is damaged and thinned, and the receptivity of endometrium is reduced, so that embryo implantation failure or early abortion after pregnancy is caused, and the fertility of women is seriously influenced. Thin endometrial symptoms have resulted in long term infertility and negative home fate.
There are many reports on thin endometrial therapy, but there is no specific method at present. Aspirin is thought to increase uterine and ovarian blood perfusion, thereby improving embryo planting rate and clinical pregnancy rate, and is widely applied to clinic, however, the curative effect is still controversial, in recent years, researches show that aspirin cannot improve clinical pregnancy rate, live rate, decrease miscarriage rate and the like, and patients have adverse reactions such as bleeding after taking aspirin during pregnancy. In recent years, researches also find that sildenafil citrate tablets can reduce the blood flow resistance of endometrium and increase the blood vessel count of endometrium, thereby improving the blood flow perfusion of endometrium, promoting the growth of endometrium, being beneficial to embryo implantation and improving the clinical pregnancy rate, however, the clinical application of the sildenafil citrate tablets can cause adverse reactions such as headache, flushing, dyspepsia, nasal obstruction, abnormal visual function and the like, no clinical evidence proves whether the sildenafil citrate tablets have adverse effects on fetus, and a part of patients using the sildenafil citrate tablets still have unsatisfactory curative effects. Although current hormone therapy can improve fertility in some women, these responses are still unsatisfactory due to the complex endometrial microenvironment damage and infection. Therefore, the search for a treatment method for improving the thin endometrium with reliable effect and small side effect is urgent in clinical application in recent years.
The research shows that the mesenchymal stem cells have the potential of strong proliferation and multidirectional differentiation capacity, immunoregulation, angiogenesis promotion and the like. Bone marrow-derived stem cells (BMSCs) and fat-derived stem cells (ADSCs) have been studied as a method for regenerating endometrium due to their ability to differentiate into endometrial epithelial cells and stromal cells. The stem cell therapy has great application prospect in the fields of regenerative medicine and intimal repair, however, a plurality of clinical prospective researches find that the long-term clinical effect of stem cell transplantation is poor. The problems of low efficiency, low survival rate and the like of adipose-derived stem cell transplantation are bottlenecks which restrict the application of clinical stem cell transplantation. In addition, research shows that the stem cell transplantation may have safety problems of microvascular embolism, in-vivo tumor formation and the like, and the clinical application of the stem cell transplantation is also limited. Current stem cell therapy is limited by: the requirement for a sustained source of epitopically stable cells, the risk of immune-mediated rejection of the ductal cells, the high cost and technical difficulties of these processes, and the potential risk of cancer or ectopic tissue development.
Recently, membrane-type microvesicle exosomes secreted by adipose-derived stem cells attract extensive attention and attention of scholars, and become a hot spot in the field of stem cell research. The adipose-derived stem cell exosome can simulate the biological function of mesenchymal stem cells, and plays roles in promoting angiogenesis, improving blood flow perfusion of ischemic tissues, repairing tissues, regulating immunity and the like. And the exosome does not have a cell nucleus structure, cannot be amplified in a host body, has no heteroploidy risk, can penetrate a plasma membrane, has lower possibility of immunological rejection, has better safety in the clinical application process, does not block a micro-vessel like an adipose-derived stem cell, breaks through more problems in adipose-derived stem cell treatment, and has wider clinical application prospect.
Recent studies have shown that exosomes have a clear role as mediators of endometrial regeneration. Stem cell-derived exosomes can deliver proteins, mrnas, miRNAs and other molecules to proximal or distal cells, effectively propagating stem cell-induced regenerative signals. Although functionally similar to stem cells, these exosomes are unlikely to induce an immune response in the treated host, thereby minimizing the risk of rejection. Therefore, exosome-based therapies are expected to be ideal tools to drive endometrial regeneration. However, animal experiments demonstrated that exosomes injected directly into the uterine cavity were only able to persist in the uterine cavity for minutes to hours, and studies showed that less than 5% of the stem cells reached the site of injury by the intravenous route, with most of the stem cells apoptotic within hours after administration, even though only a small number of the stem cells that reached the target tissue remained at the site of injury for a long period of time. Because the regeneration of endometrium is a long process and needs continuous tissue growth, a novel biocompatible scaffold is designed, which can support the continuous release of exosome in uterine cavity, thereby prolonging the bioactivity of the exosome and accelerating the regeneration of endometrium, and becomes a new direction for research in the field.
Hydrogels are a class of biomaterials commonly used in tissue repair applications because they are biocompatible and tunable solutions that can mimic the structure of the extracellular matrix to provide a scaffold more suitable for cell proliferation and regeneration. Hydrogels may also mediate the sustained release of drugs in tissue repair, making them promising candidates for exosome endometrial repair applications. However, several factors limit the use of hydrogels in this clinical setting. First, over time, constitutive tissue movement may lead to hydrogel abrasion. Furthermore, different ways of cross-linking the exosomes with the hydrogel matrix (including chemical cross-linking, aldehyde cross-linking, thermal cross-linking, photo-cross-linking) can also damage these exosomes, thereby reducing their therapeutic effect. Hydrogel injection also increases the risk of local bacterial infection, thereby inhibiting tissue regeneration.
Under the background, how to design a novel multifunctional hydrogel to realize a mild coordination-based exosome crosslinking method, and show beneficial self-healing, antibacterial and drug-loading performances, so as to be used for better repairing and treating thin endometrium, restoring fertility and improving pregnancy and birth rate, becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to solve the technical problems and provides a multifunctional microenvironment-protected exosome hydrogel as well as a preparation method and application thereof. Compared with the reported method of directly using adipose-derived stem cell exosomes to promote endometrial regeneration and restoration of fertility, and compared with the reported method of using adipose-derived stem cell exosomes to restore, the multifunctional microenvironment protection exosome hydrogel provided by the invention shows better restoration effect, has better capacity of promoting neovascularization and regeneration of endometrial tissues compared with the prior art, can better help to restore fertility and improve pregnancy and birth rate.
One of the purposes of the invention is to provide a preparation method of a multifunctional microenvironment protection exosome hydrogel, which comprises the following steps:
(1) Separating adipose-derived mesenchymal stem cells from an animal tissue sample, digesting to obtain primary cells, performing subculture, and taking the adipose-derived mesenchymal stem cells for later use;
(2) Taking adipose-derived mesenchymal stem cells, culturing the mesenchymal stem cells in a DMEM culture medium for a period of time, then extracting exosomes released by the mesenchymal stem cells by an ultracentrifugation method, and diluting the obtained exosomes for use;
(3) Dissolving sulfhydryl-polyethylene glycol into the adipose-derived stem cell exosomes obtained in the step (2) to obtain a mixture, wherein the mass-volume ratio of the sulfhydryl-polyethylene glycol to the adipose-derived stem cell exosomes is 30mg:100 μ L, then AgNO 3 The aqueous solution is diluted by the adipose-derived stem cell exosomes and then added into the mixture to form transparent multifunctional microenvironment protective exosome hydrogel after several seconds.
The method designs the multifunctional microenvironment protection exosome hydrogel, and the in-situ microinjection is adopted to promote the regeneration of endometrium and the restoration of fertility. The exosome hydrogel is prepared by formulating Ag + The dynamic coordination between S and the fusion of the adipose stem cell exosomes produces an injectable formulation and greatly reduces the risk of infection, and the cells with the activities of adipose stem cell antigens and paracrine signals can promote the regeneration of the endometrial microenvironment. In vitro, the exosome hydrogel has a remarkable effect of promoting angiogenesis, so that the proliferation and angiogenesis of human umbilical vein endothelial cells can be increased by 1.87 times and 2.2 times respectively. In vivo, this microenvironment-protected exosome hydrogel was also shown to promote neovascular and tissue regeneration, while inhibiting local tissue fibrosis. Importantly, the regenerated endometrial tissue is more likely to conceive embryos and give rise to healthy newborns. The exosome hydrogel system protected by the microenvironment provides a convenient, safe and noninvasive method for repairing thin endometrium and restoring fertility.
Further, the DMEM medium in the step (2) contains 10% fetal calf serum and 1% streptomycin/penicillin.
Further, the culture conditions in step (2) were a culture at 37 ℃ for 2 weeks.
Further, the ultracentrifugation method in the step (2) comprises the steps of: the collected culture supernatant was centrifuged at 800 Xg for 5 minutes, at 2000 Xg for 10 minutes twice, and the supernatant was filtered, at 100000 Xg, at 4 ℃ for 90 minutes.
Further, the exosome was diluted to 10 μ g/mL in step (2) for use.
Further, the molecular weight of the thiol-polyethylene glycol in the step (3) is in the range of 1000-2000Da.
Further, the AgNO in the step (3) 3 The concentration of the aqueous solution was 0.1mol/L.
Further, the AgNO in the step (3) 3 The volume of the aqueous solution and the adipose-derived stem cell exosomes is 3.
The invention also aims to provide the multifunctional microenvironment protective exosome hydrogel prepared by the method. The hydrogel shows excellent capability of promoting regeneration of endometrium and restoration of fertility, and is greatly improved compared with the prior art.
The invention also aims to provide application of the multifunctional microenvironment protection exosome hydrogel, which is application of the exosome hydrogel in promoting endometrial regeneration and fertility restoration.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a multifunctional microenvironment protection exosome hydrogel which can promote endometrial regeneration and fertility recovery by adopting in-situ microinjection;
(2) The exosome hydrogel has obvious angiogenesis promoting effect, enables the proliferation of endothelial cells of umbilical veins and the formation of blood vessels of a human body to be increased by 1.87 times and 2.2 times respectively, and shows better repairing effect compared with the prior method of adopting adipose-derived stem cell exosomes to directly promote the regeneration of endometrium and restore the fertility and compared with the method of using the adipose-derived stem cell hydrogel for repairing;
(3) The exosome hydrogel system protected by the microenvironment has the advantages of convenience, safety and no wound in the aspects of repairing thin endometrium and recovering fertility, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of the synthesis of a multifunctional microenvironment-protected exosome hydrogel of the present invention;
FIG. 2 shows characterization results of adipose-derived stem cells and adipose-derived stem cell exosomes. (A) Immunofluorescent staining patterns of adipose stem cells (ADSCs), scale: 50 μm; (B) analyzing the result of the ADSCs by flow cytometry; (C) Particle size value of adipose-derived stem cell exosome (ADSC-exo); (D) SEM image of adipose-derived stem cell exosomes (scale: 200 nm); (E) Detecting a result graph of the adipose-derived stem cell exosomes on CD63 and CD9 by using a flow cytometer; (F) Westernblotting test result graphs of adipose-derived stem cell exosomes for CD63 and CD 9; (G) The result graph of the release of the adipose-derived stem cell exosomes from the PEG-Ag hydrogel; (H) The daily release curve of the adipose-derived stem cell exosome of the PEG-Ag hydrogel; data are presented as mean SD (n =3 per group).
FIG. 3 shows the identification result of the adipose-derived stem cell exosome hydrogel. (a) self-healing and injectable mechanisms; (B) Four-arm sulfhydryl polyethylene glycol, agNO 3 And adipose stem cell exosomes; (C) SEM images of hydrogel and adipose-derived stem cell exosome hydrogel formulations; (D) images of the hydrogel after it has entered the distilled water; (E) In vitro degradation curves of hydrogel and adipose-derived stem cell exosome hydrogel; (F) Strain scan measurements of the PEG-Ag hydrogel storage modulus (G' and G "correspond to elastic modulus and loss modulus, respectively) expressed in kPa; (G) Strain scanning measurement of storage modulus of ADSCs-exo @ hydrogel; (H) The relation between the measured viscosity parameter and time, taking seconds as a unit, the strain shear rate is between 0.05 and 500 percent, and the shear time is 100s; (I) After two 1000% step strain cycles, the G' recovery rates of various hydrogels; (J) The release behavior of Ag ions from hydrogels and adipose stem cell hydrogels; (K) The in vitro cytotoxicity and the living cells of the adipose-derived stem cell exosome hydrogel are shown in the specification 1,Results of 3 and 5 days quantitative analysis.
FIG. 4 shows a free adipose-derived stem cell exosome (ADSCs-exo) or hydrogel mixed stem cell exosome group (AgNO) 3 + ADSCs-exo) on HUVEC proliferation, migration and tube formation. (A) The detection of Ki67 by immunofluorescence staining was performed in the indicated treatment group (scale: 50 μm) as an index for measuring the proliferation of HUVEC; (B) quantifying the number of Ki67 positive cells in a given group; (C) Transwell migration experiments (scale bar: 200 μm) were performed on HUVECs of each treatment group; (D) The number of the transferred HUVECs in the Transwell experiment is quantified, and the transfer is enhanced after the ADSCs-exo or AgNO3+ ADSCs-exo is treated; (E) In vitro tube formation assays using HUVEC cells in the indicated treatment groups; (F) Quantification of the number of tubes per field indicates improved HUVEC tube formation after treatment with ADSCs-exo or AgNO3+ ADSCs-exo (scale: 200 μm); the error is calculated based on three samples (. About.P)<0.05，**P<0.01)。
Fig. 5 is the result of evaluation of morphological change of uterus in related treatments. (A) The operation method for establishing a rat model with uterine horn injury comprises the steps of making a belly median incision on the uterine horn, and then injecting absolute ethyl alcohol into uterus; (B) Uterine specimens, arrows indicate uterus after endometrial injury, uterine tissue samples were submitted to H & E staining scale bars: 1000 μm, 200 μm; (C) thickness of endometrium of each group; (D) number of glands in each group; data are mean standard error, n =5, <0.05, <0.01.
FIG. 6 shows the use of ADSCs-exo or AgNO 3 + ADSCs-exo results in promotion of endometrial neovascularization, uterine muscle regeneration and reduction of endometrial collagen deposition. (A) Immunohistochemical staining showed vascular visualization of CD 31-endothelial cells in the treated group (scale: 100 μm), whereas α -SMA protein staining was used to examine smooth muscle to assess myometrial regeneration (scale: 100 μm) and Masson trichrome staining was used to assess endometrial fibrosis (blue, consistent scar formation) to show the treated group (scale: 100 μm). (B, C and D) quantitatively detecting the expression levels of CD31, -SMA and collagen. Data are mean standard error of triplicate samples,. P<0.05，**P<0.01。
FIG. 7 is an evaluation of in vivo antimicrobial activity of hydrogels. (A) Staphylococcus aureus immunofluorescent staining (red); (B) quantitative testing of Staphylococcus aureus in each treatment group; data are mean standard error, n =5, <0.05, <0.01.
Figure 8 is a graph of the effect of hydrogel transplantation on endometrial receptivity and angiogenesis marker expression. (A) The qRT-PCR method was used to assess the expression of endometrial receptivity markers (HOXA-1, LIF, ER, PR, integrin beta 3, IGF-1) and angiogenesis (VEGF, bFGF), beta-actin as normalization control; (B) Westernblotting detects the expression conditions of LIF, VEGF and IGF-1 proteins in each group; (C) Western blotting data for LIF, VEGF, IGF-1 levels for different treatment groups; data are mean standard error, n =5, # P <0.05, # P <0.01.
Figure 9 is a graph of the effect of hydrogel treatment on fertility recovery. (A) Pregnancy of different treatment groups (non-injury group, model group, hydrogel group, stem cell exosome group, hydrogel mixed stem cell exosome group) after 8 weeks of operation; (B) The embryos and neonates of SD rats in the hydrogel mixed stem cell exosome group develop normally and are consistent with the development of rats in the undamaged group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.
Example 1
(1) Isolation and culture of adipose-derived stem cells:
taking a C57BL/6 mouse with the age of 4 weeks, treating the animal in the experiment process according to the guidance opinion on the animal to be tested at present of 2006 science and technology, taking the mouse after the neck of the mouse is removed and killed, placing the mouse into ethanol for soaking for 10 minutes, taking a inguinal subcutaneous adipose tissue sample, separating mesenchymal stem cells (ADSCs) from the adipose tissue, shearing the mouse, placing the cut mouse into a mixed solution of 0.2% NB4 and 0.05% neutral protease for digestion and primary culture, continuing to culture and passage, and taking a 3 rd generation adipose stem cell for later use.
Example 2
(2) Obtaining of adipose-derived stem cell exosomes:
exosomes are derived from mesenchymal stem cells in adipose tissues of 4-week-old C57BL/6 mice, the adipose mesenchymal stem cells are placed in a low-glucose DMEM medium containing 10% fetal bovine serum and 1% streptomycin/penicillin and cultured for 2 weeks at 37 ℃, exosomes released by the mouse mesenchymal stem cells are extracted by an ultracentrifugation method, collected culture medium supernatant is centrifuged at 800 Xg for 5 minutes, centrifuged at 2000 Xg for 10 minutes twice, the supernatant is filtered, centrifuged at 100000 Xg at 4 ℃ for 90 minutes, and then the obtained exosomes are diluted to 10 mu g/mL.
Example 3
(3) Adipose-derived stem cell exosome-AgNO 3 Preparation of hydrogel (multifunctional microenvironment-protected exosome hydrogel):
30mg of mercapto-polyethylene glycol (molecular weight 1000-2000Da, batch number: TZQ09095, creative PEG Works Co., ltd.) was weighed and dissolved in 100. Mu.L of adipose-derived stem cell exosomes, and 75. Mu.L of LAgNO was added 3 (batch No. 20170808, national Chemicals Ltd.) in an aqueous solution (AgNO) 3 Concentration of 0.1 mol/L), diluted with 100. Mu.L of adipose-derived stem cell exosome, and mixed with thiol-polyethylene glycol exosome solution, and the mixture forms transparent adipose-derived stem cell exosome-AgNO after a few seconds 3 -a hydrogel.
Experimental example 1
Establishing an animal model:
all experiments were performed according to the national institute of health, "guidelines for laboratory animal Care and use" (NIH publication No. 80-23) and approved by the institutional animal Care and use Committee of Shanghai university of transportation. SD rats (200-250 g in weight) of 8 weeks old were selected, placed in a climate controlled laboratory, and food and water were freely supplied, and thin endometrial injury models were constructed by injecting 95% absolute ethanol into the uterus to injure the bilateral uterus of the rats. The experimental animals were operated again after molding, one end of the uterus was clamped, the adipose-derived stem cell exosome hydrogel formulation was injected into the uterine cavity of rat with the syringe at the other end, after 3 estrus cycles after hydrogel injection, the rat was euthanized, uterine tissue was excised and sliced or frozen for downstream analysis.
Experimental example 2
Grouping design and treatment experiment:
50 rats were randomly divided into 5 treatment groups (10 per group): (1) Uninjured group (Sham operation), injected with PBS (200 μ L) in the uterine horn; (2) A natural repair group, which is injected with 95% ethanol into uterine horn according to the method; (3) Stem cell exosome group (ADSCs-exo), adipose stem cell exosomes (20 μ g,200 μ LPBS) were injected through uterine horn 30 minutes after ethanol-mediated injury induction; (4) Hydrogel group (hydrogel) injected with 200 μ L of AgNO 30min after ethanol induced injury 3 -hydrogel (0.1 mol/L); (5) Hydrogel mixed stem cell exosome group (ADSCs-exo @ hydrogel) was injected with 200. Mu.L of adipose-derived stem cell exosome (20. Mu.g) and 200. Mu.L of LAgNO in a single injection 30 minutes after ethanol-induced injury 3 -a hydrogel. After 3 estrus cycles after hydrogel injection, rats were euthanized and uterine tissue was excised and sectioned or frozen for downstream analysis.
Test example (I) test of physical and chemical Properties of hydrogel
TRPS (tunable resistance pulse sensing) analysis was used to assess the size of exosome particles; adipose-derived stem cell exosome-AgNO 3 The viscoelasticity of the hydrogels was evaluated using an AR2000 rheometer (TA Instruments, DE, USA); the elastic modulus (G ') and loss modulus (G') values of the hydrogels were tested at 37 ℃ and 1 Hz; the oscillatory strain test is performed for two cycles at 0.1% -100%.
0.5mL of the hydrogel was weighed, added to 5mLPBS, and incubated at 37 ℃ with stirring at 150rpm to investigate the biodegradability of the hydrogel. The remaining weight of the hydrogel (Wt) was weighed every 2 days, and the degree of biodegradation was determined by the Wt/W0 equation (W0 is the initial weight of the hydrogel). In addition, the detection of Ag in hydrogel by PBS method + The amount of (a) released. 200L of the hydrogel were placed in 2000L of PBS and soaked at 37 deg.C, 200L of the solution was removed at regular intervals (2, 4,6,8, 10, 12 days) and measured by inductively coupled plasma mass spectrometry (Nexion 2000, USA)Ag + The amount of (a) released.
(II) scanning Electron microscopy analysis
Hydrogel and exosome samples were taken, freeze-dried and analysed with a scanning electron microscope (SU-8010, hitachi, japan), loaded on conductive tapes for SEM testing at an accelerating voltage of 3kV and gold-coated for 60 seconds using a suitable instrument (criceton scientific instrument, waltford, uk).
(III) test tube test
Evaluation of adipose Stem cell exosomes-AgNO Using in vitro angiogenesis kit (Kurabo) 3 Hydrogel-induced angiogenesis. Contacting endothelial cells with AgNO 3 Hydrogel, adipose stem cell exosomes or both were incubated for 12 days, then endothelial tubes were stained with anti-CD 31 (Abcam) followed by secondary alkaline phosphatase binding to target anti-mouse IgG (Abcam). The number of endothelial vessels meeting the kit exclusion criteria was then quantified by microscopy and all experiments were performed three times independently.
(IV) qRT-PCR fluorescence assay
Total RNA was isolated from uterine tissue samples using TRIzol kit, then cDNA was prepared using PrimeScript RT reagent (TaKaRa Biotech, japan), and then amplified by 3' RACE (rapid amplification of cDNA ends) PCR according to the instructions provided, primers for amplification are shown in Table S1, and phosphate dehydrogenase (GAPDH) was used as a normalization control in assessing relative gene expression.
TABLE S1
Figure BDA0002908038880000131
Figure BDA0002908038880000141
(V) proliferation, migration, and lumen formation of human umbilical vein endothelial cells
Human umbilical vein endothelial cells (HUVECs; purchased from ATCC) were used in a coculture System with Transwell analysis filters (pore size 8 μm, corning, usa) evaluated proliferation, migration and tube formation ability. Adding HUVECs (4X 10) into the inferior cavity 4 ) In the upper cavity, agNO is added 3 Hydrogel 200. Mu.L and/or 20. Mu.g adipose-derived stem cell exosomes were subjected to proliferation and tube formation experiments.
Proliferation of HUVECs was assessed by Ki67 immunofluorescence staining. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% triton X-100, stained with anti-Ki 67 (1; the cells were then examined with an AF 488-binding secondary antibody (Abcam), counterstained with DAPI, and the number of Ki 67-positive cells was determined using ImageJ software.
In migration experiments, HUVECs (4X 10) 4 ) 100 μ L of AgNO added to the upper chamber of a Transwell laboratory Filter 3 -hydrogel, 10 μ g of adipose stem cell exosomes, or a combination of both, is added to the lower chamber. The cells were then incubated at 37 ℃ for 18h, after which the cells on the upper chamber membrane were fixed, stained with 0.5% crystal violet and then quantified microscopically.
To assess the effect of adipose stem cell exosomes on HUVEC tube formation, HUVECs were reduced with growth factor reducing matrix (BD Biosciences, USA) in 24-well plates (1 × 10) 4 Individual cells/well) using AgNO 3 Hydrogel or adipose stem cell exosome mixed hydrogel culture for 16 hours. Tubes in five random fields were then evaluated by microscopy and ImageJ analysis was performed using an angiogenesis analyzer.
In a thin endometrial animal model, penicillin is injected intraperitoneally for 3-5 days to prevent bacterial infection. However, these antibiotics were not used in model rats both before and after surgery. After 1 week of operation, detecting Ag by adopting staphylococcus aureus immunofluorescence staining method + The antibacterial activity of (1). This analysis showed the use of AgNO relative to animals treated with adipose stem cell exosomes (51.5 bacteria per spot) 3 Mixed adipose stem cell exosome treated animals showed a large reduction in bacteria (6 ± 1 bacteria per spot).
(VI) fertility test
Endometrial receptivity is assessed by the ability of the endometrium to allow implantation of a fertilized egg and to provide proper nutrition to the developing fetus. The oestrus cycle status of the rats was assessed by vaginal smear. Female rats (20 in each of the above 5 treatment groups) were mated with sexually mature male rats at a ratio of 1.
(VII) tissue and immunohistochemical staining
Excised tissue sections were first fixed in 4% paraformaldehyde overnight, then desalted with 16% ethylenediaminetetraacetic acid, embedded in OCT, and evaluated for endometrial fibrosis. Slicing according to the provided description H&E and Masson trichrome staining. Endometrial fibrosis and angiogenesis levels were assessed by immunohistochemical staining. Sections 20 μm thick were blocked with 5% bsa and then incubated overnight at 4 ℃ with rabbit anti-CD 31 (1, 100 abcam) and rabbit anti- α -SMA (1. PBS was washed 3 times and incubated for 1h at room temperature with the addition of a second rabbit anti-IgG. In addition, prepared AgNO was assayed using staphylococcus aureus immunofluorescent staining 3 Antibacterial property of the mixed adipose-derived stem cell hydrogel. Fresh endometrial tissue samples were also stored at-80 ℃ and then subjected to qRT-PCR and Westernblotting analysis.
(eight) immunoblotting
After lysis of the cells with lysis buffer (200. Mu.L), 20mg of protein was separated from each sample by 10% SDS-PAGE and transferred to PVDF membrane. Specific adipose stem cell exosome antibodies (CD 63, CD 9) or biomarkers of endometrial receptivity and angiogenesis, including rabbit anti-LIF (1 5000 abcam), rabbit anti-IGF-1 (1 1000 abcam), rabbit anti-VEGF (1. Second, assays were performed with a second rabbit antibody (1. ImageJ software (national institute of health, usa) was used for density analysis of protein bands.
(nine) flow cytometry
The adipose stem cell exosomes were suspended in separation buffer, mixed with CD9 immunomagnetic beads for 10 minutes, incubated overnight at 4 ℃ and incubated for 1 hour at room temperature. Unbound exosomes were then separated using a magnet. The remaining bead exosomes were washed twice in 200. Mu.L of separation buffer and then resuspended in 200. Mu.L of separation buffer. Incubate with anti-CD 63 and anti-CD 9 (Abcam) for 30min at room temperature and centrifuge at 8000 Xg for 1 min. The supernatant was then discarded, and the labeled exosomes were resuspended in PBS and then labeled with the appropriate AF488 conjugate secondary antibody (Abcam). The samples were then spun at 8000 Xg for an additional 1 minute, then the supernatant was discarded, washed with 100L PBS resuspended twice in PBS, and then exosome staining was assessed using a FACS Canto II flow cytometer (BD Biosciences).
(ten) statistical analysis
Data were evaluated using SPSS 22.0 using. + -. SD (standard deviation). Data were compared using one-way analysis of variance, followed by a graph-based multiple comparison test, with P <0.05 indicating significant differences.
Results example (I) characterization of exosomes of adipose-derived stem cells
Adipose-derived mesenchymal stem cells were first isolated from inguinal adipose tissue samples, and it was observed that the isolated adipose-derived mesenchymal stem cells rapidly expanded, in a spindle-shaped, fibroblast-like growth pattern (see fig. 2A). Immunofluorescent staining showed that 90% of adipose mesenchymal stem cells showed positive SOX2 pluripotency labeling ECM markers fibronectin and laminin. Flow cytometry analysis showed that these markers CD90 (99.7% ± 4.3%), CD105 (95.8% ± 4.7%) and CD44 (98% ± 5.3%) were all positive (as in figure 2B), while CD11B, CD31, CD34, CD83, CD133 and MHC-II were all negative (data not shown).
Exosomes from these adipose mesenchymal stem cells were then isolated by differential centrifugation and characterized by Scanning Electron Microscopy (SEM), immunoblotting (western blotting), flow Cytometry (FCM) and Tunable Resistance Pulse Sensing (TRPS). TRPS measurements showed that these adipose stem cell exosomes were 50-100nm in size (see fig. 2C), SEM showed that they were cupped or rounded in appearance, 50-100nm in size (see fig. 2D), consistent with the results for TRPS. FCM confirmed that these exosomes were highly pure, positive for both the exosome surface markers CD63 and CD9 (as in fig. 2E), and Western blotting also showed high expression of CD9 and CD63 by the exosomes (as in fig. 2F). These data indicate that adipose-derived stem cell exosomes have been successfully prepared. In addition, an exosome release profile (fig. 2G-H) was established, and it was demonstrated that these bioactive exosomes were effectively encapsulated in PEG hydrogel, promoting the adipose-derived stem cell exosomes to be released continuously for 14 days, with a release rate of 95%.
Characterization of the hydrogels
The preparation of PEG-Ag hydrogel is shown in FIG. 3A. PEG is subjected to Ag-S coordination and Ag + crosslinking to obtain injectable PEG-Ag, and the injectable PEG-Ag can load adipose stem cell exosomes. Since the thiol-PEG solution has good fluidity (FIG. 3B), when thiol-PEG, agNO 3 When mixed with exosome solution, they form a complex gel containing adipose stem cell exosomes, thiol-polyethylene glycol and AgNO 3 After mixing, a covalent bond between S and Ag is formed, and this bonding simultaneously produces an interaction between Ag and Ag, inducing the formation of a hydrogel. Under strong external shear forces, these bonds are broken, but the forces are removed and the crosslinked network is regenerated. Therefore, we hypothesized that these hydrogels are capable of self-repair after injection. The hydrogel surface was evaluated by SEM (fig. 3C), and no significant difference was found between the hydrogels with and without adipose stem cell exosomes, indicating that the adipose stem cell exosomes were successfully bound to the PEG hydrogel. We also demonstrated that these prepared hydrogels were injectable and able to retain their shape in deionized water (DI) solution (fig. 3D). In PBS solution, the exosome hydrogel could be stable for 4 days, with the exosome hydrogel degrading faster on day 4 and only 10% of the mass below the level after 10 days (figure 3E). This degradation behavior provides a stable structure for adipose stem cell exosomes to promote regeneration of damaged endometrial tissue and modulate the local microenvironment in vivo.
The prepared adipose-derived stem cell exosome hydrogel was further evaluated for its ability to withstand external stress. The loss modulus (G ") can withstand a pressure increase of nearly 60%, while the elastic modulus (G') continues to decrease (fig. 3F and G). The recoverability of the hydrogel is evaluated by a rheometer, and the result shows that the adipose-derived stem cell exosome hydrogel can keep a colloidal state and can keep a colloidal state before a high shear rateViscosity 7.5X 10 5 Pa.s, at high shear rate, the hydrogel structure is destroyed and the viscosity drops to 0.7X 10 5 Pa · s. However, within a few seconds after the removal of the high shear rate, the hydrogel recovered to a state similar to that before the application of the shear force (fig. 3H). The G 'of these PEG hydrogels decreased significantly over the two-step strain cycle, while the G' of the prepared ADSC-exo hydrogels was essentially unchanged (fig. 3I). In FIG. 3J, ADSCs-exo @ hydrogel group releases Ag cumulatively + 46.2 + -5.13% on day 2, increased to 97.5 + -11.31% on day 12, higher than the PEG-Ag hydrogel group at day 12 for Ag + The release ratio of (89.7. + -. 7.49%). Meanwhile, after co-culturing with ADSCs-exo hydrogel for 1, 3 and 5 days, live/dead detection and CCK-8 detection are carried out. As shown in fig. 3K, live/dead staining showed that stem cells were almost alive at different time periods after 5 days of culture, with only a few dead cells, and that live cells were not significantly different in the quantitative analysis at 1, 3, and 5 days.
(III) in vitro evaluation of biocompatibility of adipose-derived stem cell exosome and Human Umbilical Vein Endothelial Cell (HUVEC)
Angiogenesis refers to the process by which endothelial cells proliferate, migrate and form tubes to form new blood vessels to support tissue repair. Here, we evaluated the ability of the prepared adipose stem cell exosome hydrogels to affect the proliferation of HUVECs in vitro based on immunofluorescence Ki67 staining after a 3-day treatment period. Analysis showed that most cells exhibited Ki67 positivity in the adipose stem cell exosome hydrogel and adipose stem cell exosome treated groups, whereas Ki67 positive cells were less in the other treated groups (fig. 4A), and Ki67 positivity was highest in the adipose stem cell exosome hydrogel group relative to the other treated groups (fig. 4B). Thus, these data demonstrate that adipose stem cell exosome hydrogel formulations are able to promote sustained proliferation of endothelial cells in vitro, underscoring their potential in promoting angiogenesis in vivo.
Proliferation after treatment of HUVECs with adipose stem cell exosomes (25, 50, 75 or 100. Mu.g/mL) was evaluated. The Ki67 assay showed that HUVEC proliferation increased in a dose-dependent manner, with proliferation most pronounced after treatment with 100 μ g/mL adipose stem cell exosomes. Therefore, for patients who clinically use the adipose stem cell exosomes, the same adipose stem cell exosome dose can be used as a single therapeutic unit. To reduce the risk of potential side effects, only 1/5 of adipose stem cell exosomes were initially used. Since no side effects were observed, the unit dose was gradually increased to promote endometrial recovery.
The effect of adipose stem cell exosome hydrogel or exosome pretreatment on the in vitro migration and tube-forming activity of HUVECs was further evaluated. The Transwell migration assay showed that adipose stem cell exosome treatment significantly enhanced HUVEC migration, an effect that could further potentiate adipose stem cell exosome hydrogel therapeutic cells (fig. 4C and D). Consistent with these results, maximal tube formation and more complete tubular structure observed adipose stem cell exosome hydrogel treated HUVECs versus the other tested treatment groups (fig. 4E and F). Taken together, these data indicate that PEG hydrogel-mediated sustained release of adipose stem cell exosomes can promote the angiogenic response of HUVECs over time.
(IV) in vivo analysis of endometrial thickness and glandular hyperplasia
An in vivo model of endometrial injury in rats was established by direct injection of absolute ethanol into the uterine horn. After three estrus cycles, endometrial lesions were assessed by appropriate histological analysis. Damaged uterine tissue shows significant thinning of the endometrial layer and is indistinguishable in some areas from the myometrium. However, in rats injected with the adipose stem cell exosome hydrogel formulation, significant endometrial regeneration was observed, demonstrating a uniform cell distribution and more visible neovascularization (fig. 5A). In addition, stromal tissue is well organized in treated animals with appropriate epithelial and secretory gland tissue. The thickness of the endometrium of the undamaged group is 500 mu m, and the thickness of the endometrium of the natural repair group is obviously reduced to 170 mu m. The thickness of all experimental treatment groups increased, and although the final thickness was smaller in all cases than in the uninjured group, the maximum thickness of the hydrogel mixed stem cell exosome group-treated rats was 410m, indicating a strong regeneration potential of this hydrogel formulation (fig. 5C). The trend of the number of glands in the endothelial tissue was consistent with the trend of the endometrial thickness for the different treatment groups. Specifically, there were 12.5 glands on average in the rat hydrogel mixed stem cell exosome group, which reached 80% of the intact group and 625% of the natural repair group (fig. 5D), indicating that the exosome hydrogel of the present invention was able to greatly promote tissue regeneration.
As it is reported to date, in rats, the intrauterine exosome injection can only be performed after abdominal incision formation, and the treatment of human patients with thinner endometrium with mesenchymal stem cell-derived exosomes would be a simple, non-invasive procedure. The adipose-derived stem cell exosome hydrogel can be injected to the cervix through a soft-tip catheter and then gently placed into the uterus. Ultrasound guidance during injection allows visualization of the motion of the catheter within the endometrial cavity, thereby minimizing the risk of endometrial damage, and large-scale clinical trials will be necessary to evaluate the efficacy of this treatment in the future.
(V) evaluation of hydrogel formulations for angiogenesis and regenerative Activity in vivo
Considering that endometrial regeneration is dependent on angiogenesis in order to provide adequate oxygen and nutrient supply to the regenerating tissue, it was investigated whether the adipose stem cell exosome hydrogel-mediated neovasculature observed in vitro (fig. 3) is associated with endometrial angiogenesis in vivo. For this reason, staining of endometrial tissue with the endothelial cell marker CD31 was performed, and it was found that staining of endometrial tissue samples of the stem cell exosome group and the hydrogel mixed stem cell exosome group was significantly increased compared to the natural repair group (fig. 6A), wherein staining was highest with the hydrogel mixed stem cell exosome group. The corresponding immunohistochemical staining results also show that the adipose-derived stem cell exosome hydrogel obviously enhances the in-vivo neovascularization (fig. 6A).
To assess myometrial regeneration, tissue sections were stained with anti- α -SMA (fig. 6A). Although the stem cell exosome group had a clear thin and continuous layer of circular myofibers in the 3 postsurgical estrus cycles, this was not the case for the natural repair group or the hydrogel group. In contrast, the number of muscle bundles was significantly increased in the animals of the stem cell exosome group compared to the hydrogel mixed stem cell exosome group (fig. 6B). The quantification shows that the staining area of the hydrogel mixed stem cell exosome group a-SMA is obviously higher than that of the stem cell exosome group (FIG. 6B). The normal muscle fiber morphology of the uterine horn is shown in fig. 5B. Taken together, these results indicate that the adipose stem cell exosome hydrogel of the present invention can promote the healing and regeneration of the myometrium.
Masson trichrome staining was further performed to assess collagen deposition in isolated uterine sections and adipose stem cell exosomes were found to significantly reduce endometrial fibrosis and fibrosis, rather than promote endometrial and myofibrillar growth. These data also indicate that the anti-fibrotic activity of the stem cell exosome group was significantly reduced compared to the hydrogel mixed stem cell exosome group treatment according to the percentage of collagen deposition in each group. The result shows that the adipose-derived stem cell exosome hydrogel can effectively inhibit inflammation and fibrosis by mediating the sustained release of adipose-derived stem cell exosomes, so that the vascular proliferation and the glandular proliferation are promoted under the condition of rat endometrial injury.
(VI) evaluation of in vivo antibacterial Activity of hydrogel preparation
Previous data suggest that intrauterine adhesion (IUA) is usually caused by infection, suggesting that our adipose stem cell exosome hydrogel may also alleviate intrauterine infection. To evaluate the antimicrobial activity of the hydrogel, an immunofluorescent staining of staphylococcus aureus was performed (fig. 7A). Animals treated with the stem cell exosome group showed 51 ± 5 bacteria per spot, while animals treated with the hydrogel mixed stem cell exosome group showed 6 ± 1 bacteria per spot, showing more excellent antibacterial performance.
(VII) evaluation of the ability of the prepared hydrogel to promote endometrial regeneration in vivo
The expression of ER and PR, which are closely related to endometrial regeneration, and the expression of bFGF and VEGF, are evaluated to promote proliferation of new blood vessels and endothelial cells by mediating vascular permeability, optimizing blastocyst implantation. At the mRNA level, ADSCs-exo treatment was associated with significantly elevated VEGF, LIF, av β 3 and IGF-1 expression (FIG. 8A), while ADSCs-exo @ hydrogel treatment group further elevated expression of these markers. The same trends were also observed for VEGF and IGF-1 protein levels in these treatment groups (fig. 8B and C).
The effect of this exosome hydrogel on endometrial regeneration was evaluated and embryo implantation rates were collected by each panel at 18 and 20 days of gestation (fig. 9). The pregnancy and implantation rates of the non-injured group were the highest, while those of the stem cell exosome group and the hydrogel mixed stem cell exosome group were both significantly increased (fig. 9A). The result shows that the adipose-derived stem cell exosome hydrogel can well promote the functional regeneration of endometrium and is beneficial to the restoration of fertility. The hydrogel mixed stem cell exosome group embryos and newborns developed normally, indicating that the endometrium morphology and function of this treatment group were normal (fig. 9B).
Comparative example 1
In order to examine the influence of the preparation of the hydrogel on the performance of the hydrogel, the mass-to-volume ratio of the mercapto-polyethylene glycol (molecular weight 3000 Da) to the adipose-derived stem cell exosomes in example 3 was adjusted to be 30mg:50 mu L of the prepared hydrogel is used as a hydrogel mixed stem cell exosome group to be compared with a control 1 to examine the influence on the implantation and pregnancy experimental results.
Comparative example 2
Considering the influence of the preparation of the hydrogel on the performance of the hydrogel, the mass-to-volume ratio of the sulfhydryl-polyethylene glycol (with a molecular weight of 5000 Da) to the adipose-derived stem cell exosomes in example 3 is specifically adjusted to 30mg:150 μ L, and the prepared hydrogel was examined as a hydrogel mixed stem cell exosome group for control 2 for its effect on the results of implantation and pregnancy experiments.
The implantation and pregnancy data of each treatment group and comparative example were counted, and the results are shown in table 1. From table 1, it can be seen that the hydrogel obtained by the present invention is a multifunctional hydrogel, which can promote the control of exosome release and mediate endometrial regeneration.
TABLE 1
Figure BDA0002908038880000241

Claims (4)

1. A preparation method of a multifunctional microenvironment protection exosome hydrogel is characterized by comprising the following steps:
(1) Separating adipose-derived mesenchymal stem cells from an animal tissue sample, digesting to obtain primary cells, performing subculture, and taking the adipose-derived mesenchymal stem cells for later use;
(2) Taking adipose-derived mesenchymal stem cells, placing the mesenchymal stem cells into a DMEM culture medium, culturing for 2 weeks at 37 ℃, and then extracting exosomes released by the mesenchymal stem cells by adopting an ultracentrifugation method, wherein the ultracentrifugation method comprises the following steps: centrifuging the collected culture medium supernatant at 800 Xg for 5 min, centrifuging at 2000 Xg for 10 min twice, filtering the supernatant, and centrifuging at 100000 Xg at 4 deg.C for 90 min; diluting the obtained exosome to 10 mu g/mL for use;
(3) Dissolving sulfydryl-polyethylene glycol into the adipose-derived stem cell exosomes obtained in the step (2) to obtain a mixture, wherein the molecular weight of the sulfydryl-polyethylene glycol ranges from 1000 Da to 2000Da, and the mass-volume ratio of the sulfydryl-polyethylene glycol to the adipose-derived stem cell exosomes is 30mg: 100. mu L; then AgNO with the concentration of 0.1mol/L 3 Diluting the water solution by using adipose-derived stem cell exosomes, wherein the AgNO is 3 And (3) adding the aqueous solution and the adipose-derived stem cell exosomes into the mixture at a volume ratio of 3.
2. The method according to claim 1, wherein the DMEM medium in step (2) contains 10% fetal bovine serum and 1% streptomycin or penicillin.
3. A multifunctional microenvironment-protected exosome hydrogel prepared by the method of claim 1 or 2.
4. Use of a multifunctional microenvironment-protective exosome hydrogel according to claim 3 in the preparation of a medicament for promoting endometrial regeneration and fertility restoration.
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