Preparation method of an IVK8-RGD polypeptide and its hydrogel, and composition after loading human umbilical cord-derived mesenchymal stem cells

Abstract

The present invention discloses a method for preparing an IVK8-RGD polypeptide and a hydrogel thereof, as well as a composition loaded with human umbilical cord-derived mesenchymal stem cells. The IVK8-RGD polypeptide is functionally modified using the bioactive sequence RGD on a self-assembling polypeptide IVK8 having eight amino acids, IEVEIRVK. Glycine and aspartic acid are selected as connecting groups between the bioactive sequence RGD and the self-assembling polypeptide IVK8. The charge of the entire peptide chain is arranged in a -++ pattern. The hydrogel can self-assemble into a gel after incubation at 37°C for 15 minutes, and has adjustable mechanical strength and long-term stability. The IVK8-RGD polypeptide hydrogel loaded with human umbilical cord-derived mesenchymal stem cells can effectively inhibit excessive endometrial fibrosis, reduce inflammatory responses, promote endometrial proliferation and vascular remodeling, promote endometrial repair, effectively treat endometrial damage and intrauterine adhesions, and promote fertility recovery.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for preparing an IVK8-RGD polypeptide and its hydrogel, and a composition loaded with human umbilical cord-derived mesenchymal stem cells. Background Art

[0002] The uterus is one of the most important reproductive organs for women. The two main factors for successful embryo implantation are receptive endometrium and normal embryonic development. However, when the endometrium is physically damaged (induced abortion, frequent uterine surgery) and biochemically damaged (infection, endocrine disruptors), it will cause damage to the endometrium, causing adhesions in the uterine cavity, affecting embryo implantation, and leading to female infertility or recurrent miscarriage. At present, there are still huge challenges in promoting the repair and regeneration of endometrial damage. Traditional treatment methods mainly include surgery, intrauterine barrier, endocrine regulation, and increasing endometrial blood perfusion. However, the above strategies are difficult to effectively repair the uterus and restore fertility.

[0003] Stem cells are multipotent and can differentiate into a variety of tissue cells. They also have the ability to release growth factors and regulate inflammation. They have shown great potential in the field of regenerative medicine for treating various injuries and diseases. Mesenchymal stem cells (MSCs) are pluripotent stem cells with multidirectional differentiation potential and can be isolated from a variety of tissues, such as bone marrow, placenta, umbilical cord, fat, and endometrium. Currently, a large number of animal studies have shown that human umbilical cord-derived mesenchymal stem cells (UC-MSCs) can improve and repair damaged endometrium and increase pregnancy rates. Intravenous injection of UC-MSCs can also increase endometrial thickness and glandular number, promote blood vessel growth and endothelial cell proliferation, restore endometrial structure and function, and improve embryo implantation rates. Furthermore, UC-MSCs have the characteristics of low immunogenicity, strong self-replication ability, and non-invasive collection, which can promote the regeneration and repair of damaged tissues.

[0004] However, the clinical application of stem cell therapy still has some defects, such as adverse reactions such as tumorigenicity, thrombosis, and fever. At the same time, simple local stem cell injection therapy is significantly limited in application due to shortcomings such as cell extravasation from target tissues and organs and low cell survival rate. The survival rate and long-term activity of stem cells after entering the body are the key to successful treatment. Hydrogel, as a polymer network with water as its main component, is an ideal tissue engineering material. It can not only protect cells from membrane rupture caused by injection, but also simulate the extracellular matrix to achieve long-term survival of stem cells in target tissues and organs and continue to exert paracrine effects.

[0005] Self-assembling peptide hydrogels (SAPs), first discovered in 1993, have garnered widespread attention for their excellent biocompatibility and biodegradability, low toxicity, batch-to-batch consistency, and excellent optical clarity. The classic RADA16-I peptide (RADARADARADARADA) has been developed into clinical hemostatic materials such as Purastat (3D Matrix) and Pura Matrix (Corning), a 3D cell culture matrix. In the cell culture field, the bioactivity of the RADA16-I peptide is limited. For example, Pura Matrix requires mixing with bioactive molecules to achieve optimal cell growth and differentiation. Several studies have also shown that the bioactivity of the RADA16-I peptide has been enhanced by modifying its termini with bioactive short peptides. Common active peptides include the RGD peptide sequence common to various ECM proteins, the laminin-derived peptide sequences IKVAV and YIGSR, the bone marrow homing peptide-derived sequences PSFFTKT and SKPPGTSS, and the vascular endothelial growth factor (VEGF) mimetic peptide sequence KLT. On the other hand, the RADA16-I peptide is weakly acidic and can damage cells when directly mixed with cell suspensions to form a gel. Therefore, the Pura Matrix instructions specifically state that the gel must be immediately rinsed with a large amount of culture medium to neutralize the solution. This issue has created inconvenience in the application of the RADA16 peptide in cell culture.

[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for preparing an IVK8-RGD polypeptide and its hydrogel, and a composition loaded with human umbilical cord-derived mesenchymal stem cells, which can avoid the defects of many adverse reactions in clinical stem cell treatment and the problem of cell damage caused by the mixing of RADA16-I peptide and cell suspension into gel.

[0008] To achieve the above objectives, the present invention provides an IVK8-RGD polypeptide, which uses the biologically active sequence RGD to functionally modify the self-assembling polypeptide IVK8 having 8 amino acids IEVEIRVK, and selects glycine and aspartic acid as the connecting groups between the biologically active sequence RGD and the self-assembling polypeptide IVK8.

[0009] The present invention also provides a method for preparing an IVK8-RGD polypeptide hydrogel, comprising the following steps:

[0010] (1) synthesizing a self-assembling polypeptide IVK8 from the eight amino acids IEVEIRVK, functionalizing the self-assembling polypeptide IVK8 with the biologically active sequence RGD, and selecting glycine and aspartic acid as linking groups between the biologically active sequence RGD and the self-assembling polypeptide IVK8 to obtain an IVK8-RGD polypeptide;

[0011] (2) Preparation of hydrogel: The IVK8-RGD polypeptide was dissolved in a 300 mM sterile sucrose solution at a concentration of 1.0% w / v, sonicated to promote dissolution, the pH was adjusted to 7.4, and mixed and defoamed by shaking and centrifugation. The solution was mixed with DMEM at a ratio of 4:1 (volume) and placed in an incubator for incubation for 15-30 minutes to form an IVK8-RGD polypeptide hydrogel.

[0012] The present invention also provides a composition of an IVK8-RGD polypeptide hydrogel loaded with human umbilical cord-derived mesenchymal stem cells, wherein the IVK8-RGD polypeptide hydrogel is prepared using the above-mentioned method for preparing the IVK8-RGD polypeptide hydrogel.

[0013] Furthermore, the composition of IVK8-RGD polypeptide hydrogel loaded with human umbilical cord-derived mesenchymal stem cells is used to promote endometrial cell proliferation and vascular remodeling to repair the endometrium, treat endometrial damage and intrauterine adhesions, and promote the recovery of fertility.

[0014] Compared with the prior art, the preparation method of the IVK8-RGD polypeptide and its hydrogel according to the present invention, and the composition loaded with human umbilical cord-derived mesenchymal stem cells have the following beneficial effects:

[0015] The self-assembling peptide IVK8 (IEVEIRVK) is an ion-complementary peptide with eight amino acids. It contains alternating hydrophobic and hydrophilic amino acids. The four hydrophobic amino acids are two isoleucines and two valines, and the four hydrophilic amino acids are two glutamics, which are negatively charged at physiological pH, and arginine and lysine, which are positively charged. R has good gel-forming properties, while the combination of arginine and lysine has good biocompatibility. As a result, the charge of the entire peptide chain is arranged in a -++ pattern. This arrangement enables it to self-assemble into a gel after incubation at 37°C for 15 minutes, and achieves adjustable mechanical strength and long-term stability, solving the problems of insufficient biological activity and susceptibility to cell damage at low pH values that exist in conventional self-assembling peptides.

[0016] The bioactive sequence RGD was selected to functionalize the self-assembling peptide IVK8, and glycine and aspartic acid were used as linker groups to adjust the isoelectric point and hydrophilicity of the entire peptide chain, resulting in the IVK8-RGD peptide that can stably form a gel under neutral conditions.

[0017] After loading human umbilical cord-derived mesenchymal stem cells (UC-MSCs), IVK8-RGD hydrogel protected the activity of UC-MSCs and enhanced their repair effect. It is non-toxic to the human body and cells. IVK8-RGD hydrogel loaded with UC-MSCs can effectively inhibit excessive endometrial fibrosis, reduce inflammatory response, promote endometrial cell proliferation and vascular remodeling, thereby repairing the endometrium, effectively treating endometrial damage and intrauterine adhesions, and promoting the recovery of fertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 (a) is a schematic diagram of the structure of the IVK8-RGD polypeptide according to one embodiment of the present invention, (b) is a schematic diagram of the molecular model of the self-assembly of the IVK8-RGD polypeptide and a TEM image obtained after assembly (scale bar 1 μm), (c) a schematic diagram of the gelation of the IVK8 polypeptide and the IVK8-RGD polypeptide at physiological pH, (d) a circular dichroism spectrum image of the IVK8 polypeptide and the IVK8-RGD polypeptide, (e) a schematic diagram of the rheological properties of the IVK8 polypeptide and the IVK8-RGD polypeptide, (f) a schematic diagram of the adhesion and injectability of the IVK8-RGD polypeptide hydrogel at pH 7.4 to different mouse organs;

[0019] Figure 2 Schematic diagram comparing the cell activity of IVK8 polypeptide hydrogel and IVK8-RGD polypeptide hydrogel according to one embodiment of the present invention, (a) is a schematic diagram comparing the activity of UC-MSCs cultured in 2D on the surface, (b) is a live / dead staining image, (c) is a schematic diagram comparing the activity of UC-MSCs cultured in 3D, and (d) is a live / dead image.

[0020] Figure 3 Schematic diagram of the characterization of IVK8-RGD polypeptide hydrogels with different concentrations and 3D culture cell activity according to one embodiment of the present invention, (a) is a SEM image of IVK8-RGD polypeptide hydrogels with different concentrations (scale 20 μm), (b) is a schematic diagram of strain scanning and frequency scanning for rheological properties testing, (c) is a schematic diagram of MTT detection of cell activity, and (d) is a confocal image of live / dead staining of 3D cell culture.

[0021] Figure 4 Schematic diagram of HE staining of a rat endometrial injury model after human umbilical cord-derived mesenchymal stem cells were loaded with IVK8-RGD polypeptide hydrogel according to one embodiment of the present invention, (a) schematic diagram of HE staining of both sides of the rat uterus after unilateral left injury, (b) schematic diagram of HE staining of both sides of the rat uterus after unilateral left injury and treatment, (c) schematic diagram of the number of pregnancies in rats after unilateral left injury, (d) schematic diagram of the number of pregnancies in rats after unilateral left injury and treatment. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0023] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0024] According to a preferred embodiment of the present invention, a method for preparing an IVK8-RGD polypeptide and its hydrogel, and a composition loaded with human umbilical cord-derived mesenchymal stem cells, the IVK8-RGD polypeptide hydrogel is a self-assembling polypeptide IVK8 functionalized with the bioactive sequence RGD, and glycine (G) and aspartic acid (D) are selected as linking groups to obtain an IVK8-RGD polypeptide that can stably form a gel under neutral conditions. Figure 1 (a) and Figure 1 As shown in (b), the self-assembling polypeptide IVK8 is an ion-complementary peptide IEVEIRVK with 8 amino acids, which contains alternating hydrophobic amino acids and hydrophilic amino acids. The four hydrophobic amino acids are two isoleucines (I) and two valines (V), and the four hydrophilic amino acids are two glutamic acids (E) with negative charges at physiological pH and arginine (R) and lysine (K) with positive charges. Among them, R has good gel-forming properties, and the combination of K and R has good biocompatibility. Therefore, the charge of the entire peptide chain is arranged in a (--++) pattern. This arrangement enables it to self-assemble into a gel after incubation at 37°C for 15 minutes, and achieve adjustable mechanical strength and long-term stability. Compared with the RADA16-I peptide, the ion-complementary peptide IVK8 has only eight amino acids, significantly reducing production costs. However, simple self-assembling peptide sequences also have the disadvantages of insufficient activity and being weakly acidic and easily damaging cells. Therefore, the bioactive sequence RGD was selected to functionalize the IVK8 peptide, and glycine (G) and aspartic acid (D) were selected as linking groups to adjust the isoelectric point and hydrophilicity of the entire peptide chain, enabling the IVK8-RGD peptide to stably form a gel under neutral conditions.

[0025] The preparation method of IVK8-RGD polypeptide hydrogel comprises the following specific steps:

[0026] (1) The IVK8 peptide was synthesized from the 8 amino acids IEVEIRVK, and the IVK8-RGD peptide was obtained by functionally modifying the IVK8 peptide with the biologically active sequence RGD. Both were synthesized by Shanghai Sangon Biotechnology Co., Ltd., with a purity greater than 95%. The IVK8 peptide and IVK8-RGD peptide were desalted separately.

[0027] (2) Preparation of hydrogel: The self-assembling polypeptide IVK8 and IVK8-RGD polypeptide were dissolved in 300mM sterile sucrose solution at a concentration of 1.0% w / v, and ultrasonic dissolution was used to promote dissolution. The pH of the obtained IVK8 polypeptide solution was 5-6, and no pH adjustment was required. The obtained IVK8-RGD polypeptide solution was adjusted to 7.4 using precise pH test paper, and was quickly mixed by shaking and centrifugation and became a mucus state after defoaming. After filtration and sterilization using a 0.22μm vacuum filter, it can be stored at 4°C for a long time if not used temporarily. When used, it was mixed with high-glucose DMEM at a ratio of 4:1 (volume) and placed in an incubator for incubation for 15 to 30 minutes to form stable hydrogels IVK8 polypeptide hydrogel and IVK8-RGD polypeptide hydrogel.

[0028] like Figure 1 As shown in (c), the prepared IVK8-RGD polypeptide hydrogel and self-assembling polypeptide IVK8 can stably form gel under neutral conditions. Figure 1 As shown in (f), the IVK8-RGD polypeptide hydrogel at pH 7.4 has certain adhesion properties and can effectively adhere to mouse tissues and organs. At the same time, SYSU can be written by squeezing the IVK8-RGD polypeptide hydrogel using a syringe, indicating that the IVK8-RGD polypeptide hydrogel at pH 7.4 is injectable.

[0029] like Figure 1 As shown in (d), circular dichroism spectroscopy was used to study the secondary structure of the IVK8-RGD polypeptide hydrogel. The IVK8 polypeptide solution and the IVK8-RGD polypeptide solution were diluted to 0.5 mg / ml with ultrapure water and adjusted to neutrality. The protein secondary structure was determined using a circular dichroism spectrometer (Chirascan) in the range of 190 nm to 260 nm. A micro-cuvette with a path length of 1 mm was used. Pro-Data was used for buffer subtraction, and the Savitsky-Golay mode was selected as the smoothing mode. The Dichroweb database was used for the qualitative analysis of the protein secondary structure.

[0030] The results showed that the polypeptide solutions before and after grafting RGD showed a typical β-folded structure, which was manifested as a strong positive peak at 195-198 nm and a negative peak at 217-218 nm.

[0031] like Figure 1As shown in (e), the mechanical properties of IVK8-RGD polypeptide hydrogel were measured using a rheometer. On the one hand, the amplitude scanning results showed that there was an intersection between the storage modulus (G') and the loss modulus (G"), indicating gelation. On the other hand, by setting a constant strain for frequency scanning, the results showed that the IVK8 polypeptide hydrogel was not stable, and the storage modulus (G') of the IVK8-RGD polypeptide hydrogel was greater than the loss modulus (G") in the entire frequency scanning range, indicating that it maintained a stable gel scaffold structure.

[0032] For cell viability experiments, human umbilical cord-derived mesenchymal stem cells were purchased from cyagen (HUXUC-01001) and cultured in complete medium (cyagen, HUXUC-90011) at 37°C in a 5% CO2 incubator. The medium was changed every 2-3 days. When the cells reached 90% confluence, they were digested with 0.25% trypsin and then perfused with medium. The cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh complete medium to obtain a concentration of 2.5×10 6 / ml human umbilical cord-derived mesenchymal stem cell suspension.

[0033] For the surface culture experiment, IVK8 peptide solution and IVK8-RGD peptide solution were mixed with complete culture medium at a ratio of 4:1, and then added to a 96-well plate at 100 μl per well. The plates were incubated in an incubator for 30 minutes. After complete gelation, 5,000 cells were added to each well. The activity of the cells was determined by CCK8 at the 2nd and 12th hours, and the cells were stained with the Calcein / PI cell activity and cytotoxicity detection kit and photographed.

[0034] (2) 3D culture experiments

[0035] The IVK8 peptide solution and the IVK8-RGD peptide solution were directly mixed with a suspension of human umbilical cord-derived mesenchymal stem cells at a 4:1 ratio (volume) to a final cell density of 500 cells per μL of hydrogel. 10 μL of the cell-hydrogel mixture was added to each well of a 96-well plate and incubated for 15 to 30 minutes. 100 μL of complete culture medium was then added to each well. To measure cell viability, we quantified cell viability using MTT assays on days 1, 3, 5, and 7. Cells were also stained using a Calcein / PI cell viability and cytotoxicity assay kit and then scanned in 3D using the Z-stack function of a two-photon laser confocal scanning microscope.

[0036] The adhesion effect of IVK8-RGD polypeptide hydrogel on UC-MSCs was detected. Cells were cultured on the surface of IVK8-RGD polypeptide hydrogel and the cell number was monitored at 2 hours and 8 hours. Figure 2As shown in (a), compared with the control group IVK8 polypeptide hydrogel, the number of cells adhering to the surface of IVK8-RGD polypeptide hydrogel increased significantly. The results of live / dead staining images are shown in Figure 2 As shown in (b), compared with the IVK8 polypeptide hydrogel group, the UC-MSCs cultured in the IVK8-RGD polypeptide hydrogel group were in good condition, and most of them showed an extended morphology. Figure 2 (c) and Figure 2 As shown in Figure (d), cells were further cultured in 3D within the two peptide hydrogels. The cells were then encapsulated in the peptide hydrogels for long-term culture. The results showed that the proliferation rate of UC-MSCs in the IVK8-RGD peptide hydrogel group was significantly higher than that in the IVK8 peptide hydrogel group on day five of culture. Furthermore, the test results of the IVK8-RGD peptide hydrogel group on days three and five of culture were significantly different from those on day one. These results suggest that IVK8 peptide hydrogels grafted with the RGD bioactive peptide can effectively promote the adhesion and proliferation of UC-MSCs.

[0037] To further explore the optimal concentration of IVK8-RGD polypeptide hydrogel, 0.8%, 1.0%, and 1.2% IVK8-RGD polypeptide hydrogel solutions were quickly frozen with liquid nitrogen and cut into thin slices. After freeze-drying, they were sprayed with gold and photographed using an EVO scanning electron microscope (Zeiss). Figure 3 (a), Scanning electron microscopy image, showing that the pore size of IVK8-RGD polypeptide hydrogel decreases with increasing concentration, and its pore size ranges from 50 to 200 nm.

[0038] The rheology of the hydrogel was studied by setting the operating temperature to 25°C and using a rotational rheometer (Thermo HAAKE RotationRheometer Mars 40) in the plate mode. The hydrogel was subjected to amplitude scanning and frequency scanning one hour after preparation. The amplitude scanning was set to 0.1% to 1000% strain to find the linear viscoelastic region of the hydrogel. The appropriate stress was selected in the linear viscoelastic region (Strain = 5% in the experiment), and then the frequency scanning was performed. The frequency scanning range was set to 0.1 to 100 rad / s to study the performance of IVK8-RGD polypeptide hydrogel at different frequencies. Figure 3 (b), the rheological results show that its mechanical properties increase with increasing concentration and remain stable in the angular frequency range of 0.1 to 100 rad / s. Figure 3 (c) Further exploration of the long-term 3D cell activity of IVK8-RGD polypeptide hydrogels with different concentrations was conducted. The activity was detected by MTT kit on days 1, 3, 5, and 7. The results showed that the cells grown in 0.8% IVK8-RGD polypeptide hydrogels had the best activity, indicating that large-pore hydrogels are more conducive to cell diffusion and proliferation. Figure 3(d) UC-MSCs adhered and proliferated in 0.8% IVK8-RGD polypeptide hydrogels on days 1, 3, 5, and 7. Z-stack images taken after live / dead staining showed that UC-MSCs exhibited a better cell morphology in 0.8% IVK8-RGD polypeptide hydrogels, gradually transforming from a spherical state to an extended state, which may be due to the increase of cell adhesion sites by RGD active peptide.

[0039] The above research results show that IVK8-RGD polypeptide hydrogel has good biocompatibility, neutral pH, rapid gelation, easy use, and good optical transparency for easy observation. Therefore, it can be used as a matrix for long-term 3D culture of stem cells. At the same time, it has good biological adhesion to mouse organs.

[0040] An IVK8-RGD polypeptide solution is mixed with a cell suspension and then incubated for 15 to 30 minutes to form a gel. The composition is used for repairing the endometrium.

[0041] To verify the effectiveness of the IVK8-RGD peptide hydrogel-loaded human umbilical cord mesenchymal stem cell-loaded composition on endometrial restoration, a severe rat endometrial injury model was established using mechanical injury. Adult female rats (6-8 weeks old) were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital, and the uterine horns were exposed through an abdominal incision. The proximal and distal uteri were then gently clamped with two vascular clamps. A small incision was made near the uterine horns. A roughened 1.8 mm diameter tool was inserted through the opening and repeatedly massaged in and out until the uterus became red, swollen, and bleeding occurred. 95% ethanol was then injected into the uterine cavity for 5 minutes. The clamps were then released, and the remaining ethanol was gently squeezed to remove it. The injured area was then washed with PBS to further remove any remaining ethanol. The uterine wound was closed with absorbable sutures. Different drugs were injected bilaterally into the uterus according to the group, and finally, the myometrium and skin were closed with non-absorbable sutures.

[0042] In the preliminary experiment, rats were divided into two groups. One group was the injury group, in which the left uterus of the rats was unilaterally injured, and the right uterus was used as a self-control; the other group was the treatment group, in which the left uterus of the rats was injured and injected with IVK8-RGD polypeptide hydrogel loaded with human umbilical cord stem cells for treatment. At the same time, the right uterus was used as a self-control. The uterine tissue was fixed with 4% paraformaldehyde solution overnight and embedded into wax blocks. Then all tissues were cut into 7um slices, stained with hematoxylin and eosin, and observed and photographed under a microscope. Figure 4 ,The results showed that the rat injury model was successfully established, e.g. Figure 4 As shown in (a), the endometrium on the damaged side is much thinner than that on the normal side. Figure 4 As shown in (c), the pregnancy rate of the damaged uterus is much lower than that of the normal uterus. Figure 4 As shown in (b), after IVK8-RGD polypeptide hydrogel was injected into the injured side, the endometrium was significantly restored to the same thickness as the normal side. Figure 4 As shown in (d), the uterine fertility of the treated side was also slightly improved. The results show that IVK8-RGD peptide hydrogel loaded with human umbilical cord mesenchymal stem cells can effectively promote the regeneration of the endometrium, thereby promoting the recovery of the uterus.

[0043] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An IVK8-RGD polypeptide, characterized in that The self-assembling polypeptide IVK8 having 8 amino acids IEVEIRVK was functionally modified using the biologically active sequence RGD. Glycine and aspartic acid were selected as connecting groups between the biologically active sequence RGD and the self-assembling polypeptide IVK8. The biologically active sequence RGD was used to adjust the isoelectric point and hydrophilicity of the entire peptide chain to obtain an IVK8-RGD polypeptide that can stably form a gel under neutral conditions. The sequence of the IVK8-RGD polypeptide is IEVEIRVK-GD-RGDS.

2. A method for preparing an IVK8-RGD polypeptide hydrogel, characterized in that: The following steps are involved: (1) A self-assembling polypeptide IVK8 was synthesized from the 8 amino acids IEVEIRVK, and the self-assembling polypeptide IVK8 was functionally modified using the biologically active sequence RGD. Glycine and aspartic acid were selected as the linking groups between the biologically active sequence RGD and the self-assembling polypeptide IVK8 to obtain an IVK8-RGD polypeptide. The sequence of the IVK8-RGD polypeptide was IEVEIRVK-GD-RGDS. (2) Preparation of hydrogel: The IVK8-RGD polypeptide was dissolved in a 300 mM sterile sucrose solution at a concentration of 1.0% w / v, sonicated to promote dissolution, and the pH was adjusted to 7.

4. The solution was mixed and defoamed by shaking and centrifugation. The solution was mixed with DMEM at a volume ratio of 4:1 and incubated in an incubator for 15-30 minutes to form an IVK8-RGD polypeptide hydrogel.

3. A composition of IVK8-RGD polypeptide hydrogel loaded with human umbilical cord-derived mesenchymal stem cells, characterized in that: The IVK8-RGD polypeptide hydrogel is prepared using the preparation method of the IVK8-RGD polypeptide hydrogel according to claim 2.

4. A use of the IVK8-RGD polypeptide hydrogel-loaded human umbilical cord-derived mesenchymal stem cell composition according to claim 3 in the preparation of a medicine, characterized in that: Used to prepare drugs that promote endometrial cell proliferation and vascular remodeling to repair the endometrium, treat endometrial damage and intrauterine adhesions, and promote fertility recovery.

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