A gel for repairing endometrial damage and its preparation method and application

Abstract

The present invention belongs to the field of biomaterials, and specifically relates to a gel for repairing endometrial damage, and a preparation method and application thereof. The present invention provides a gel for repairing endometrial damage, and the gel comprises recombinant humanized type I collagen, antimicrobial peptides and mesenchymal stem cells. The gel of the present invention promotes the proliferation of endometrial cells, repairs the endometrial microenvironment, promotes endometrial regeneration, effectively repairs damaged endometrium, and has development prospects for clinical application.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a gel for repairing endometrial damage and a preparation method and application thereof. Background Art

[0002] Endometrial damage is a very common tissue lesion in obstetrics and gynecology, usually caused by surgical injury or inflammation, such as curettage, abortion, endometritis, etc. Because the long-term inflammatory environment damages the endometrium, the endometrial environment is destroyed, and the endometrial receptivity is reduced. Although endometrial inflammation can be alleviated after treatment with oral antibiotics, intrauterine perfusion, mechanical stimulation, etc., the existing treatment methods are prone to recurrence and the treatment effect is not ideal.

[0003] Collagen is the most abundant protein in the human body, accounting for 25-30% of the total protein. As the most abundant protein in the extracellular matrix, collagen maintains the integrity of the cell structure and various physiological functions. Collagen has good biocompatibility, low immunogenicity and tissue degradability and absorption. It is an ideal medical biomaterial in terms of wound hemostasis, healing, filling, repair and reconstruction of defective tissues.

[0004] The prior art has disclosed the use of collagen gel as a scaffold to carry corresponding active substances to treat endometrial damage. However, collagen is currently mainly derived from animal tissues, and naturally extracted collagen is a mixture of collagens of various molecular weights, which is insoluble in water and has poor biocompatibility. The traditional method of producing collagen is to use acid, alkali, and enzymatic hydrolysis to treat animal-derived tissues to extract collagen derivatives. The collagen extracted by these methods has lost its original biological activity and cannot be applied to the biomedical field to play a real function. In addition, the prior art is also particularly limited in improving the microenvironment of the endometrium. Therefore, in view of the shortcomings of the prior art, it is necessary to provide a gel with good biocompatibility and the ability to improve the microenvironment of the endometrium to promote the repair of endometrial damage. Summary of the invention

[0005] The main purpose of the present invention is to provide a gel for endometrial repair, which contains recombinant humanized collagen, antimicrobial peptides and mesenchymal stem cells. The recombinant humanized collagen is used as a scaffold, and the antimicrobial peptides that improve the endometrial microenvironment and mesenchymal stem cells that have the ability to promote the repair of damaged endometrium are used to construct the gel, which has the effect of repairing damaged endometrium.

[0006] One aspect of the present invention is to provide a recombinant humanized collagen, specifically a recombinant type I humanized collagen, whose amino acid sequence is shown in SEQ ID NO.1.

[0007] Among them, recombinant humanized type I collagen is based on human type I collagen, and is obtained by artificially inserting, deleting, replacing, and other changes at certain amino acid sites.

[0008] Optionally, an amino acid sequence having more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1, and having the same biological function as the amino acid sequence shown in SEQ ID NO.1.

[0009] Optionally, an amino acid sequence in which one or more amino acid residues are substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO.1 has the same biological function as the amino acid sequence shown in SEQ ID NO.1.

[0010] One aspect of the present invention is to provide a nucleic acid encoding recombinant humanized type I collagen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] One aspect of the present invention is to provide a vector comprising a nucleic acid having a nucleotide sequence as shown in SEQ ID NO.2.

[0012] One aspect of the present invention is to provide a cell, wherein the cell comprises the above-mentioned vector. Preferably, the cell is Pichia pastoris.

[0013] One aspect of the present invention is to provide a method for preparing recombinant humanized type I collagen, specifically, the nucleic acid shown in SEQ ID NO.2 is introduced into a plasmid and then transformed into Pichia pastoris, the Pichia pastoris is cultured to suitable conditions for inducing expression, and finally the recombinant humanized type I collagen is obtained by separation and purification. Preferably, the purification is performed by cation exchange chromatography, and the protein corresponding to the elution peak is eluted and collected to obtain the recombinant humanized type I collagen.

[0014] Another aspect of the present invention is to provide an antimicrobial peptide. Specifically, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.3.

[0015] Among them, antimicrobial peptides are derived from the α chain of human hemoglobin, which have inhibitory effects on Escherichia coli, Staphylococcus aureus and Candida albicans, providing a strong guarantee for a good microenvironment of the endometrium. Among them, antimicrobial peptides are directly synthesized.

[0016] Another aspect of the present invention also provides a mesenchymal stem cell, specifically, the mesenchymal stem cell promotes endometrial and vascular regeneration and endometrial repair by secreting a large amount of cytokines. The preparation method of the mesenchymal stem cell comprises the following steps:

[0017] (1) P5 mesenchymal stem cells were cultured at 1×106 The density of cells was inoculated into cell culture flasks, and then complete medium was added. The cells were cultured in a 37°C, 5% CO2 incubator, and the medium was changed regularly until the cells grew to 85%-90% fusion.

[0018] (2) Digest the mesenchymal stem cells with trypsin and pass them to the P10 generation. Collect the mesenchymal stem cell suspension, centrifuge it, and dilute it with complete culture medium to obtain the mesenchymal stem cell suspension.

[0019] Another aspect of the present invention is to provide a method for preparing a gel for repairing endometrial damage, which specifically comprises the following steps:

[0020] (1) Prepare recombinant humanized type I collagen and store it at 4°C for future use;

[0021] (2) Directly synthesize antimicrobial peptides according to SEQ ID NO.3;

[0022] (3) Cultivate and expand mesenchymal stem cells;

[0023] (4) Mixing the antimicrobial peptide and mesenchymal stem cells and storing them at 4°C for future use;

[0024] (5) Collagen, antimicrobial peptides and a mixture of mesenchymal stem cells are mixed at 4°C to prepare a gel, and the gel is stored at 4°C.

[0025] Another aspect of the present invention also provides an application of a gel for repairing endometrial damage. Specifically, the prepared gel is used as a biological material and is directly injected into a damaged uterus to repair the endometrium.

[0026] Beneficial technical effects of the present invention:

[0027] The gel for repairing endometrial damage of the present invention comprises recombinant humanized type I collagen, antimicrobial peptides and mesenchymal stem cells, wherein the recombinant humanized type I collagen serves as a scaffold of the gel, has a complete triple helix structure and biological activity, has low immunogenicity, and improves the biosafety and effectiveness of the gel; the antimicrobial peptides play an important role in maintaining a good microenvironment of the uterus; and the mesenchymal stem cells can secrete a large amount of cytokines, promote endometrial and vascular regeneration, and promote endometrial repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 : Gel-forming state for repairing endometrial damage.

[0030] Figure 2 : HE staining of endometrium in each group.

[0031] Figure 3 : Western blot of the protein expressions of pro-inflammatory factor TNF-α and anti-inflammatory factor IL-10 in each group. DETAILED DESCRIPTION

[0032] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1: Preparation of recombinant humanized type I collagen

[0034] The gene fragment was synthesized according to the nucleotide sequence shown in Seq ID NO.2, and the synthesized gene fragment was inserted between the restriction sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0035] The recombinant plasmid was mixed with the GS115 competent cells, and after ice bath, the cells were electroporated, and then a sorbitol solution was added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted and cultured at 30°C for 2d-5d until a single colony grew;

[0036] Scrape off the His on the MD solid plate + After the transformant is diluted, it is spread on a YPD plate containing G418, and inverted and cultured at 30°C until a single colony appears, and then the single colony on the YPD plate is selected and transferred to a 96-well culture plate containing YPD medium, and cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0037] The recombinant yeast engineering bacteria are selected and inoculated into BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0;

[0038] After centrifugation at 3000 rpm for 10 min, the cells were collected and re-inoculated into BMGY induction medium until the OD600nm absorbance reached 2.0. The culture was continued at 30°C and 220 rpm, and 0.5% methanol was added every 24 h. After induction, the induced supernatant was collected by centrifugation.

[0039] The induced supernatant is purified by a cation exchange chromatography column, and the protein corresponding to the elution peak is eluted and collected to obtain recombinant type I humanized collagen with a purity of 98%.

[0040] Example 2: Antibacterial effect of antimicrobial peptides

[0041] The company directly synthesized the antimicrobial peptide based on SEQ ID NO.3, and dissolved the antimicrobial extract in PBS solution to obtain antimicrobial peptide solutions with concentrations of 1%, 0.5% and 0.2%, respectively, and carried out agarose diffusion antibacterial experiments.

[0042] The results showed that different concentrations of antimicrobial peptides had significant inhibitory effects on Escherichia coli, Staphylococcus aureus and Candida albicans, with the best inhibitory effect being at a concentration of 1%. (Table 1)

[0043]

[0044] Note: "+" indicates antibacterial effect, the more "+" there are, the better the antibacterial effect is.

[0045] Example 3: Preparation of gel for repairing endometrial damage

[0046] The recombinant humanized type I collagen of Example 1 was diluted with pure water to a concentration of 5%, the pH was adjusted to 7, and the solution was stored at 4°C for later use. The antimicrobial peptide of Example 2 was dissolved with PBS solution to obtain an antimicrobial peptide suspension with a concentration of 1%, and the solution was stored at 4°C for later use. The P5 mesenchymal stem cells were cultured at 1×10 6 The cells were inoculated at a density of 100 μg / cm2 into cell culture flasks, and then complete culture medium was added. The cells were cultured in a 37°C, 5% CO2 incubator with regular medium changes until the cells grew and merged to 85%-90%. The mesenchymal stem cells were digested with trypsin and passed to the P10 generation. The mesenchymal stem cell suspension was collected, centrifuged and diluted with complete culture medium to obtain the mesenchymal stem cell suspension for later use.

[0047] The antimicrobial peptide and mesenchymal stem cells were mixed and stored at 4°C for later use; collagen was mixed with the antimicrobial peptide and mesenchymal stem cell mixture at 4°C to prepare a gel, which was stored at 4°C, and the state of the gel was observed at room temperature.

[0048] The results showed that the gelation was good at room temperature ( Figure 1 ).

[0049] Example 4: Evaluation of the effect of the gel for repairing endometrial damage on repairing endometrial damage

[0050] SPF female rats were divided into 4 groups, namely the model group, gel 1 (recombinant humanized type I collagen + antimicrobial peptide) treatment group, gel 2 (recombinant humanized type I collagen + mesenchymal stem cells) treatment group and gel 3 (recombinant humanized type I collagen + antimicrobial peptide + mesenchymal stem cells) treatment group, with 6 rats in each group. The mechanical endometrial injury model of rats was established by mechanical curettage: SPF female SD rats weighing 250-300g were taken in a sterile environment, and the vaginal smears of the rats were observed daily. Female rats in estrus were used for model construction. The experiment was anesthetized with 10% chloral hydrate, and the lower abdomen was disinfected with 75% alcohol. The skin was cut 1 cm above the urethra to enter the abdominal cavity, the uterus was exposed, and an incision was made at one-third of the right Y-shaped uterus. The endometrium was scraped with a homemade scraper to simulate the clinical human endometrial curettage, causing pathological endometrial damage, and the surgical incision was sutured after surgery. After the rats woke up from anesthesia, they were placed in the animal room for breeding. The breeding environment was changed to clean cages for individual feeding. The bedding was changed frequently and kept dry. The rats were allowed to drink water and eat freely, and their nutrition was enhanced.

[0051] On the second day after surgery, 200 μl PBS was injected into the uterus of the model group, and 200 μl of the corresponding gel was injected into the uterus of the gel 1-3 treatment groups. The rats were killed on the 14th day after surgery, and the uterine tissues of 3 rats in each group were fixed with paraformaldehyde, sectioned with paraffin, and stained with HE. The number of endometrial glands was counted, and the endometrial structure was analyzed ( Figure 2 ). Three more mice were selected from each group for protein extraction, and the protein expressions of anti-inflammatory factor IL-10 and pro-inflammatory factor TNF-α were analyzed by WB to analyze the inflammatory status of the uterine microenvironment.

[0052] The results showed that the number of glands was: Gel 3 treatment group > Gel 2 treatment group > Gel 1 treatment group > Model group (Table 2); Inflammation: The Model group had a more severe inflammatory reaction, followed by the Gel 2 treatment group. Due to the addition of antimicrobial peptides to the Gel 1 and Gel 3 treatment groups, the inflammatory reaction of Gel 1 was milder, while there was almost no inflammatory reaction in the Gel 3 treatment group ( Figure 3 ). It can be seen that the gel composed of recombinant humanized type I collagen, antimicrobial peptides and mesenchymal stem cells promotes the regeneration of endometrial cells and blood vessels, and promotes the repair of endometrial damage.

[0053]

[0054] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention's specification and drawings, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A gel for repairing endometrial damage, characterized in that: The gel contains collagen, antimicrobial peptides and mesenchymal stem cells. The collagen is recombinant type I humanized collagen, and its amino acid sequence is shown in SEQ ID NO.1; the antimicrobial peptide is derived from the α chain of human hemoglobin, and its amino acid sequence is shown in SEQ ID NO.

3.

2. The gel for repairing endometrial damage according to claim 1, characterized in that: The method for preparing the recombinant humanized type I collagen comprises the following steps: introducing the nucleic acid shown in SEQ ID NO.2 into a plasmid and then transforming it into Pichia pastoris, culturing Pichia pastoris, inducing expression, and finally obtaining the recombinant humanized type I collagen by separation and purification.

3. The gel for repairing endometrial damage according to claim 1, characterized in that: The method for preparing mesenchymal stem cells comprises the following steps: (1) P5 mesenchymal stem cells were cultured at 1×10 6 The density of cells was inoculated into cell culture flasks, and then complete medium was added. The cells were cultured in a 37°C, 5% CO2 incubator, and the medium was changed regularly until the cells grew to 85%-90% fusion. (2) Digest the mesenchymal stem cells with trypsin and pass them to the P10 generation. Collect the mesenchymal stem cell suspension, centrifuge it, and dilute it with complete culture medium to obtain the mesenchymal stem cell suspension.

4. A method for preparing the gel for repairing endometrial damage according to claim 1, characterized in that: The method comprises the following steps: (1) Prepare recombinant humanized type I collagen and store it at 4°C for future use; (2) Directly synthesize antimicrobial peptides according to SEQ ID NO.3; (3) Cultivate and expand mesenchymal stem cells; (4) Mixing the antimicrobial peptide and mesenchymal stem cells and storing them at 4°C for future use; (5) Collagen is mixed with antimicrobial peptides and a mixture of mesenchymal stem cells at 4°C to prepare a gel, and the gel is stored at 4°C.

5. Use of the gel for repairing endometrial damage according to claim 1 in preparing a biological material for repairing endometrial damage.

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