A protein solution for promoting the repair of endometrial injury and its application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a protein solution and application thereof for promoting the repair of endometrial damage. The protein solution comprises the following raw materials: a linker polypeptide-collagen, PDGF, a hyaluronic acid-carrageenan composite material, and normal saline; the present invention designs and synthesizes a linker polypeptide capable of connecting PDGF and collagen to form a PDGF-linker-collagen, which can prolong the attachment time of PDGF in the endometrium. The present invention prepares a hyaluronic acid-carrageenan composite material, which is a cross-linked mesh structure, can load PDGF-linker-collagen, effectively protects its activity, prevents aggregation, has good biocompatibility, is non-toxic to the human body and cells, and can further promote the repair of endometrial damage. The present invention also provides a protein solution for promoting the repair of endometrial damage, and the prepared protein solution can effectively promote the repair of endometrial damage without potential toxic effects on cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a protein solution for promoting the repair of endometrial damage and application thereof. Background Art

[0002] The endometrium is the basis for embryo development. A relatively thick endometrium has a better bearing effect. If the endometrium is thin, the implantation rate and pregnancy rate after implantation will be affected. A recognized sign of endometrial receptivity is the thickness of the endometrium. The minimum endometrial thickness for successful embryo transfer is 7 mm. When it is less than this value, the success rate of embryo transfer will be greatly affected. In particular, when the endometrial thickness is less than 5 mm, embryo transfer is usually not possible, and the survival rate of the embryo after transfer will also be significantly reduced. The continuous thinning of the endometrium during the reproductive cycle may be related to a lower implantation rate and a higher miscarriage rate. Endometrial damage caused by various reasons such as artificial abortion, curettage, intrauterine surgery, infection, etc. may cause endometrial regeneration disorders, leading to reduced menstruation, amenorrhea, recurrent miscarriage and infertility. It is reported that the incidence of endometrial damage in infertile patients is about one-fifth. At present, progesterone is mostly used to repair endometrial damage, but the effect is poor. Platelet-derived growth factor (PDGF) plays an important role in angiogenesis and cell mitosis, and participates in important physiological processes such as angiogenesis and wound repair. PDGF can promote the proliferation and invasion of endometrial stromal cells, promote angiogenesis, and has a certain effect on the repair of endometrial damage, but simple PDGF solution or gel preparation cannot be attached to the endometrial surface for a long time, and the effect is limited, making it difficult to achieve endometrial repair. Therefore, finding a method that can enhance PDGF to repair endometrial damage and promote the thickening of the damaged endometrium to normal levels is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention proposes a protein solution and its application for promoting the repair of endometrial damage. A linker polypeptide capable of connecting PDGF and collagen was designed and synthesized. The N-terminus of the linker polypeptide simulated the PDGF receptor structure and could bind to PDGF. The C-terminus of the linker polypeptide was rich in lysine residues and could be covalently cross-linked to collagen by transglutaminase to form PDGF-linker-collagen. A protein solution capable of promoting the repair of endometrial damage was prepared, which formed a stable repair microenvironment on the surface of the endometrium and promoted the repair of endometrial damage.

[0004] The present invention is achieved through the following technical solutions:

[0005] A protein solution for promoting the repair of endometrial damage, comprising the following raw materials: linker polypeptide-collagen, PDGF, and physiological saline; the linker polypeptide-collagen is made from the following raw materials: PBS buffer, linker polypeptide, human type III collagen, and transglutaminase; the linker polypeptide is any one of the following:

[0006] (a) the amino acid sequence shown in SEQ ID No.1;

[0007] (b) A polypeptide having more than 80% identity with the polypeptide described in (a) and having the same biological function as the polypeptide described in (a) obtained by substitution and / or deletion and / or insertion of amino acid residues in the polypeptide described in (a).

[0008] Preferably, a protein solution for promoting the repair of endometrial damage comprises the following raw materials: linker polypeptide-collagen, PDGF, hyaluronic acid-carrageenan composite material, and physiological saline.

[0009] Furthermore, the PDGF is preferably PDGF-bb.

[0010] Furthermore, the linker polypeptide-collagen is made from the following raw materials: PBS buffer, linker polypeptide, human type III collagen and transglutaminase.

[0011] Furthermore, the human type III collagen is preferably recombinant human collagen. For example, the human type III collagen of the present invention may be human type III collagen obtained by the method disclosed in Chinese patent CN103102407A.

[0012] Further, the linker polypeptide is any one of the following:

[0013] (a) The amino acid sequence shown in SEQ ID No.1; SEQ ID No.1: tlmcivignpvvnfpwtyprpsgrlvppvdflldmpyhirsilhipsapptllgnssppsqlptnvtwpppqpfpvvstlrlqhvdrplsvr ctlrvdpmqlpydswplprdqlvgrtlgsgafgqvvpamvpfpirppfsqlvlllprllgpmlkwkqkkkpkrkykpkikkrkwkkkvipks;

[0014] (b) A polypeptide having more than 80% identity with the polypeptide described in (a) and having the same biological function as the polypeptide described in (a) obtained by substitution and / or deletion and / or insertion of amino acid residues in the polypeptide described in (a).

[0015] Furthermore, the linker polypeptide is synthesized in a directed manner according to the polypeptide sequence using amino acids as raw materials using conventional polypeptide synthesis methods such as solid phase synthesis and liquid phase synthesis.

[0016] Furthermore, the preparation method of the linker polypeptide-collagen comprises the following steps:

[0017] (1) Add linker polypeptide, human type III collagen and transglutaminase to a 10 mM PBS buffer at pH 7.4, and stir at 180 rpm for 20-30 min to obtain a mixed solution;

[0018] (2) heating the mixed solution obtained in step (1) to 50° C. for 2-3 h, then heating to 70° C. for 15-20 min, and naturally cooling to room temperature to obtain a post-reaction solution;

[0019] (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography, and freeze-dried to obtain linker polypeptide-collagen.

[0020] Furthermore, in step (1), the dosage ratio of the PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL: 10 mg: 4 mg: 0.1 mg.

[0021] Furthermore, the preparation method of the hyaluronic acid-carrageenan composite material comprises the following steps:

[0022] (i) dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid solution, dissolving fumaric acid and succinic acid in distilled water to obtain a fumaric acid-succinic acid solution, adding the fumaric acid-succinic acid solution to the hyaluronic acid solution at a rate of 0.8-1 mL / min, stirring at 180 rpm for 4-5 h, dialyzing in deionized water for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da, and freeze-drying to obtain modified hyaluronic acid;

[0023] (ii) adding carrageenan to deionized water, stirring and dissolving at 50°C to obtain a carrageenan solution, dissolving poloxamer 407 in 10 mM phosphate buffer at a pH of 8, adding curcumin to obtain a poloxamer 407 solution, and mixing the carrageenan solution and the poloxamer 407 solution evenly, stirring at 25°C and 200 rpm for 2-3 h to obtain a modified carrageenan;

[0024] (iii) adding the modified hyaluronic acid obtained in step (i) to the modified carrageenan obtained in step (ii), and then adding calcium chloride, stirring at 40° C. and 200 rpm for 60-80 min, and freeze-drying to obtain a hyaluronic acid-carrageenan composite material.

[0025] Furthermore, in step (i), the ratio of hyaluronic acid to deionized water is 1 g:20 mL.

[0026] Furthermore, in step (i), the usage ratio of fumaric acid, succinic acid and distilled water is 0.2 g:0.8 g:50 mL.

[0027] Furthermore, in step (i), the volume ratio of the hyaluronic acid solution to the fumaric acid-succinic acid solution is 5:1.

[0028] Furthermore, in step (ii), the ratio of carrageenan to deionized water is 3 g:50 mL.

[0029] Furthermore, in step (ii), the dosage ratio of poloxamer 407, phosphate buffer and curcumin is 1 g:25 mL:0.3 g.

[0030] Furthermore, in step (ii), the volume ratio of the carrageenan solution to the poloxamer 407 solution is 3:1.

[0031] Furthermore, in step (iii), the mass ratio of the modified hyaluronic acid, modified carrageenan and calcium chloride is 5:20:2.

[0032] The present invention also provides a method for preparing the protein solution for promoting the repair of endometrial damage, comprising the following steps: adding linker polypeptide-collagen and PDGF to physiological saline, stirring at 80 rpm for 30-40 min, adding hyaluronic acid-carrageenan composite material, stirring at 100-150 rpm for 40-60 min, to obtain a protein solution for promoting the repair of endometrial damage.

[0033] Furthermore, the dosage ratio of the physiological saline, linker polypeptide-collagen, PDGF and hyaluronic acid-carrageenan composite material is 1 L: 0.5 g: 0.1 g: 4 g.

[0034] Furthermore, the present invention also provides the use of the protein solution for promoting endometrial damage repair in promoting endometrial thickening and endometrial damage repair.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention designs and synthesizes a linker polypeptide that can connect PDGF and collagen. The N-terminus of the linker polypeptide simulates the PDGF receptor structure and can bind to PDGF. The C-terminus of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen by transglutaminase to form PDGF-linker-collagen, which can prolong the attachment time of PDGF in the endometrium and effectively promote endometrial thickening and endometrial damage repair.

[0037] 2. The present invention prepares hyaluronic acid-carrageenan composite material, which is a cross-linked network structure, can load PDGF-connector-collagen, effectively protect its activity, prevent aggregation, and hyaluronic acid-carrageenan composite material itself has persistent antibacterial ability, good biocompatibility, non-toxic to human body and cells, can further promote endometrial damage repair, reduce inflammatory response, and hyaluronic acid-carrageenan composite material is relatively slow to degrade in vivo compared with raw materials such as hyaluronic acid and gelatin, and the degradation time can be significantly extended, which helps to enhance the regeneration and repair of endometrial damage, and plays a good anti-adhesion effect at the same time. A kind of protein solution for promoting endometrial damage repair provided by the present invention can form a stable repair microenvironment on the endometrial surface, promote endometrial damage repair, can promote the recovery of the number of endometrial glands in which intrauterine adhesion occurs, reduce fibrosis ratio, promote the thickening of thin endometrium, have high tissue compatibility, simple preparation, obvious improvement effect and other advantages, no potential toxicity effect on cells, with good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or 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 in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 The cytotoxicity test diagram of the protein solution described in Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0040] Figure 2 The graph is a test result of the effect of the protein solution of Examples 1-3 and Comparative Examples 1-3 of the present invention on the retention time of PDGF in the endometrium;

[0041] Figure 3 The uterine section staining images of the sham operation group, model group and Example 1 group described in Experimental Example 3 of the present invention;

[0042] Figure 4 This is a test result diagram of the effect of the protein solution described in Examples 1-3 of the present invention and Comparative Examples 1-3 on the number of endometrial glands and the proportion of endometrial fibrosis;

[0043] Figure 5 This is a test result diagram of the effect of the protein solution described in Examples 1-3 of the present invention and Comparative Examples 1-3 on the thickness of the endometrium;

[0044] Figure 6 This is a scanning electron microscope image of the hyaluronic acid-carrageenan composite material described in Example 1 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0046] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0047] Example 1: This example provides a protein solution for promoting the repair of endometrial damage, comprising the following raw materials: linker polypeptide-collagen, PDGF-bb, hyaluronic acid-carrageenan composite material, and physiological saline; the linker polypeptide-collagen is made of the following raw materials: PBS buffer, linker polypeptide, human type III collagen and transglutaminase; the linker polypeptide is the amino acid sequence shown in SEQ ID No.1, and is synthesized in a directed manner according to the polypeptide sequence using amino acids as raw materials using a solid phase synthesis method; the human type III collagen is recombinant human collagen, and the human type III collagen in this example is human type III collagen obtained by the method disclosed in Chinese patent CN103102407A.

[0048] The preparation method of the linker polypeptide-collagen comprises the following steps:

[0049] (1) Adding linker polypeptide, human type III collagen and transglutaminase to a pH 7.4 10 mM PBS buffer, the amount ratio of PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL:10 mg:4 mg:0.1 mg, stirring at 180 rpm for 30 min to obtain a mixed solution;

[0050] (2) heating the mixed solution obtained in step (1) to 50° C. for 3 h, then heating to 70° C. for 20 min, and naturally cooling to room temperature to obtain a post-reaction solution;

[0051] (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography, and freeze-dried to obtain linker polypeptide-collagen.

[0052] The preparation method of the hyaluronic acid-carrageenan composite material comprises the following steps:

[0053] (i) dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid solution, wherein the ratio of hyaluronic acid to deionized water is 1 g:20 mL; dissolving fumaric acid and succinic acid in distilled water to obtain a fumaric acid-succinic acid solution, wherein the ratio of fumaric acid, succinic acid and distilled water is 0.2 g:0.8 g:50 mL; adding the fumaric acid-succinic acid solution to the hyaluronic acid solution at a rate of 1 mL / min, wherein the volume ratio of the hyaluronic acid solution to the fumaric acid-succinic acid solution is 5:1; stirring at 180 rpm for 5 h; dialyzing the mixture in deionized water for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da; and freeze-drying the mixture to obtain modified hyaluronic acid;

[0054] (ii) adding carrageenan to deionized water, stirring and dissolving at 50°C to obtain a carrageenan solution, wherein the amount ratio of carrageenan to deionized water is 3 g:50 mL; dissolving poloxamer 407 in 10 mM phosphate buffer having a pH value of 8, adding curcumin to obtain a poloxamer 407 solution, wherein the amount ratio of poloxamer 407, phosphate buffer and curcumin is 1 g:25 mL:0.3 g; mixing the carrageenan solution and the poloxamer 407 solution evenly, wherein the volume ratio of the carrageenan solution to the poloxamer 407 solution is 3:1, and stirring at 25°C and 200 rpm for 3 h to obtain a modified carrageenan;

[0055] (iii) adding the modified hyaluronic acid obtained in step (i) to the modified carrageenan obtained in step (ii), and then adding calcium chloride, wherein the mass ratio of the modified hyaluronic acid, the modified carrageenan and the calcium chloride is 5:20:2, stirring at 40° C. and 200 rpm for 80 min, and freeze-drying to obtain a hyaluronic acid-carrageenan composite material, as shown in the scanning electron microscope image. Figure 6 As shown, it is a three-dimensional network structure.

[0056] This embodiment also provides a method for preparing the protein solution for promoting the repair of endometrial damage, comprising the following steps: adding linker polypeptide-collagen and PDGF to physiological saline, stirring at 80 rpm for 40 min, adding hyaluronic acid-carrageenan composite material, the amount ratio of physiological saline, linker polypeptide-collagen, PDGF and hyaluronic acid-carrageenan composite material is 1 L:0.5 g:0.1 g:4 g, stirring at 150 rpm for 60 min, to obtain a protein solution for promoting the repair of endometrial damage.

[0057] Example 2: This example provides a protein solution for promoting the repair of endometrial damage, comprising the following raw materials: linker polypeptide-collagen, PDGF-bb, hyaluronic acid-carrageenan composite material, and normal saline; the linker polypeptide-collagen is made of the following raw materials: PBS buffer, linker polypeptide, human type III collagen and transglutaminase; the linker polypeptide is the amino acid sequence shown in SEQ ID No.1, and is synthesized in a directed manner according to the polypeptide sequence using amino acids as raw materials using a liquid phase synthesis method; the human type III collagen is recombinant human collagen, and the human type III collagen in this example is human type III collagen obtained by the method disclosed in Chinese Patent CN103102407A.

[0058] The preparation method of the linker polypeptide-collagen comprises the following steps:

[0059] (1) Adding linker polypeptide, human type III collagen and transglutaminase to a pH 7.4 10 mM PBS buffer, the amount ratio of PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL:10 mg:4 mg:0.1 mg, stirring at 180 rpm for 20 min to obtain a mixed solution;

[0060] (2) heating the mixed solution obtained in step (1) to 50° C. and maintaining it for 2 h, then heating it to 70° C. and maintaining it for 15 min, and naturally cooling it to room temperature to obtain a post-reaction solution;

[0061] (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography, and freeze-dried to obtain linker polypeptide-collagen.

[0062] The preparation method of the hyaluronic acid-carrageenan composite material comprises the following steps:

[0063] (i) dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid solution, wherein the ratio of hyaluronic acid to deionized water is 1 g:20 mL; dissolving fumaric acid and succinic acid in distilled water to obtain a fumaric acid-succinic acid solution, wherein the ratio of fumaric acid, succinic acid and distilled water is 0.2 g:0.8 g:50 mL; adding the fumaric acid-succinic acid solution to the hyaluronic acid solution at a rate of 0.8 mL / min; the volume ratio of the hyaluronic acid solution to the fumaric acid-succinic acid solution is 5:1; stirring at 180 rpm for 4 h; dialyzing in deionized water for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da; and freeze-drying to obtain modified hyaluronic acid;

[0064] (ii) adding carrageenan to deionized water, stirring and dissolving at 50°C to obtain a carrageenan solution, wherein the ratio of carrageenan to deionized water is 3 g:50 mL; dissolving poloxamer 407 in 10 mM phosphate buffer at a pH of 8, adding curcumin to obtain a poloxamer 407 solution, wherein the ratio of poloxamer 407, phosphate buffer and curcumin is 1 g:25 mL:0.3 g; mixing the carrageenan solution and the poloxamer 407 solution evenly, wherein the volume ratio of the carrageenan solution to the poloxamer 407 solution is 3:1, and stirring at 25°C and 200 rpm for 2 h to obtain a modified carrageenan;

[0065] (iii) adding the modified hyaluronic acid obtained in step (i) to the modified carrageenan obtained in step (ii), and then adding calcium chloride, wherein the mass ratio of the modified hyaluronic acid, the modified carrageenan and the calcium chloride is 5:20:2, stirring at 40° C. and 200 rpm for 60 min, and freeze-drying to obtain a hyaluronic acid-carrageenan composite material.

[0066] This embodiment also provides a method for preparing the protein solution for promoting the repair of endometrial damage, comprising the following steps: adding linker polypeptide-collagen and PDGF to physiological saline, stirring at 80 rpm for 30 min, adding hyaluronic acid-carrageenan composite material, the amount ratio of physiological saline, linker polypeptide-collagen, PDGF and hyaluronic acid-carrageenan composite material is 1 L:0.5 g:0.1 g:4 g, stirring at 100-150 rpm for 40 min, to obtain a protein solution for promoting the repair of endometrial damage.

[0067] Example 3: This example provides a protein solution for promoting the repair of endometrial damage, comprising the following raw materials: linker polypeptide-collagen, PDGF-bb, hyaluronic acid-carrageenan composite material, and normal saline; the linker polypeptide-collagen is made of the following raw materials: PBS buffer, linker polypeptide, human type III collagen and transglutaminase; the linker polypeptide is the amino acid sequence shown in SEQ ID No.1, and is synthesized in a directed manner according to the polypeptide sequence using amino acids as raw materials using a solid phase synthesis method; the human type III collagen is recombinant human collagen, and the human type III collagen in this example is human type III collagen obtained by the method disclosed in Chinese Patent CN103102407A.

[0068] The preparation method of the linker polypeptide-collagen comprises the following steps:

[0069] (1) Adding linker polypeptide, human type III collagen and transglutaminase to a pH 7.4 10 mM PBS buffer, the amount ratio of PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL:10 mg:4 mg:0.1 mg, stirring at 180 rpm for 25 min to obtain a mixed solution;

[0070] (2) heating the mixed solution obtained in step (1) to 50° C. and maintaining it for 2.5 h, then heating it to 70° C. and maintaining it for 18 min, and naturally cooling it to room temperature to obtain a post-reaction solution;

[0071] (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography, and freeze-dried to obtain linker polypeptide-collagen.

[0072] The preparation method of the hyaluronic acid-carrageenan composite material comprises the following steps:

[0073] (i) dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid solution, wherein the ratio of hyaluronic acid to deionized water is 1 g:20 mL; dissolving fumaric acid and succinic acid in distilled water to obtain a fumaric acid-succinic acid solution, wherein the ratio of fumaric acid, succinic acid and distilled water is 0.2 g:0.8 g:50 mL; adding the fumaric acid-succinic acid solution to the hyaluronic acid solution at a rate of 0.9 mL / min; the volume ratio of the hyaluronic acid solution to the fumaric acid-succinic acid solution is 5:1; stirring at 180 rpm for 4.5 h; dialyzing in deionized water for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da; and freeze-drying to obtain modified hyaluronic acid;

[0074] (ii) adding carrageenan to deionized water, stirring and dissolving at 50°C to obtain a carrageenan solution, wherein the ratio of carrageenan to deionized water is 3 g:50 mL; dissolving poloxamer 407 in 10 mM phosphate buffer at a pH of 8, adding curcumin to obtain a poloxamer 407 solution, wherein the ratio of poloxamer 407, phosphate buffer and curcumin is 1 g:25 mL:0.3 g; mixing the carrageenan solution and the poloxamer 407 solution evenly, wherein the volume ratio of the carrageenan solution to the poloxamer 407 solution is 3:1, and stirring at 25°C and 200 rpm for 2.5 h to obtain a modified carrageenan;

[0075] (iii) adding the modified hyaluronic acid obtained in step (i) to the modified carrageenan obtained in step (ii), and then adding calcium chloride, wherein the mass ratio of the modified hyaluronic acid, the modified carrageenan and the calcium chloride is 5:20:2, stirring at 40° C. and 200 rpm for 70 min, and freeze-drying to obtain a hyaluronic acid-carrageenan composite material.

[0076] This embodiment also provides a method for preparing the protein solution for promoting the repair of endometrial damage, comprising the following steps: adding linker polypeptide-collagen and PDGF to physiological saline, stirring at 80 rpm for 35 min, adding hyaluronic acid-carrageenan composite material, the amount ratio of physiological saline, linker polypeptide-collagen, PDGF and hyaluronic acid-carrageenan composite material is 1 L:0.5 g:0.1 g:4 g, stirring at 120 rpm for 50 min, to obtain a protein solution for promoting the repair of endometrial damage.

[0077] The only difference between Comparative Example 1 and Example 1 is that the linker polypeptide and the hyaluronic acid-carrageenan composite material are not added.

[0078] The difference between Comparative Example 2 and Example 1 is that no linker polypeptide is added.

[0079] The only difference between Comparative Example 3 and Example 1 is that no hyaluronic acid-carrageenan composite material is added.

[0080] Experimental Example 1: The method for preparing a protein solution for promoting the repair of endometrial damage was respectively prepared according to Examples 1-3 and Comparative Examples 1-3, and the physiological saline was replaced with MEM culture medium containing 10% fetal bovine serum to obtain an experimental solution. L-929 cells (NCTCclone 929) were cultured in MEM culture medium containing 10% fetal bovine serum and antibiotics (penicillin 100 U / mL, streptomycin 100 µg / mL) and cultured in a 37°C, 5% CO2 incubator. The cells were digested with 0.25% trypsin, centrifuged at 200 g for 3 min, and then the cells were redispersed in fresh culture medium and the cell density was adjusted to 2×10 5cells / mL cell suspension; inoculate the above cell suspension into a 96-well culture plate, 100 μL per well, place in a carbon dioxide incubator (5% CO2, 37°C, humidity>90%) for 24 h, then add 100 μL of experimental solution to each well as the experimental group, set up a well with only 200 μL of MEM medium containing 10% fetal bovine serum as the blank control group, and a well with 100 μL of medium + 100 μL of cells as the negative control. After adding the experimental solution, continue to culture for 5 days, add 20 μL of MTT (5 mg / mL prepared in PBS) to each well in the dark every day, and continue to culture for 4 hours after adding MTT on the last day, remove all the liquid in the well, add 150 μL of DMSO solution to each well, shake at low speed for 15 min in an ELISA reader, and measure its OD value at λ=490 nm. The cytotoxicity was determined by calculating the relative growth rate (RGR, %): RGR = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%. The specific toxicity grading standards are: Level 0: RGR ≥ 100%; Level 1: 99% ≥ RGR ≥ 75; Level 2: 74 ≥ RGR ≥ 50; Level 3: 49 ≥ RGR ≥ 25; Level 4: 24 ≥ RGR ≥ 1; Level 5 = 0. Levels 2 and above can be considered as cytotoxic reactions. The results are shown in Figure 1 shown.

[0081] Figure 1 The results showed that the RGR of the comparative example 1-3 group was lower than that of the embodiment 1-3, but the toxicity levels of the embodiment 1-3 and the comparative example 1-3 group were both 0, indicating that the protein solution for promoting the repair of endometrial damage of the present invention is non-cytotoxic and has high biosafety.

[0082] Experimental Example 2: 60 female SD rats aged 8-10 weeks and weighing 220-260 g were selected. After one week of adaptive feeding, they were randomly divided into 6 groups, with 10 rats in each group. The model was induced by mechanical injury and ethanol perfusion. The rats were fasted and deprived of water for 12 h before the operation. After intraperitoneal injection of 2% sodium pentobarbital, they were fixed in the supine position. The uterine horns were exposed by a transverse incision in the abdomen. A longitudinal incision of about 4 mm was made about 0.5 cm below the uterine horns. The middle and upper endometrium was scraped with an endometrial scraper with a diameter of 2.5 mm. The curettage was stopped when the four walls of the uterus were rough. The uterine horns were clamped with a vascular clamp, and 95% ethanol was extracted with a 1 mL syringe and injected downward from the incision until the uterine cavity was filled. Then the incision was temporarily clamped with a vascular clamp. After 3 minutes, the vascular clamp at the incision was loosened to discharge the residual ethanol, and the uterine surface liquid was dried with sterile gauze. After modeling, each group was immediately given 0.5 mL of the protein solution prepared in Example 1-3 and Comparative Example 1-3 to perfuse the uterine cavity. After the operation was completed, the abdominal cavity was flushed with sterile saline. After the uterus was repositioned, the abdomen was closed layer by layer, and the incision was disinfected with 75% alcohol. After surgery, 2 mL of gentamicin was injected intramuscularly every day to prevent infection. One rat in each group was killed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after surgery, and the uterus was collected. The drug concentration of PDGF-bb in the uterus was detected by ELISA. The results are shown in Figure 2. Figure 2 shown.

[0083] Figure 2 The results showed that the concentration of PDGF-bb in Example 1-3 was significantly higher than that in Comparative Examples 1-3, and the rate of decrease was also lower than that in Comparative Examples 1-3. Among them, the PDGF-bb in Comparative Example 2 was lower than that in Comparative Example 3, indicating that the linker polypeptide can effectively prolong the action time of PDGF, and the addition of hyaluronic acid-carrageenan composite material can further prolong the action time of PDGF factor in the body and promote the repair of uterine damage.

[0084] Experimental Example 3: 80 female SD rats aged 8-10 weeks and weighing 220-260 g were selected. After one week of adaptive feeding, they were randomly divided into 8 groups, each with 10 rats, including 6 experimental groups, 1 sham operation group, and 1 model group. The experimental group and the model group were induced by mechanical injury and ethanol perfusion. The modeling method was the same as that of Experimental Example 2. The sham operation group was anesthetized and operated laparotomy. The uterine horn was fully exposed to the outside of the body and then retracted. No other operations were performed. The uterine surface liquid was dried with sterile gauze after the physiological saline was rinsed and cleaned. After modeling, each experimental group was immediately given 0.5 mL of the protein solution prepared in Example 1-3 and Comparative Example 1-3 to perfuse the uterine cavity. The sham operation group and the model group were given an equal amount of physiological saline. After the operation was completed, the abdominal cavity was rinsed with sterile physiological saline. After the uterus was repositioned, the abdomen was closed layer by layer. The incision was disinfected with 75% alcohol. After the operation, 2 mL of gentamicin was given intramuscularly for 3 consecutive days to prevent infection. The mice in each group were killed 14 days after surgery, and the uterine tissues were collected, fixed with paraformaldehyde, and sectioned with paraffin, and stained with HE and Masson staining. Figure 3 The number of endometrial glands and the proportion of endometrial fibrosis were statistically analyzed, and the statistical results are shown in Figure 4 shown.

[0085] Figure 3 and Figure 4 The results showed that the sham operation group had normal endometrial structure, a large number of glands, and a normal fibrosis ratio; the model group had severe intrauterine adhesions, a significantly reduced number of glands, and an increased fibrosis ratio; the experimental group had lower intrauterine adhesions than the model group, the number of glands recovered, and the fibrosis ratio was lower than the model group, and close to the sham operation group. The above results show that the protein solution for promoting endometrial damage repair of the present invention has a good repair effect on intrauterine adhesions, and the effect is better than using PDGF and collagen alone. The linker polypeptide can effectively improve the repair effect of endometrial damage and promote endometrial damage repair.

[0086] Experimental Example 4: 60 patients with thin endometrium, aged 35-41 years old, with a previous endometrial thickness of 3.5 mm in the late follicular phase, were selected on a voluntary basis and randomly divided into 6 groups, with 10 patients in each group. The protein solutions of Examples 1-3 and Comparative Examples 1-3 were injected into the uterine cavity, 3 mL at a time. The results were as follows: Figure 5 shown.

[0087] Figure 5 The results showed that the endometrial thickness of Example 1-3 and Comparative Example 3 increased to more than 8 mm, which was better than Comparative Examples 1-2, indicating that the protein solution of the present invention for promoting the repair of endometrial damage can effectively promote endometrial thickening in patients with thin endometrium, and the linker polypeptide can significantly enhance the effect of the protein solution in promoting endometrial thickening.

[0088] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0089] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a protein solution for promoting the repair of endometrial damage, characterized in that: The following steps are involved: Adding linker polypeptide-collagen and PDGF to physiological saline, stirring at 80 rpm for 30-40 min, adding hyaluronic acid-carrageenan composite material, stirring at 100-150 rpm for 40-60 min, to obtain a protein solution that promotes endometrial damage repair; The amino acid sequence of the linker polypeptide is shown in SEQ ID No. 1; The preparation method of the linker polypeptide-collagen comprises the following steps: (1) adding a linker polypeptide, human type III collagen and transglutaminase to a PBS buffer solution and stirring to obtain a mixed solution; (2) heating the mixed solution obtained in step (1) to 50° C. for 2-3 h, then heating to 70° C. for 15-20 min, and cooling to obtain a post-reaction solution; (3) separating and purifying the reaction solution in step (2) by molecular sieve chromatography, and freeze-drying to obtain a linker polypeptide-collagen; The preparation method of the hyaluronic acid-carrageenan composite material comprises the following steps: (i) dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid solution, dissolving fumaric acid and succinic acid in distilled water to obtain a fumaric acid-succinic acid solution, adding the fumaric acid-succinic acid solution to the hyaluronic acid solution, stirring, dialyzing, and freeze-drying to obtain modified hyaluronic acid; (ii) adding carrageenan to deionized water and stirring to dissolve, dissolving poloxamer 407 in phosphate buffer, adding curcumin to obtain a poloxamer 407 solution, and mixing the carrageenan solution and the poloxamer 407 solution uniformly and stirring to obtain modified carrageenan; (iii) adding the modified hyaluronic acid obtained in step (i) to the modified carrageenan obtained in step (ii), and then adding calcium chloride, stirring, and freeze-drying to obtain a hyaluronic acid-carrageenan composite material.

2. The method for preparing a protein solution for promoting endometrial damage repair according to claim 1, characterized in that: The dosage ratio of the physiological saline, linker polypeptide-collagen, PDGF and hyaluronic acid-carrageenan composite material is 1L: 0.5 g: 0.1 g: 4 g.

3. The method for preparing a protein solution for promoting endometrial damage repair according to claim 1, characterized in that: The usage ratio of the PBS buffer, linker polypeptide, human type III collagen and transglutaminase in step (1) is 1 mL: 10 mg: 4 mg: 0.1 mg.

4. The method for preparing a protein solution for promoting endometrial damage repair according to claim 3, characterized in that: The dosage ratio of the hyaluronic acid to deionized water in step (i) is 1 g:20 mL; the dosage ratio of fumaric acid, succinic acid and distilled water in step (i) is 0.2 g:0.8 g:50 mL; the volume ratio of the hyaluronic acid solution to the fumaric acid-succinic acid solution in step (i) is 5:

1.

5. The method for preparing a protein solution for promoting endometrial damage repair according to claim 4, characterized in that: The dosage ratio of the carrageenan to deionized water in step (ii) is 3 g:50 mL; the dosage ratio of poloxamer 407, phosphate buffer and curcumin in step (ii) is 1 g:25 mL:0.3 g; the volume ratio of the carrageenan solution to the poloxamer 407 solution in step (ii) is 3:

1.

6. The method for preparing a protein solution for promoting endometrial damage repair according to claim 5, characterized in that: In step (iii), the mass ratio of the modified hyaluronic acid, modified carrageenan and calcium chloride is 5:20:

2.

7. A protein solution for promoting the repair of endometrial damage prepared by the method according to any one of claims 1 to 6.

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