An insulin-like growth factor-1 linked polypeptide and its application in repairing endometrial damage

Abstract

The present invention discloses an insulin-like growth factor-1-linker polypeptide-collagen, and the linker polypeptide has an amino acid sequence as shown in SEQ ID No.1. The N-terminus of the linker polypeptide designed and synthesized by the present invention simulates the IGF-1 receptor structure, can be combined with IGF-1, and the C-terminus of the linker polypeptide is rich in lysine residues, which can be covalently cross-linked to collagen by transglutaminase, prolonging the attachment time of IGF-1 in the endometrium, and effectively promoting endometrial thickening and endometrial damage repair. A protein solution for promoting endometrial damage repair provided by the present invention can form a stable repair microenvironment on the surface of the endometrium, promote the recovery of the number of endometrial glands with intrauterine adhesions, reduce the proportion of fibrosis, and promote the thickening of thin endometrium, with the advantages of high tissue compatibility, simple preparation, obvious improvement effect, no potential toxic effects on cells, and good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an insulin-like growth factor-1 linked polypeptide and an application thereof in repairing endometrial damage. 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, especially 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. It is reported that the incidence of endometrial damage in infertile patients is about one in five. At present, progesterone is mostly used to repair endometrial damage, but the effect is poor.

[0003] Insulin-like growth factor 1 (IGF-1) is a protein encoded by the human gene IGF1. In the 1970s, IGF-1 was also called "acid sulfation factor" and its function is "uninhibited islet-like activity". Literature reports that IGF-1 can promote the proliferation and differentiation of female endometrial cells, but IGF-1 has a small molecular weight and cannot act on the endometrium for a long time when used alone, resulting in a short action time and no obvious effect, which limits the application of this factor. Therefore, finding a method to enhance IGF-1 to repair endometrial damage and promote the thickening of the damaged endometrium to normal levels is an urgent problem to be solved. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention designs and synthesizes a linker polypeptide that can connect IGF-1 and collagen. The N-terminus of the linker polypeptide simulates the IGF-1 receptor structure and can bind to IGF-1; the C-terminus of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen by transglutaminase to form IGF-1-linker polypeptide-collagen. A protein solution that can promote the repair of endometrial damage is further prepared, which can form a stable repair microenvironment on the surface of the endometrium and promote the repair of endometrial damage.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] An insulin-like growth factor-1-linker polypeptide-collagen, wherein the linker polypeptide has an amino acid sequence as shown in SEQ ID No. 1; the N-terminus of the linker polypeptide binds to insulin-like growth factor-1, the C-terminus of the linker polypeptide contains a lysine residue, there are glutamine residues on the collagen, and the C-terminus of the linker polypeptide binds to the collagen through covalent cross-linking by transglutaminase.

[0007] Preferably, the collagen is human type I, type II, type III, type V or type XI collagen.

[0008] In addition, the collagen in the present application may also be other types of proteins. For example, as long as there are glutamine residues on the side chains of the above-mentioned proteins, the above-defined proteins are also within the protection scope of the present application.

[0009] Those skilled in the art will appreciate that the above-mentioned methods of bonding and covalent cross-linking can be performed with reference to the prior art. For example, the linker polypeptide is synthesized using conventional polypeptide synthesis methods such as solid phase synthesis and liquid phase synthesis with amino acids as raw materials according to the polypeptide sequence.

[0010] The present invention also provides a method for preparing a protein solution containing the insulin-like growth factor-1-linker polypeptide-collagen, comprising the following steps:

[0011] S1. Add the linker polypeptide, collagen and transglutaminase to a 10 mM PBS buffer having a pH of 7.4 to obtain a mixed solution; wherein the amount ratio of the PBS buffer, the linker polypeptide, the collagen and the transglutaminase is 1 mL: 1-50 mg: 1-100 mg: 0.01-0.5 mg;

[0012] S2. The mixed solution obtained in step S1 is heated to 50°C for 2-3 h, then heated to 70°C for 15-20 min, and naturally cooled to room temperature to obtain a reaction solution;

[0013] S3. Purifying the reaction solution in step S2 and freeze-drying the solution to obtain a linker polypeptide-collagen;

[0014] S4. Add IGF-1 and the linker polypeptide-collagen obtained in step S3 to physiological saline, and stir to obtain a protein solution containing insulin-like growth factor-1-linker polypeptide-collagen as an active ingredient; wherein the dosage ratio of physiological saline, linker polypeptide-collagen and IGF-1 is 1 L: 0.1~10 g: 0.01~1 g.

[0015] Preferably, in step S3, purification is performed by separation through G25 molecular sieve chromatography.

[0016] Preferably, after adding each raw material in step S1, stirring is carried out at 180 rpm for 30 min.

[0017] Preferably, in step S4, a protein solution containing insulin-like growth factor-1-linker polypeptide-collagen as an active ingredient is obtained after stirring at 80 rpm for 30 min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention designs and synthesizes a linker polypeptide capable of connecting IGF-1 and collagen. The N-terminus of the linker polypeptide simulates the IGF-1 receptor structure and can bind to IGF-1. The C-terminus of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen by transglutaminase to form IGF-1-linker-collagen, which can prolong the attachment time of IGF-1 in the endometrium and effectively promote endometrial thickening and endometrial damage repair.

[0020] The protein solution provided by the present invention for promoting the repair of endometrial damage can form a stable repair microenvironment on the surface of the endometrium, promote the repair of endometrial damage, promote the recovery of the number of endometrial glands with intrauterine adhesions, reduce the proportion of fibrosis, and promote the thickening of thin endometrium. It has the advantages of high tissue compatibility, simple preparation, obvious improvement effect, etc., has no potential toxic effects on cells, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The cytotoxicity test diagram of the protein solution described in Example 1 and Comparative Examples 1-9 of the present invention;

[0022] Figure 2 This is a test result diagram of the effect of the protein solution described in Example 1 of the present invention and Comparative Examples 1-5 on the retention time of IGF-1 in the endometrium;

[0023] 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;

[0024] Figure 4 This is a test result diagram of the effect of the protein solution described in Example 1 of the present invention and Comparative Example 1 on the number of endometrial glands;

[0025] Figure 5 This is a test result diagram of the effect of the protein solution described in Example 1 of the present invention and Comparative Example 1 on the proportion of endometrial fibrosis. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the comparative examples. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0028] The applicant's prior patent CN118593690A discloses a method for preparing a corresponding protein solution, and the preparation method disclosed above can be referred to. Among them, human type III collagen is also disclosed in CN118593690A.

[0029] Example 1

[0030] The present embodiment provides a linker polypeptide-collagen, and the linker polypeptide-collagen is made of the following raw materials: PBS buffer, a linker polypeptide, human type III collagen and glutamine transaminase; 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 a recombinant human collagen, and the human type III collagen of the present embodiment is the human type III collagen disclosed in Chinese patent CN118593690A.

[0031] The preparation method of the linker polypeptide-collagen comprises the following steps:

[0032] (1) Add linker polypeptide, human type III collagen and transglutaminase to 10 mM PBS buffer at pH 7.4, the dosage ratio of PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL:10 mg:4 mg:0.1 mg, and stir at 180 rpm for 30 min to obtain a mixed solution;

[0033] (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;

[0034] (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography, and freeze-dried to obtain linker polypeptide-collagen.

[0035] This embodiment also provides a protein solution for promoting the repair of endometrial damage, comprising the following raw materials: linker polypeptide-collagen, IGF-1, and physiological saline.

[0036] This embodiment provides a method for preparing the protein solution for promoting the repair of endometrial damage, comprising the following steps: adding linker polypeptide-collagen and IGF-1 to physiological saline, stirring at 80 rpm for 30 min, wherein the amount ratio of physiological saline, linker polypeptide-collagen and IGF-1 is 1 L:1 g:0.2 g, to obtain a protein solution for promoting the repair of endometrial damage.

[0037] Comparative Example 1:

[0038] The difference from Example 1 is that no linker polypeptide is added, and only collagen, IGF-1 and physiological saline are contained.

[0039] The present invention also designed a series of polypeptides based on the factor receptor protein structure (IGF-1) as potential linkers, and conducted experimental comparisons:

[0040] Comparative Example 2:

[0041] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No. 2: EICGPGIDIRNDLWTDPVFFYVASYRFPLTVITEYLLLFRVAGGDLFPNLTVIRGWKKLFYNYALKKKVIFEMKKKKLKDIGLKKKKY.

[0042] Comparative Example 3:

[0043] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.3: LRNITRGAIRVDWSLILDAVSNNYIVGNPPECGDLCPGTMEEPMCETTINNEYNYRCWYLRLENCTVIEGYLHITTNRCMKKCGRAKKKE.

[0044] Comparative Example 4:

[0045] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.4: YRVFENFLHNSIFVPRASCESDVLEGYWTDSPSLPNYLRITYTVISNLRPFTLKYRIDIHSCNHEADPVFKKKYVAKEGKKKIELGKKKKKCSD.

[0046] Comparative Example 5:

[0047] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.5: NECCHPECLGSCSAPDWTDIENADLCWTDPVFFYVAYLSTHYYYAGVCVPACPPNTYRFEGKWRCVDRDFCANILSAESSDSEGFVIHDKCMECPSKKKRNKKMYC.

[0048] Comparative Example 6:

[0049] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.6: PCEGPCPVCEEIENADLCWTDPVFFYVAYLSTETTIDSVTSAMLGCTIFGNLLINIRRGNWTDPVKFFYVANIAEELKKKGNYSKKVLT.

[0050] Comparative Example 7:

[0051] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.7: NNLLWNFMGLIEVVTGYVIRHSHALVDWDHRNLYLHIDPVFFYVATIAGGRSGDINSELESLSFLKNLRLILGTRNNGERTKKIIITWHKKKPPDYRDL.

[0052] Comparative Example 8:

[0053] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.8: SFTVYYEAPFNVTEYDMYFAFNPLCVSEILRLENCTVIEGYLHIACGSNSWNMVDVDLPPNDVEKPGILLHWTYAVYVKKLRLENCTKKSEILYIRKKSIPLKVR.

[0054] Comparative Example 9:

[0055] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.9: SASNSSSLIVWNPPVFFYVALPPVFFYVANLSYYIVRWRPDGYLYRHNYCSDIPIRYADGTIDIEEVKTAVTLTMVENDHIRGAKKENPTEVKKCPTEAKKEEKAPI.

[0056] Experimental Example 1:

[0057] The protein solution for promoting the repair of endometrial damage was prepared according to the method of Example 1 and Comparative Examples 1-9, respectively, and the physiological saline was replaced with MEM medium containing 10% fetal bovine serum to obtain the experimental solution. L-929 cells (NCTC clone 929) were cultured in MEM medium containing 10% fetal bovine serum and antibiotics (penicillin 100 U / mL, streptomycin 100 µg / mL) at 37°C and 5% CO 2 The cells were digested with 0.25% trypsin, centrifuged at 200 g for 3 min, and then redispersed in fresh culture medium to adjust the cell density to 2 × 10 5 cells / mL of cell suspension; inoculate the above cell suspension into a 96-well culture plate, 100 µL per well, and place in a carbon dioxide incubator (5% CO 2 , 37°C, humidity>90%) for 24 h, and then 100 μL of experimental solution was added to each well as the experimental group. Another well with only 200 μL of MEM medium containing 10% fetal bovine serum was set as the blank control group, and the well with 100 μL of medium + 100 μL of cells was set as the negative control. After adding the experimental solution, the cells were cultured for 5 days. Every day, 20 μL of MTT (5 mg / mL prepared in PBS) was added to each well in the dark. On the last day, MTT was added and cultured for 4 h. After discarding all the liquid in the well, 150 μL of DMSO solution was added to each well. The cells were shaken at a low speed for 15 min in a microplate reader, and the OD value at λ=490 nm was measured. 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.

[0058] Figure 1 The results showed that the toxicity levels of Example 1 and Comparative Examples 1-5 were all 0, indicating that the relevant protein solutions were non-cytotoxic and had high biosafety. The toxicity levels of Comparative Examples 6-9 were all 1, indicating that the corresponding protein solutions had slight cytotoxicity.

[0059] Experimental Example 2:

[0060] Sixty 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 surgery. After intraperitoneal injection of 2% sodium pentobarbital, they were fixed in the supine position. The uterine horns were exposed through 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 uterine walls felt rough. The uterine horns were clamped with a vascular clamp, and 95% ethanol was drawn from 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 min, the vascular clamp at the incision was released 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 and Comparative Examples 1-5 to perfuse the uterine cavity. After the operation, 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 IGF-1 in the uterus was detected by ELISA. The results are shown in Figure 2. Figure 2 shown.

[0061] Figure 2 The results showed that the concentration of IGF-1 in Example 1 was significantly higher than that in Comparative Examples 1-5, and the rate of decline was also lower than that in Comparative Example 1, indicating that the linker polypeptide can effectively prolong the action time of IGF-1 and promote the repair of uterine damage.

[0062] Experimental Example 3:

[0063] 40 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 4 groups, 10 in each group, 2 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 rinsing and sucking with saline. After modeling, each experimental group was immediately given 0.5 mL of the protein solution prepared in Example 1 and Comparative Example 1 to perfuse the uterine cavity. The sham operation group and the model group were given an equal amount of saline. After the operation was completed, the abdominal cavity was rinsed with sterile saline. After the uterus was reset, 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 3The number of endometrial glands and the proportion of endometrial fibrosis were statistically analyzed, and the statistical results are shown in Figure 4 , Figure 5 shown.

[0064] Figure 3 , Figure 4 , Figure 5 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 less intrauterine adhesions than the model group, the number of glands recovered, and the fibrosis ratio was lower than the model group, and close to that of the sham operation group.

[0065] In summary, the protein solution for promoting the repair of endometrial damage in the present invention has a good repairing effect on intrauterine adhesions, and the effect is better than using only a mixture of IGF-1 and collagen. The linker polypeptide can effectively improve the repair effect of endometrial damage and promote the repair of endometrial damage.

[0066] The physicochemical indicators in the above embodiments are only some embodiments, but it cannot be determined that the specific implementation of the present invention is limited to these examples and these application tests. Obviously, the above embodiments of the present invention are only examples made to clearly illustrate the technical solution of the present invention, and are not limitations on the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An insulin-like growth factor-1-linker polypeptide-collagen, characterized in that: The amino acid sequence of the linker polypeptide is shown in SEQ ID No. 1; the N-terminus of the linker polypeptide binds to insulin-like growth factor-1, the C-terminus of the linker polypeptide contains a lysine residue, there are glutamine residues on the collagen, and the C-terminus of the linker polypeptide binds to the collagen through covalent cross-linking by glutamine transaminase; the collagen is type III collagen.

2. A method for preparing a protein solution comprising the insulin-like growth factor-1-linker polypeptide-collagen according to claim 1, characterized in that: The steps include: S1. Add the linker polypeptide, collagen and transglutaminase to a 10 mM PBS buffer having a pH of 7.4 to obtain a mixed solution; wherein the amount ratio of the PBS buffer, the linker polypeptide, the collagen and the transglutaminase is 1 mL: 1-50 mg: 1-100 mg: 0.01-0.5 mg; S2. The mixed solution obtained in step S1 is heated to 50°C for 2-3 h, then heated to 70°C for 15-20 min, and naturally cooled to room temperature to obtain a reaction solution; S3. Purifying the reaction solution in step S2 and freeze-drying the solution to obtain a linker polypeptide-collagen; S4. Add insulin-like growth factor-1 and the linker polypeptide-collagen obtained in step S3 to physiological saline, and stir to obtain a protein solution containing insulin-like growth factor-1-linker polypeptide-collagen as an active ingredient; wherein the dosage ratio of physiological saline, linker polypeptide-collagen and insulin-like growth factor-1 is 1 L: 0.1~10 g: 0.01~1 g.

3. The preparation method according to claim 2, characterized in that: In step S3, purification is performed by separation through G25 molecular sieve chromatography.

4. The preparation method according to claim 2, characterized in that: Step S1: After adding all the raw materials, stir at 180 rpm for 30 min.

5. The preparation method according to claim 2, characterized in that: Step S4: After stirring at 80 rpm for 30 min, a protein solution containing insulin-like growth factor-1-linker polypeptide-collagen as an active ingredient is obtained.

6. Use of the insulin-like growth factor-1-linker polypeptide-collagen according to claim 1 in the preparation of a preparation for promoting the repair of endometrial damage.

7. The use according to claim 6, characterized in that: The insulin-like growth factor-1-linker polypeptide-collagen can prolong the action time of insulin-like growth factor-1.

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