Epidermal growth factor connecting polypeptide and application thereof in endometrial injury repair

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses an epidermal growth factor-linker polypeptide-collagen, and the linker polypeptide has an amino acid sequence as shown in SEQ ID No.1. The linker polypeptide capable of connecting the EGF and the collagen is designed and synthesized, the N end of the linker polypeptide simulates an EGF receptor structure and can be combined with the EGF, and the linker polypeptide is connected with the EGF through non-covalent Van der Waals force, so that the structure of the EGF cannot be damaged; the C terminal of the linker polypeptide is rich in lysine residues, and can be covalently crosslinked to collagen through glutamine transaminase to form EGF-linker polypeptide-collagen. The protein solution taking EGF-linker polypeptide-collagen as active ingredients is low in cytotoxicity and high in biological safety, and meanwhile, the action time of the epidermal growth factor can be remarkably prolonged. In addition, a stable repairing microenvironment can be formed on the surface of the endometrium, and the endometrium injury repairing effect is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an epidermal growth factor-linked polypeptide and its application 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] Epidermal Growth Factor (EGF) family members are a group of glycoproteins located in the cell membrane. They are mainly expressed in the endometrium and can promote the growth of endometrial vascular epithelium, angiogenesis, and permeability. HB-EGF belongs to the growth factor family. Because it has a special heparin binding region, it is named HB-EGF (heparin-binding epidermal growth factor). It is the earliest cytokine to appear in the endometrial implantation window. It plays an important role in promoting endometrial growth and differentiation, mediating blastocyst adhesion and implantation, and maintaining placental function. HB-EGF is low in the endometrial hyperplasia phase, increases in the secretory phase, and reaches a peak in the implantation window. Reports have shown that EGF can promote endometrial regeneration. Because EGF molecules are small and not easy to attach to the endometrium for a long time, the effect of simply using EGF or gel preparations containing EGF to promote endometrial growth is not good.

[0004] Therefore, finding a method to enhance EGF's ability 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. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention proposes an active component that promotes the repair of endometrial damage: epidermal growth factor-linker polypeptide-collagen. First, the present invention designs and synthesizes a linker polypeptide that can connect EGF and collagen. The N-terminus of the linker polypeptide simulates the EGF receptor structure and can bind to EGF. The connection between the linker polypeptide and EGF is through non-covalent van der Waals forces and will not destroy the structure of EGF; the C-terminus of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen by transglutaminase to form EGF-linker polypeptide-collagen. And through the connection of the above-mentioned specific structure, a protein solution that can promote the repair of endometrial damage is prepared, forming a stable repair microenvironment on the surface of the endometrium, and promoting the repair of endometrial damage.

[0006] The present invention is achieved through the following technical solutions: An epidermal growth factor-linker polypeptide-collagen, wherein the N-terminus of the linker polypeptide is bound to the epidermal growth factor, the C-terminus of the linker polypeptide contains a lysine residue, there is a glutamine residue on the collagen, and the C-terminus of the linker polypeptide is bound to the collagen through covalent cross-linking by transglutaminase. It can be understood by those skilled in the art that the above-mentioned binding and covalent cross-linking methods can be carried out with reference to the prior art.

[0007] The linker polypeptide is any of the following: (a) the amino acid sequence shown in SEQ ID No.1; (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] Furthermore, 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. The connection synthesis of the linker polypeptide with epidermal growth factor and collagen can also be carried out according to the existing technology.

[0009] Preferably, the epidermal growth factor is heparin-binding epidermal growth factor.

[0010] 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.

[0011] Preferably, the collagen is human type I, type II, type III, type V or type XI collagen.

[0012] Specifically, the collagen used in this embodiment is human type III collagen. Further, the human type III collagen can be commercially available human type III collagen, and the recombinant human collagen used in the embodiment of the present invention can be human type III collagen obtained by the method disclosed in Chinese patent CN118593690A, but this application does not strictly limit this.

[0013] Specifically, the present invention provides a method for preparing the protein solution of epidermal growth factor-linker polypeptide-collagen, comprising the following steps: S1. Adding 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 was heated to 50°C for 3 h, then heated to 70°C for 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 EGF and the linker polypeptide-collagen obtained in step S3 to physiological saline, and stir to obtain a protein solution containing epidermal growth factor-linker polypeptide-collagen as an active ingredient; wherein the dosage ratio of physiological saline, linker polypeptide-collagen and EGF is 1 L: 0.1 ~10g: 0.01~0.5 g.

[0014] Preferably, in step S3, purification is performed by separation through G25 molecular sieve chromatography.

[0015] Preferably, after adding each raw material in step S1, stirring is carried out at 180 rpm for 30 min.

[0016] Preferably, in step S4, a protein solution containing epidermal growth factor-linker polypeptide-collagen as an active ingredient is obtained after stirring at 80 rpm for 30 min.

[0017] Preferably, the epidermal growth factor-linker polypeptide-collagen is used in the preparation of a preparation for promoting the repair of endometrial damage.

[0018] Preferably, the epidermal growth factor-linker polypeptide-collagen can prolong the action time of epidermal growth factor.

[0019] Preferably, the epidermal growth factor-linker polypeptide-collagen promotes endometrial thickening.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention designs and synthesizes a linker polypeptide capable of connecting EGF and collagen. The N-terminus of the linker polypeptide simulates the EGF receptor structure and can bind to EGF. The C-terminus of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen by transglutaminase to form EGF-linker-collagen, which can prolong the attachment time of EGF in the endometrium and effectively promote endometrial thickening and endometrial damage repair.

[0021] 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

[0022] Figure 1 The cytotoxicity test diagram of the protein solution described in Example 1 and Comparative Examples 1-9 of the present invention; 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 EGF in the endometrium; 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; 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; 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

[0023] 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.

[0024] 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.

[0025] The 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; the EGF used in the following examples is HB-EGF (heparin-binding epidermal growth factor).

[0026] Example 1

[0027] 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 can be synthesized in a directed manner according to the polypeptide sequence using amino acids as raw materials by solid phase synthesis; the human type III collagen is recombinant human collagen, and the human type III collagen of the present embodiment is the human type III collagen disclosed in Chinese patent CN118593690A.

[0028] The preparation method of the linker polypeptide-collagen comprises the following steps: (1) Adding the linker polypeptide shown in SEQ ID No. 1, human type III collagen and transglutaminase to a 10 mM PBS buffer at pH 7.4, the amount ratio of PBS buffer, linker polypeptide, human type III collagen and transglutaminase is 1 mL: 10 mg: 5 mg: 0.1 mg, stirring at 180 rpm for 30 min to obtain a mixed solution; (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; (3) The reaction solution of step (2) is separated and purified by G25 molecular sieve chromatography and freeze-dried to obtain linker polypeptide-collagen.

[0029] This embodiment also provides a protein solution for promoting the repair of endometrial damage, comprising the following raw materials: linker polypeptide-collagen, EGF, and physiological saline.

[0030] 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 EGF to physiological saline, stirring at 80 rpm for 30 min, wherein the amount ratio of physiological saline, linker polypeptide-collagen and EGF is 1 L:1 g:0.05 g, to obtain a protein solution for promoting the repair of endometrial damage.

[0031] Comparative Example 1: The difference from Example 1 is that no linker polypeptide is added, and only collagen, EGF and physiological saline are contained.

[0032] In addition, the present invention also designed a series of polypeptides based on the factor receptor protein structure (EGF) as potential linkers, and conducted experimental comparisons: Comparative Example 2: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.2: LEEVCGTSNLTLGTFEDHFLSLRMFNNSGNNLCYANTINWLFGCEVVLGNLEITYVRNYDLSFLTIEVAGYVLIAKKVEKKKNLIIRKGKKNMKKY.

[0033] Comparative Example 3: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No. 3: YENSYALAVLSNYDANTGLELPMRNLEILHGAVRFSNCGTSNLTLGTFEDHFLSLRNPALCNKVESIWRDIVSSDKKSMDFKNHLGKKSCCKKKKKDPSC.

[0034] Comparative Example 4: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.4: PNGSCWGAGEENCLTIICACSGRCRGSPSDCCHNCAAGCTGPRESDCLVCRFRDEKATCKDTCKPKKKKPLKMLYNPKKTKTYKMD.

[0035] Comparative Example 5: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.5: VNPEGYSFGATCVCPRNYVVTDHGSCVRACGALSNYDANTGLELPMRNLEIDSYEMEEDGVRCCEGPCRVCNKKKEFDSKKLSINKAKKKHFNKKKCT.

[0036] Comparative Example 6: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.6: ISGDLHILPVAFRGDSFTHTPTDHGSCVRACGALSNYDPLDPELDILTVEITGFLLIAWPENRTDLHAFENLEIIRGKRTHKKGFSLAKKVVSLKKKGLRT.

[0037] Comparative Example 7: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.7: LEISDGDVIISGNNLCYANTINWLFGTSGTIILSNYDPLDPELDILTVEITSNRGENSCATGVCHALCSPEGCKKWGPEPKKRDCVSKKKRECKKV.

[0038] Comparative Example 8: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.8: CNLLEGEPREFVENNTLVWYADAGHVCHLCHPNENSECICHPECNNLCYANTINWLFGTSGTIILSNLPKAMKKNKKKKITCKKTGKKKR.

[0039] Comparative Example 9: The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.9: PDNCICAHYIDGPHCVTCPAGVMGENNTLPRNYVVTDHGSCVRACGALSNVWYADAGHVCHLCHPNCTKKYGCTGKPKGLEKGCPTNKKGPI.

[0040] Experimental Example 1: The method for preparing a protein solution for promoting the repair of endometrial damage was respectively prepared according to Example 1 and Comparative Examples 1-9, and the physiological saline was replaced with MEM medium containing 10% fetal bovine serum to obtain an 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) 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 medium and the cell density was adjusted to 2×10 5cells / mL of 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 h 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 enzyme reader, and measure its OD value at λ=490 nm. The toxicity of the cells is determined by calculating the relative growth rate (RGR, %) of the cells: RGR = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%, the specific toxicity classification standard is: 0: RGR ≥ 100%; 1: 99% ≥ RGR ≥ 75%; 2: 74% ≥ RGR ≥ 50%; 3: 49% ≥ RGR ≥ 25%; 4: 24% ≥ RGR ≥ 1%; 5 = 0. Among them, 2 or above can be regarded as the presence of cytotoxic reaction. The calculation and statistical results are as follows Figure 1 shown.

[0041] 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 had no cytotoxicity and high biological safety. The toxicity level of Comparative Examples 6-9 was 1, indicating that the relevant protein solutions had weak toxicity and were not suitable for use as in vivo protein solutions after combination.

[0042] Experimental Example 2: 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 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 EGF in the uterus was detected by ELISA. The results are shown in Figure 2. Figure 2 shown.

[0043] Figure 2 The results show that the concentration of EGF in Example 1 is significantly higher than that in Comparative Examples 1-5, and the rate of decrease is also lower than that in Comparative Examples 1-5, indicating that the specific structure linker polypeptide in Example 1 can effectively prolong the action time of EGF.

[0044] In combination with Experimental Examples 1 and 2, the epidermal growth factor-linker polypeptide-collagen composed of the specific linker polypeptide provided in Example 1 of the present application is used to prepare the corresponding protein solution, which not only has low cytotoxicity and high biosafety, but also can significantly prolong the action time of epidermal growth factor.

[0045] Experimental Example 3: Forty female SD rats aged 8-10 weeks and weighing 220-260 g were selected and randomly divided into 4 groups after one week of adaptive feeding, with 10 rats in each group, including 2 experimental groups, 1 sham operation group and 1 model group.

[0046] Among them, the experimental group and the model group were induced to establish models by mechanical injury and ethanol perfusion. The modeling method was the same as that in Experimental Example 2. The sham operation group was anesthetized and performed laparotomy. The uterine horns were fully exposed to the body and then retracted. No other operations were performed. The uterine horns were rinsed and cleaned with saline, and the liquid on the surface of the uterus was dried with sterile gauze. 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, the abdominal cavity was rinsed with sterile saline. After the uterus was repositioned, the abdomen was closed layer by layer, and the incision was disinfected with 75% alcohol. After the operation, 2 mL of gentamicin was given intramuscularly for 3 consecutive days to prevent infection. Each group was killed 14 days after the operation, and the uterine tissue was taken, fixed with paraformaldehyde, sectioned with paraffin, stained with HE, and stained with Masson. The results are shown as follows: 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 , Figure 5 shown.

[0047] 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.

[0048] The above results show that the protein solution for promoting the repair of endometrial damage of the present invention has a good repair effect on intrauterine adhesions, and the effect is better than the combined use of EGF and collagen, indicating that the addition of linker polypeptides can effectively improve the repair effect of endometrial damage and promote the repair of endometrial damage.

[0049] 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.

[0050] 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. An epidermal growth factor-linker polypeptide-collagen, characterized in that: The linker polypeptide has an amino acid sequence as shown in SEQ ID No. 1; the N-terminus of the linker polypeptide binds to epidermal growth factor, 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.

2. The epidermal growth factor-linker polypeptide-collagen according to claim 1, characterized in that: The epidermal growth factor is heparin-binding epidermal growth factor.

3. The epidermal growth factor-linker polypeptide-collagen according to claim 1, characterized in that: The collagen is human type I, type II, type III, type V or type XI collagen.

4. A method for preparing a protein solution comprising the epidermal growth factor-linker polypeptide-collagen according to claim 1, characterized in that: The steps include: S1. Adding 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 EGF and the linker polypeptide-collagen obtained in step S3 to physiological saline, and stir to obtain a protein solution containing epidermal growth factor-linker polypeptide-collagen as an active ingredient; wherein the dosage ratio of physiological saline, linker polypeptide-collagen and EGF is 1 L: 0.1 ~10g: 0.01~0.5 g.

5. The preparation method according to claim 4, characterized in that: In step S3, purification is performed by separation through G25 molecular sieve chromatography.

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

7. The preparation method according to claim 4, characterized in that: Step S4: After stirring at 80 rpm for 30 min, a protein solution containing epidermal growth factor-linker polypeptide-collagen as an active ingredient is obtained.

8. Use of the epidermal growth factor-linker polypeptide-collagen according to any one of claims 1 to 3 in the preparation of a preparation for promoting the repair of endometrial damage.

9. The use according to claim 8, characterized in that: The epidermal growth factor-linker polypeptide-collagen can prolong the action time of epidermal growth factor.

10. The use according to claim 8, characterized in that: The epidermal growth factor-linker polypeptide-collagen promotes endometrial thickening.

Images