An epidermal growth factor-linked polypeptide and its application in the repair of endometrial injury

Abstract

The present invention discloses an epidermal growth factor - linker polypeptide - collagen, and the linker polypeptide has an amino acid sequence as shown in SEQ ID No.1. In the present invention, a linker polypeptide capable of connecting EGF and collagen is designed and synthesized. The N - terminal of the linker polypeptide mimics the EGF receptor structure and can bind to EGF, and the connection between the linker polypeptide and EGF is through non - covalent van der Waals forces, which will not destroy the structure of EGF; the C - terminal of the linker polypeptide is rich in lysine residues and can be covalently cross - linked to collagen through transglutaminase to form EGF - linker polypeptide - collagen. The protein solution with the above - mentioned EGF - linker polypeptide - collagen as the active ingredient not only has low cytotoxicity and high biological safety, but also can significantly prolong the action time of epidermal growth factor. In addition, it can form a stable repair microenvironment on the surface of the endometrium and significantly improve the repair effect of endometrial injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an epidermal growth factor-linked polypeptide and its application in the repair of endometrial injury. Background Art

[0002] The endometrium is the basis for embryo implantation. A relatively thick endometrium has a better bearing effect. If the endometrium is thin, the implantation rate and the pregnancy rate after implantation will be affected. A recognized marker 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 usually cannot be carried out, and the survival rate of the embryo after transplantation will also be significantly reduced. It is reported that among infertility patients, the incidence of endometrial injury is about one-fifth. At present, estrogen and progesterone are mostly used for the repair of endometrial injury, but the effect is poor.

[0003] Members of the epidermal growth factor (EGF) family are a group of glycoproteins located on the cell membrane, mainly expressed in the endometrium, and can promote the growth of endometrial vascular epithelium, angiogenesis, and the formation of permeability. HB-EGF belongs to the growth factor family and is named HB-EGF (heparin-binding epidermal growth factor) because it has a special heparin-binding region. It is the earliest cytokine to appear in the endometrial implantation window and 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 proliferative phase of the endometrium, increases in the secretory phase, and reaches a peak in the implantation window phase. It is reported that EGF can promote endometrial regeneration. Since the EGF molecule is small and not easy to adhere to the endometrium for a long time, the effect of promoting endometrial growth by simply using EGF or a gel preparation containing EGF is not good.

[0004] Therefore, finding a method to enhance the repair of endometrial injury by EGF and promoting the thickening of the damaged endometrium to the normal level is a technical problem to be solved urgently. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides an active component for promoting the repair of endometrial injury: epidermal growth factor-linker polypeptide-collagen. First, the present invention designed and synthesized a linker polypeptide capable of connecting EGF and collagen. The N-terminal of the linker polypeptide mimics 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, which will not damage the structure of EGF. The C-terminal of the linker polypeptide is rich in lysine residues and can be covalently cross-linked to collagen through transglutaminase to form EGF-linker polypeptide-collagen. And a protein solution capable of promoting the repair of endometrial injury is prepared through the connection of the above specific structure, forming a stable repair microenvironment on the surface of the endometrium and promoting the repair of endometrial injury.

[0006] The present invention is achieved through the following technical solutions:

[0007] An epidermal growth factor-linker polypeptide-collagen, wherein the N-terminal of the linker polypeptide binds to epidermal growth factor, the C-terminal of the linker polypeptide contains lysine residues, glutamine residues exist on the collagen, and the C-terminal of the linker polypeptide is covalently cross-linked to collagen through transglutaminase. Those skilled in the art can understand that the above binding and covalent cross-linking methods can be referred to the prior art.

[0008] The linker polypeptide is any one of the following:

[0009] (a) The amino acid sequence shown in SEQ ID No.1;

[0010] (b) A polypeptide obtained by substitution and / or deletion and / or insertion of amino acid residues in the polypeptide described in (a) and having more than 80% identity with the polypeptide described in (a) and having the same biological function.

[0011] Furthermore, the linker polypeptide is directionally synthesized from amino acids by conventional polypeptide synthesis methods such as solid-phase synthesis method and liquid-phase synthesis method according to the polypeptide sequence. The connection synthesis of the linker polypeptide with epidermal growth factor and collagen can also be referred to the prior art.

[0012] Preferably, the epidermal growth factor is heparin-binding epidermal growth factor.

[0013] In addition, the collagen in the present application can also be other types of proteins. For example, it only needs to satisfy that glutamine residues exist on the side chains of the above proteins, and the proteins defined above are also within the protection scope of the present application.

[0014] Preferably, the collagen is human-derived type I, type II, type III, type V or type XI collagen.

[0015] Specifically, the collagen used in this embodiment is human type III collagen. Further, any commercially available human type III collagen can be used. In the embodiments of the present invention, the recombinant human collagen used can be the human type III collagen obtained by the method disclosed in Chinese Patent CN118593690A, but this application does not impose strict restrictions on this.

[0016] Specifically, the present invention provides a method for preparing a protein solution of the epidermal growth factor-linker polypeptide-collagen, comprising the following steps:

[0017] S1. Add the linker polypeptide, collagen, and transglutaminase to a 10 mM PBS buffer solution with a pH of 7.4 to obtain a mixed solution; wherein, the dosage ratio of the PBS buffer solution, linker polypeptide, collagen, and transglutaminase is 1 mL: 1-50 mg: 1-100 mg: 0.01-0.5 mg;

[0018] S2. Heat the mixed solution obtained in step S1 to 50 °C and maintain it for 3 h, then heat it to 70 °C and maintain it for 20 min, and naturally cool it to room temperature to obtain a post-reaction solution;

[0019] S3. Purify and lyophilize the post-reaction solution obtained in step S2 to obtain the linker polypeptide-collagen;

[0020] 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 the active ingredient; wherein, the dosage ratio of physiological saline, linker polypeptide-collagen, and EGF is 1 L: 0.1-10 g: 0.01-0.5 g.

[0021] Preferably, in step S3, purification is carried out by G25 molecular sieve chromatography separation.

[0022] Preferably, after adding each raw material in step S1, stir at 180 rpm for 30 min.

[0023] Preferably, in step S4, stir at 80 rpm for 30 min to obtain a protein solution containing epidermal growth factor-linker polypeptide-collagen as the active ingredient.

[0024] Preferably, the application of the epidermal growth factor-linker polypeptide-collagen in the preparation of a preparation for promoting the repair of endometrial injury.

[0025] Preferably, the epidermal growth factor-linker polypeptide-collagen can prolong the action time of epidermal growth factor.

[0026] Preferably, the epidermal growth factor-linker polypeptide-collagen promotes endometrial thickening.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention designs and synthesizes a linker polypeptide capable of connecting EGF and collagen. The N-terminus of the linker polypeptide mimics 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 through transglutaminase to form EGF-linker-collagen, which can extend the attachment time of EGF to the endometrium and effectively promote endometrial thickening and repair of endometrial injury.

[0029] A protein solution for promoting the repair of endometrial injury provided by the present invention can form a stable repair microenvironment on the endometrial surface, promote the repair of endometrial injury, promote the restoration of the number of endometrial glands with intrauterine adhesions, reduce the fibrosis ratio, promote the thickening of thin endometrium, has the advantages of high tissue compatibility, simple preparation, obvious improvement effect, etc., has no potential toxic effect on cells, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a cytotoxicity test chart of the protein solutions described in Example 1 and Comparative Examples 1-9 of the present invention;

[0031] Figure 2 It is a test result chart of the effect of the protein solutions described in Example 1 and Comparative Examples 1-5 of the present invention on the retention time of EGF in the endometrium;

[0032] Figure 3 It is a uterine section staining chart of the sham operation group, model group and Example 1 group described in Experimental Example 3 of the present invention;

[0033] Figure 4 It is a test result chart of the effect of the protein solutions described in Example 1 and Comparative Example 1 of the present invention on the number of endometrial glands;

[0034] Figure 5 It is a test result chart of the effect of the protein solutions described in Example 1 and Comparative Example 1 of the present invention on the endometrial fibrosis ratio. DETAILED DESCRIPTION OF THE INVENTION

[0035] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments and comparative examples of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Unless otherwise specified, the test methods used in the following examples are conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] The corresponding method for preparing the protein solution is disclosed in the prior patent CN118593690A, 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 all HB-EGF (heparin-binding epidermal growth factor).

[0038] Example 1

[0039] This example provides a linker polypeptide - collagen, which is made from the following raw materials: PBS buffer, linker polypeptide, human type III collagen, and transglutaminase; the linker polypeptide has the amino acid sequence shown in SEQ ID No.1 and can be directionally synthesized from amino acids by solid-phase synthesis method according to the polypeptide sequence; the human type III collagen is recombinant human collagen, and the human type III collagen in this example is the human type III collagen disclosed in Chinese Patent CN118593690A.

[0040] The preparation method of the linker polypeptide - collagen includes the following steps:

[0041] (1) Add the linker polypeptide shown in SEQ ID No.1, human type III collagen, and transglutaminase to a 10 mM PBS buffer with a pH of 7.4. The dosage ratio of PBS buffer, linker polypeptide, human type III collagen, and transglutaminase is 1 mL:10 mg:5 mg:0.1 mg, and stir at 180 rpm for 30 min to obtain a mixed solution;

[0042] (2) Heat the mixed solution obtained in step (1) to 50°C and maintain for 3 h, then heat to 70°C and maintain for 20 min, and naturally cool to room temperature to obtain the post-reaction solution;

[0043] (3) Separate and purify the post-reaction solution described in step (2) by G25 molecular sieve chromatography and freeze-dry to obtain the linker polypeptide - collagen.

[0044] This example also provides a protein solution for promoting the repair of endometrial injury, which includes the following raw materials: linker polypeptide - collagen, EGF, and physiological saline.

[0045] This embodiment provides a method for preparing the protein solution for promoting the repair of endometrial injury, comprising the following steps: adding linker polypeptide - collagen and EGF to physiological saline, stirring at 80 rpm for 30 min, and the dosage ratio of physiological saline, linker polypeptide - collagen and EGF is 1 L:1 g:0.05 g to obtain the protein solution for promoting the repair of endometrial injury.

[0046] Comparative Example 1:

[0047] The difference from Example 1 is that no linker polypeptide is added, and it only contains collagen, EGF, and physiological saline.

[0048] In addition, the present invention also designed a series of polypeptides based on the factor receptor protein structure (EGF), which are used as potential linkers, and experimental comparisons were carried out:

[0049] Comparative Example 2:

[0050] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.2: LEEVCGTSNLTLGTFEDHFLSLRMFNNSGNNLCYANTINWLFGCEVVLGNLEITYVRNYDLSFLTIEVAGYVLIAKKVEKKKNLIIRKGKKNMKKY.

[0051] Comparative Example 3:

[0052] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.3: YENSYALAVLSNYDANTGLELPMRNLEILHGAVRFSNCGTSNLTLGTFEDHFLSLRNPALCNKVESIWRDIVSSDKKSMDFKNHLGKKSCCKKKKKDPSC.

[0053] Comparative Example 4:

[0054] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.4: PNGSCWGAGEENCLTIICACSGRCRGSPSDCCHNCAAGCTGPRESDCLVCRFRDEKATCKDTCKPKKKKPLKMLYNPKKTKTYKMD.

[0055] Comparative Example 5:

[0056] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.5: VNPEGYSFGATCVCPRNYVVTDHGSCVRACGALSNYDANTGLELPMRNLEIDSYEMEEDGVRCCEGPCRVCNKKKEFDSKKLSINKAKKKHFNKKKCT.

[0057] Comparative Example 6:

[0058] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.6: ISGDLHILPVAFRGDSFTHTPTDHGSCVRACGALSNYDPLDPELDILTVEITGFLLIAWPENRTDLHAFENLEIIRGKRTHKKGFSLAKKVVSLKKKGLRT.

[0059] Comparative Example 7:

[0060] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.7: LEISDGDVIISGNNLCYANTINWLFGTSGTIILSNYDPLDPELDILTVEITSNRGENSCATGVCHALCSPEGCKKWGPEPKKRDCVSKKKRECKKV.

[0061] Comparative Example 8:

[0062] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.8: CNLLEGEPREFVENNTLVWYADAGHVCHLCHPNENSECICHPECNNLCYANTINWLFGTSGTIILSNLPKAMKKNKKKKITCKKTGKKKR.

[0063] Comparative Example 9:

[0064] The difference from Example 1 is that the amino acid sequence of the linker polypeptide is SEQ ID No.9: PDNCICAHYIDGPHCVTCPAGVMGENNTLPRNYVVTDHGSCVRACGALSNVWYADAGHVCHLCHPNCTKKYGCTGKPKGLEKGCPTNKKGPI.

[0065] Experimental Example 1:

[0066] The method for preparing the protein solution for promoting the repair of endometrial injury according to Example 1 and Comparative Examples 1-9 was used. 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) in an incubator at 37°C and 5% CO2. The cells were digested with 0.25% trypsin, centrifuged at 200 g for 3 min, and then the cells were redispersed in fresh medium. The cell density was adjusted to 2×10 5 cells / mL cell suspension; The above cell suspension was inoculated into a 96-well culture plate, 100 μL per well, and cultured in a carbon dioxide incubator (5% CO2, 37°C, humidity > 90%) for 24 h. Then 100 μL of the experimental solution was added to each well as the experimental group; Another group with only 200 μL of MEM medium containing 10% fetal bovine serum was set as the blank control group, and the wells with 100 μL of medium + 100 μL of cells were used 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 with PBS) was added to each well in the dark. After adding MTT on the last day, the cells were cultured for another 4 h. After discarding all the liquid in the wells, 150 μL of DMSO solution was added to each well, and it was shaken gently at a low speed in an enzyme-linked immunosorbent assay (ELISA) reader for 15 min, and the optical density (OD) value at λ = 490 nm was measured. The cytotoxicity of the cells was determined by calculating the relative growth rate (RGR, %) of the cells:

[0067] RGR = [(ODexperimental group - ODblank group) / (ODnegative control group - ODblank group)] × 100%. The specific classification criteria for toxicity are as follows: Grade 0: RGR ≥ 100%; Grade 1: 99% ≥ RGR ≥ 75%; Grade 2: 74% ≥ RGR ≥ 50%; Grade 3: 49% ≥ RGR ≥ 25%; Grade 4: 24% ≥ RGR ≥ 1%; Grade 5 = 0. Among them, a toxicity level of 2 or above can be regarded as having a cytotoxic reaction. The calculation results of the statistics are as Figure 1 shown.

[0068] Figure 1 The results showed that the toxicity grades of Example 1 and Comparative Examples 1-5 were all 0, indicating that the related protein solution had no cytotoxicity and high biosafety. The toxicity grades of Comparative Examples 6-9 were 1, indicating that the related protein solution had weak toxicity and was not suitable for use as an in vivo protein solution after combination.

[0069] Experimental Example 2:

[0070] Sixty female Sprague-Dawley 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 of 10 rats each. A model was induced by mechanical injury and ethanol perfusion. The rats were fasted and water-deprived for 12 h before surgery. After anesthesia with intraperitoneal injection of 2% sodium pentobarbital, they were fixed in the supine position. A transverse abdominal incision was made to expose the uterine horns. A longitudinal incision about 4 mm long was made about 0.5 cm below the uterine horns. The middle and upper segments of the endometrium were scraped with an endometrial curette with a diameter of 2.5 mm. The curettage was stopped when the four walls of the uterus felt rough. The uterine horns were clamped with a vascular clamp. 1 mL of 95% ethanol was aspirated with a syringe and injected downward from the incision until the uterine cavity was filled. Then the incision was also temporarily clamped with a vascular clamp. After 3 min, the vascular clamp at the incision was released, and the residual ethanol was drained. The uterus was rinsed and suctioned with normal saline and dried with sterile gauze. After modeling, each group was immediately perfused with 0.5 mL of the protein solution prepared in Example 1 and Comparative Examples 1-5 into the uterine cavity. After the operation, the abdominal cavity was rinsed with sterile normal 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 every day to prevent infection. One rat from each group was sacrificed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after the operation, and the uterus was collected. The drug concentration of EGF in the uterus was detected by ELISA method. The results were as Figure 2 shown.

[0071] Figure 2 The results in

[0072] showed that the concentration of EGF in the group of Example 1 was significantly higher than that in Comparative Examples 1-5, and the decline rate was also lower than that in Comparative Examples 1-5, indicating that the specific structural linker polypeptide of Example 1 could effectively prolong the action time of EGF.

[0073] Experimental Example 3:

[0074] Forty female Sprague-Dawley 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 of 10 rats each, including 2 experimental groups, 1 sham operation group, and 1 model group.

[0075] Among them, the experimental group and the model group were induced to establish models by mechanical injury and ethanol perfusion methods. The modeling method was the same as that in Experimental Example 2. The sham operation group was anesthetized and laparotomy was performed. After the uterine horns were fully exposed outside the body, they were replaced, and no other operations were taken. The abdominal cavity was rinsed with normal saline and suctioned clean, and the surface liquid of the uterus was dried with sterile gauze. After modeling, each experimental group was immediately perfused with 0.5 mL of the protein solution prepared in Example 1 and Comparative Example 1 into the uterine cavity. The sham operation group and the model group were given an equal amount of normal saline. After the operation was completed, the abdominal cavity was rinsed with sterile normal saline, and the abdomen was closed layer by layer after the uterus was reset. The incision was disinfected with 75% alcohol, and gentamicin 2 mL was intramuscularly injected continuously for 3 days after the operation to prevent infection. Each group was sacrificed on the 14th day after the operation, and uterine tissues were taken, fixed with paraformaldehyde, paraffin sectioned, HE stained, and Masson stained. The results were as Figure 3 shown. The number of endometrial glands and the proportion of endometrial fibrosis were statistically analyzed. The statistical results were as Figure 4 , Figure 5 shown.

[0076] Figure 3 , Figure 4 , Figure 5 The results showed that the endometrial structure of the sham operation group was normal, the number of glands was large, and the fibrosis ratio was normal; severe intrauterine adhesions occurred in the model group, the number of glands decreased significantly, and the fibrosis ratio increased; the intrauterine adhesion situation in the experimental group was reduced compared with the model group, the number of glands recovered, and the fibrosis ratio was lower than that in the model group and was close to that in the sham operation group.

[0077] The above results indicate that the protein solution for promoting the repair of endometrial injury in 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 the linker polypeptide can effectively improve the effect of endometrial injury repair and promote the repair of endometrial injury.

[0078] Those of ordinary skill in the art should understand that the discussion of any embodiment above is only exemplary and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples; under the idea of the present invention, the technical features between the above embodiments or different embodiments can also be combined, and the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity.

[0079] The present invention aims 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 principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An epidermal growth factor-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 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 binds to the collagen through covalent cross-linking by transglutaminase; The N-terminus of the linker polypeptide simulates the receptor structure of epidermal growth factor and binds to the epidermal growth factor. The connection between the linker polypeptide and the epidermal growth factor is through non-covalent van der Waals force. The collagen is human type III collagen.

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. 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 a linker polypeptide, collagen and transglutaminase to a 10 mM PBS buffer solution at a pH of 7.4 to obtain a mixed solution; wherein the amount ratio of the PBS buffer solution, 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 epidermal growth factor and the linker polypeptide-collagen obtained in step S3 to physiological saline, and obtain a protein solution containing epidermal growth factor-linker polypeptide-collagen as an active ingredient after stirring; wherein the dosage ratio of physiological saline, linker polypeptide-collagen and epidermal growth factor is 1 L: 0.1 ~10g: 0.01~0.5 g.

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

5. The preparation method according to claim 3, characterized in that: Step S1: After adding all the raw materials, stir at 180 rpm for 30 min.

6. The preparation method according to claim 3, 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.

7. Use of the epidermal growth factor-linker polypeptide-collagen according to any one of claims 1 or 2 in the preparation of a preparation for promoting the repair of endometrial damage.

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

9. The use according to claim 7, characterized in that: The epidermal growth factor-linker polypeptide-collagen promotes endometrial thickening.

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