A polypeptide promoting epithelial cell adhesion, a functional collagen material, and a preparation method and application thereof

Abstract

The present invention provides a polypeptide that promotes epithelial cell adhesion, a functional collagen material, and a preparation method and application thereof. The polypeptide that promotes epithelial cell adhesion includes a collagen material binding unit, a connecting unit, and an epithelial cell binding unit that are connected in sequence. The present invention also provides a functional collagen material, that is, the polypeptide that promotes epithelial cell adhesion is incubated with a collagen material, so that the functional peptide sequence is specifically bound to the collagen material to form the polypeptide functionalized collagen material. The functional collagen material prepared by the polypeptide that promotes epithelial cell adhesion of the present invention can quickly recruit endogenous epithelial cells at the site of injury and increase the number of adhesion thereof; and the functional collagen material can not only supplement the functional cells in the damaged area, but also promote the secretion of a variety of growth factors and signal molecules, etc., to provide a microenvironment that is conducive to damage repair, thereby enhancing the effect of tissue and organ damage repair.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a polypeptide promoting epithelial cell adhesion, a functional collagen material, and a preparation method and application thereof. Background Art

[0002] Epithelial cells perform important functions in human tissues and organs, such as gas exchange in the lungs, filtration in the kidneys, detoxification and binding in the liver, secretion of insulin by pancreatic islet cells, and resistance to harmful links in the skin. Therefore, epithelial cells play a key role in the repair of tissue and organ damage. However, after tissue and organ damage occurs, an unfavorable microenvironment is often formed at the site of damage, which hinders the rapid epithelialization of tissues and organs. In addition, epithelial cells that are specifically differentiated by human organs are difficult to culture in vitro, which limits the introduction of exogenous epithelial cells for treatment.

[0003] CN110404110A discloses a suture-resistant collagen-based corneal regeneration and repair material and its preparation method. Collagen is added to hydrochloric acid to dissolve, and then an EDC / NHS cross-linking agent is added to carry out a cross-linking reaction, heated and concentrated to obtain a cross-linked collagen solution, which is added to a culture dish to a certain thickness after degassing, and then a PCL ring is spread on the bottom of the cross-linked collagen solution, air-dried, and washed to obtain a composite film; the obtained composite film is hot-pressed and secondary-molded at 100-120°C in a mold to obtain a suture-resistant collagen-based corneal regeneration and repair material. In the invention, corneal epithelial cells can adhere and proliferate on the surface of the material, thereby realizing the rapid epithelialization of corneal epithelial cells on the implant during surgery, and ultimately achieving the purpose of corneal defect repair and corneal replacement.

[0004] CN113563452A discloses a bioactive peptide and its application with adipose stem cell exosomes in skin proliferation and repair. The bioactive peptide can simultaneously bind to VEGFR and bFGFR with high affinity to promote epithelial cell proliferation; the active peptide can be used in combination with adipose stem cell exosomes to treat skin damage, promote cytokine secretion, and assist in fat cell transplantation, inhibit immune rejection, reduce the expression of inflammatory factors, increase the level of hyaluronic acid secretion, improve skin physiological activity, maintain the normal physiological state of skin tissue, and provide new ideas for skin repair and maintenance.

[0005] CN108715600A discloses an oligopeptide for promoting the proliferation and migration of intestinal mucosal epithelial cells, and a preparation method and application thereof. The peptide component and oligopeptide prepared by the invention have a significant promoting effect on the migration of intestinal mucosal epithelial cells; and are selective for the proliferation of intestinal mucosal epithelial cells, and have no obvious promoting effect on normal cultured cells, but show a significant promoting effect under the culture conditions of bacterial lipopolysaccharide.

[0006] CN106880871A discloses a collagen dermal material for promoting endometrial repair and a preparation method thereof. The present invention provides a collagen dermal material for promoting endometrial repair and a preparation method thereof, comprising the following steps: soaking collagen in a polypeptide solution to obtain a collagen material; the polypeptide comprises a collagen binding domain, a MSCs aggregation promoting domain and a connecting peptide located between the two, and the introduced polypeptide modification can increase the binding, aggregation and differentiation of MSCs, and play an effective barrier role against intrauterine adhesion.

[0007] The polypeptides or repair materials of the above inventions still have the problem of being unable to quickly recruit endogenous epithelial cells at the site of injury and having a limited number of adhesions. Therefore, it is urgent to provide a functional polypeptide with collagen-specific binding ability and binding to epithelial cells, as well as a functionalization method with collagen materials, and an application in recruiting a variety of epithelial cells. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention aims to provide a polypeptide that promotes epithelial cell adhesion, a functional collagen material, and a preparation method and application thereof. The polypeptide is a functional polypeptide that has both collagen-specific binding ability and epithelial cell binding, as well as a functionalization method with collagen material, and an application for recruiting multiple epithelial cells.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a polypeptide for promoting epithelial cell adhesion, wherein the polypeptide for promoting epithelial cell adhesion comprises a collagen material binding unit, a connecting unit and an epithelial cell binding unit which are sequentially connected.

[0011] The polypeptide promoting epithelial cell adhesion of the present invention can quickly recruit endogenous epithelial cells at the site of injury and increase their adhesion number, which can not only replenish the functional cells in the damaged area, but also increase the secretion of various growth factors and signal molecules, etc., provide a favorable microenvironment for damage repair, thereby enhancing the effect of tissue and organ damage repair.

[0012] Preferably, the amino acid sequence of the collagen material binding unit is selected from any one of TKKTLRT, WYRGRL, WREPSFCALS, KLWVLPK or LRELHLNNN, preferably TKKTLRT.

[0013] Preferably, the amino acid sequence of the linking unit is selected from any one of GSAGA, GSAGSAAGSGG or GGGGS, preferably GSAGA.

[0014] Preferably, the amino acid sequence of the epithelial cell binding unit is selected from any one of KHPAGGHPVIWS, SNFYMPL, STGLPAGGVITIGAGA or VAPGGHPVLL, preferably KHPAGGHPVIWS and / or SNFYMPL.

[0015] In a second aspect, the present invention provides a method for preparing a polypeptide for promoting epithelial cell adhesion as described in the first aspect, the preparation method comprising the following steps:

[0016] The collagen material binding unit and the epithelial cell binding unit are respectively assembled on both sides of the connecting unit through solid phase synthesis to obtain the polypeptide promoting epithelial cell adhesion.

[0017] In a third aspect, the present invention provides a functional collagen material, comprising a collagen material and the polypeptide promoting epithelial cell adhesion; wherein the collagen material binding unit in the polypeptide promoting epithelial cell adhesion recognizes and connects with the collagen material.

[0018] The present invention also provides a method for preparing the aforementioned polypeptide functionalized collagen material, which comprises: incubating the aforementioned functionally linked polypeptide with the collagen material, allowing the functional peptide sequence to specifically bind to the collagen material, and forming the polypeptide functionalized collagen material. The aforementioned polypeptide functionalized collagen material can be used as a tissue engineering scaffold and can recruit epithelial cells (of various types).

[0019] Preferably, the collagen material is a collagen membrane.

[0020] In the present invention, the collagen membrane is any commercially available collagen membrane or a collagen membrane prepared by an existing method, and can be connected with the collagen material binding unit in the polypeptide promoting epithelial cell adhesion.

[0021] Preferably, the thickness of the collagen film is 0.01-3 mm, for example, it can be 0.01 mm, 0.1 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, etc., and the standard collagen content is 20-99.5%, for example, it can be 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99.5%, etc.

[0022] Preferably, the tensile strength of the collagen membrane is 0.001-20 MPa, for example, it can be 0.001 MPa, 1 MPa, 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 12 MPa, 14 MPa, 16 MPa, 18 MPa, 20 MPa, etc., and the suture tearing force is 0.5-20 N, for example, it can be 0.5 N, 2 N, 4 N, 6 N, 8 N, 10 N, 12 N, 14 N, 16 N, 18 N, 20 N, etc.

[0023] In a fourth aspect, the present invention provides a method for preparing the functional collagen material as described in the third aspect, wherein the method for preparing the functional collagen material comprises the following steps:

[0024] The collagen material is immersed in a solution containing the polypeptide that promotes epithelial cell adhesion, and then incubated statically to obtain the functional collagen material.

[0025] Preferably, the collagen material needs to be washed with PBS buffer for 1-10 times before infiltration, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0026] Preferably, the soaking temperature is 30-40°C, for example, 30°C, 32°C, 34°C, 36°C, 37°C, 40°C, etc.

[0027] Preferably, the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the solution is (1×10 -4 -2×10 -2 )μmol:60mm 3 , for example, it can be 1×10 -4 μmol:60mm 3 , 5×10 -4 μmol:60mm 3 , 1×10 -3 μmol:60mm 3 , 5×10 -3 μmol:60mm 3 , 1×10 -2 μmol:60mm 3 , 2×10 -2 μmol:60mm 3 wait.

[0028] Preferably, the static incubation temperature is 30-40°C, for example, it can be 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc., and the static incubation time is 0.5-5h, for example, it can be 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, etc.

[0029] Preferably, the static incubation also includes post-treatment: after the collagen material connected to the polypeptide is taken out, it is washed with PBS buffer for 1-10 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc., each time for 1-10 minutes, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.

[0030] In a fifth aspect, the present invention provides a use of the polypeptide for promoting epithelial cell adhesion as described in the first aspect and the functional collagen material as described in the second aspect in the preparation of a functional product for repairing vaginal damage.

[0031] The functional product for repairing vaginal damage has the function of promoting epithelialization at the damaged site.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The polypeptide-modified functionalized collagen material provided by the present invention specifically anchors the functional linking polypeptide on the collagen scaffold material, prolongs the action time of the functional polypeptide on the collagen material, and can bind to the action of epithelial cells, thereby increasing the residence time and number of epithelial cells on the collagen material, which is beneficial to the rapid epithelialization in the repair of tissue and organ damage, provides an emerging platform for tissue engineering, and provides a new treatment method and approach for the repair of tissue and organ damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a fluorescence image of collagen material binding unit-connection unit-epithelial cell binding unit-fluorescent molecule (C-1).

[0035] Figure 2 This is a fluorescence image of the misfolded polypeptide-fluorescent molecule (C-s1) of the collagen material binding unit-epithelial cell binding unit.

[0036] Figure 3 This is a fluorescence image of the collagen material binding unit's hamartoxylin-linker unit-epithelial cell binding unit-fluorescent molecule (sC-1).

[0037] Figure 4 This is a bar graph comparing the binding analysis of PEP1 and its corresponding misfolded peptide to vaginal epithelial cells.

[0038] Figure 5 This is a bar graph comparing the binding analysis of PEP1 and its corresponding misfolded peptide to retinal epithelial cells.

[0039] Figure 6 Figure 4 Recruitment of vaginal epithelial cells to a functional collagen matrix in vitro.

[0040] Figure 7 These are the SEM and HE results of vaginal epithelial cells cultured in vitro using functional collagen materials.

[0041] Figure 8 The results of immunostaining for the expression of cytokeratin AE1 / AE3 in vaginal epithelial cells cultured on functional collagen materials.

[0042] Fig. 9 This is a diagram showing the results of an animal experiment on repairing vaginal damage using the functional collagen membrane prepared in Application Example 1.

[0043] Fig.10 This is a diagram showing the results of an animal experiment on repairing vaginal damage using the functional collagen membrane prepared in Application Example 2. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] Example 1

[0047] This embodiment provides a polypeptide TKKTLRTGSAGAKHPAGGHPVIWS (named PEP1) for promoting epithelial cell adhesion. The polypeptide for promoting epithelial cell adhesion is prepared by the following preparation method:

[0048] (1) Put the Fmoc-protected GSAGA resin into a peptide solid phase synthesis tube, add an appropriate amount of DMF to swell for more than 4 hours; remove the DMF, use Fmoc deprotection agent to remove Fmoc, and shake for 15 minutes; remove the Fmoc deprotection agent, add DMF and DCM to wash the resin alternately for 3 times, take 10 mg of resin from the peptide solid phase synthesis tube into a test tube, add ninhydrin detection solution and heat to detect positive (the resin turns dark blue);

[0049] (2) According to the amino acid sequence of TKKTLRTGSAGAKHPAGGHPVIWS, the amino acids were selected and mixed with HBTU in a ratio of 1:1 and dissolved in the reaction solution, and then put into the peptide solid phase synthesis tube and stirred for reaction for 2 hours; a small amount of resin was taken from the peptide solid phase synthesis tube and placed in a test tube, and ninhydrin detection solution was added. The result was negative (no color change), which proved that the condensation reaction was successful, and the excess liquid was removed. DMF and DCM were added to wash the resin alternately for 4 times; the resin was then washed with methanol for 4 times, and the resin volume was concentrated to 1 / 3 of the original volume, and the resin was continued to be drained for 20 minutes to remove the methanol;

[0050] (3) Take out the synthesized peptide resin, cleave it in the cleavage solution at room temperature for 4 hours, filter the resin and evaporate it to dryness in a rotary evaporator, precipitate the polypeptide with anhydrous ether (ice bath), and remove the anhydrous ether by centrifugation; wash the polypeptide with ether several times, place it in a vacuum drying oven to dry, and obtain a crude peptide product, which is purified by preparative reverse-phase HPLC. The pure peptide is >90% pure. After the pure peptide is correctly identified by mass spectrometry, it is freeze-dried and stored at -20°C for use.

[0051] Example 2

[0052] This example provides a polypeptide TKKTLRTGSAGASNFYMPL that promotes epithelial cell adhesion, and the preparation method is the same as that of Example 1.

[0053] Example 3

[0054] This example provides a polypeptide TKKTLRTGGGGSKHPAGGHPVIWS that promotes epithelial cell adhesion. The preparation method is the same as that in Example 1.

[0055] Example 4

[0056] This example provides an epithelial cell adhesion polypeptide TKKTLRTGSAGSAAGSGGKHPAGGHPVIWS, and the preparation method is the same as that in Example 1.

[0057] Example 5

[0058] This example provides an epithelial cell adhesion polypeptide WREPSFCALSGSAGAKHPAGGHPVIWS, and the preparation method is the same as that in Example 1.

[0059] Example 6

[0060] This example provides an epithelial cell adhesion polypeptide WYRGRGSAGAKHPAGGHPVIWS, and the preparation method is the same as that in Example 1.

[0061] Example 7

[0062] This example provides an epithelial cell adhesion polypeptide KLWVLPKGSAGAKHPAGGHPVIWS, and the preparation method is the same as that in Example 1.

[0063] Example 8

[0064] This example provides an epithelial cell adhesion polypeptide LRELHLNNNGSAGAKHPAGGHPVIWS, and the preparation method is the same as that in Example 1.

[0065] Example 9

[0066] This example provides an epithelial cell adhesion polypeptide TKKTLRTGSAGASTGLPAGGVITIGAGA, and the preparation method is the same as that of Example 1.

[0067] Example 10

[0068] This example provides an epithelial cell adhesion polypeptide TKKTLRTGSAGAVAPGGHPVLL, and the preparation method is the same as that of Example 1.

[0069] Application Example 1

[0070] This application example provides a functional collagen material, which is prepared by the following method:

[0071] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs) and wash it twice with PBS; prepare the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 1 with sterile deionized water;

[0072] (2) 20 μL of the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 1 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion in the solution to the amount of the collagen material in the infiltration solution was 1×10 -3 μmol:60mm 3 , 0.5×10 -3 μmol:60mm 3 , 5×10 -3 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0073] Application Example 2

[0074] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced by the polypeptide provided in example 2 (TKKTLRTGSAGASNFYMPL).

[0075] Application Example 3

[0076] This application example provides a functional collagen material, which is prepared by the following method:

[0077] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs), wash twice with PBS; prepare the TKKTLRTGSAGASNFYMPL polypeptide solution provided in Example 2 with sterile deionized water;

[0078] (2) 20 μL of the TKKTLRTGSAGASNFYMPL polypeptide solution provided in Example 2 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0079] Application Example 4

[0080] This application example provides a functional collagen material, which differs from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced with the polypeptide provided in example 3 (TKKTLRTGGGGSKHPAGGHPVIWS).

[0081] Application Example 5

[0082] This application example provides a functional collagen material, which is prepared by the following method:

[0083] (1) The collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) was cut into the same small size (5 mm diameter discs) and washed twice with PBS; the TKKTLRTGGGGSKHPAGGHPVIWS polypeptide solution provided in Example 3 was prepared with sterile deionized water;

[0084] (2) 20 μL of the TKKTLRTGGGGSKHPAGGHPVIWS polypeptide solution provided in Example 3 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0085] Application Example 6

[0086] This application example provides a functional collagen material, which differs from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced with the polypeptide provided in example 4 (TKKTLRTGSAGSAAGSGGKHPAGGHPVIWS).

[0087] Application Example 7

[0088] This application example provides a functional collagen material, which is prepared by the following method:

[0089] (1) The collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) was cut into the same small size (5 mm diameter discs) and washed twice with PBS; the TKKTLRTGSAGSAAGSGGKHPAGGHPVIWS polypeptide solution provided in Example 4 was prepared with sterile deionized water;

[0090] (2) 20 μL of the TKKTLRTGSAGSAAGSGGKHPAGGHPVIWS polypeptide solution provided in Example 4 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0091] Application Example 8

[0092] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced by the polypeptide (WREPSFCALSGSAGAKHPAGGHPVIWS) provided in example 5.

[0093] Application Example 9

[0094] This application example provides a functional collagen material, which is prepared by the following method:

[0095] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs), wash twice with PBS; prepare the WREPSFCALSGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 5 with sterile deionized water;

[0096] (2) 20 μL of the WREPSFCALSGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 5 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0097] Application Example 10

[0098] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced with the polypeptide (WYRGR GSAGAKHPAGGHPVIWS) provided in example 6.

[0099] Application Example 11

[0100] This application example provides a functional collagen material, which is prepared by the following method:

[0101] (1) A collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) was cut into the same small size (5 mm diameter discs) and washed twice with PBS; the WYRGRGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 6 was prepared with sterile deionized water;

[0102] (2) 20 μL of the WYRGR GSAGAKHPAGGHPVIWS polypeptide solution provided in Example 6 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3, infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0103] Application Example 12

[0104] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced by the polypeptide provided in example 7 (KLWVLPKGSAGAKHPAGGHPVIWS).

[0105] Application Example 13

[0106] This application example provides a functional collagen material, which is prepared by the following method:

[0107] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs) and wash it twice with PBS; prepare the KLWVLPKGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 7 with sterile deionized water;

[0108] (2) 20 μL of the KLWVLPKGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 7 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0109] Application Example 14

[0110] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced by the polypeptide provided in example 8 (LRELHLNNNGSAGAKHPAGGHPVIWS).

[0111] Application Example 15

[0112] This application example provides a functional collagen material, which is prepared by the following method:

[0113] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs), wash twice with PBS; prepare the LRELHLNNNGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 8 with sterile deionized water;

[0114] (2) 20 μL of the LRELHLNNNGSAGAKHPAGGHPVIWS polypeptide solution provided in Example 8 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0115] Application Example 16

[0116] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced with the polypeptide provided in example 9 (TKKTLRTGSAGASTGLPAGGVITIGAGA).

[0117] Application Example 17

[0118] This application example provides a functional collagen material, which is prepared by the following method:

[0119] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs), wash twice with PBS; prepare the TKKTLRTGSAGASTGLPAGGVITIGAGA polypeptide solution provided in Example 9 with sterile deionized water;

[0120] (2) 20 μL of the TKKTLRTGSAGASTGLPAGGVITIGAGA polypeptide solution provided in Example 9 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0121] Application Example 18

[0122] This application example provides a functional collagen material, which is different from application example 1 only in that the TKKTLRTGSAGAKHPAGGHPVIWS polypeptide provided in example 1 is replaced by the polypeptide provided in example 10 (TKKTLRTGSAGAVAPGGHPVLL).

[0123] Application Example 19

[0124] This application example provides a functional collagen material, which is prepared by the following method:

[0125] (1) Cut the collagen membrane (manufacturer: Cook Medical Incorporated, model: C-SLH-4S-4X6) into the same small size (5 mm diameter discs), wash twice with PBS; prepare the TKKTLRTGSAGAVAPGGHPVLL polypeptide solution provided in Example 10 with sterile deionized water;

[0126] (2) 20 μL of the TKKTLRTGSAGAVAPGGHPVLL polypeptide solution provided in Example 10 was added to the cut collagen membrane, so that the ratio of the amount of the polypeptide promoting epithelial cell adhesion to the amount of the collagen material in the infiltration solution was 2×10 -2 μmol:60mm 3 , infiltrate the collagen membrane, and place it at 37°C in the dark for 2 h. Remove the solution, take out the collagen membrane, and wash it 5 times with PBS, 5 min each time.

[0127] Comparative application example 1

[0128] This comparative application example provides a functional collagen material, which is different from application example 1 only in that KHPAGGHPVIWS is directly connected to the collagen membrane via a coupling agent (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt).

[0129] Comparative Application Example 2

[0130] This comparative application example provides a functional collagen material, which differs from application example 1 only in that the polypeptide does not contain an epithelial cell binding unit and only connects TKKTLRTGSAGA to the collagen membrane.

[0131] Test Example 1

[0132] Test sample: the polypeptide for promoting epithelial cell adhesion provided in Examples 1-10;

[0133] Test items: The binding ability of peptides to collagen;

[0134] Test method: Cut the collagen membrane into the same small size (5mm diameter discs) and wash it with PBS 1-3 times. Prepare peptide solutions of different concentrations using sterile deionized water. Take 20 microliters of fluorescently labeled peptides and fluorescently labeled misconstructed peptides of different solubility gradients, add them to the pretreated collagen membrane, infiltrate the collagen membrane, stand it at 37℃ in the dark for 2 hours, remove the solution, wash it with PBS 5 times, 5 minutes each time, and observe the fluorescence;

[0135] The test results are shown in Table 1 below:

[0136] Table 1

[0137]

[0138] As shown in Table 1, the polypeptides for promoting epithelial cell adhesion provided by the present invention can better bind to collagen materials, among which the TKKTLRTGSAGAKHPAGGHPVIWS sequence has the strongest binding ability. Figure 1-3 As shown (taking the sequence provided in Example 1 as an example), collagen material binding unit-connection unit-epithelial cell binding unit-fluorescent molecule (C-1); collagen material binding unit-epithelial cell binding unit's misconstructed polypeptide-fluorescent molecule (C-s1); collagen material binding unit's misconstructed polypeptide-connection unit-epithelial cell binding unit-fluorescent molecule (sC-1). Figure 1-3 It can be seen that compared with (sC-1), (C-1) and (C-s1) are bound to the collagen membrane in larger quantities, indicating that the amino acids in the collagen material binding unit described in the present application have a stronger binding ability with collagen.

[0139] Test Example 2

[0140] Test sample: the polypeptide for promoting epithelial cell adhesion provided in Examples 1-10;

[0141] Test items: The ability of functional collagen materials to bind to vaginal epithelial cells;

[0142] Test method: Vaginal epithelial cells (VK2) or retinal epithelial cells (ARPE-19) were collected, washed with PBS, and resuspended with 2 mL PBS. Incubate with an antibody labeled with phycoerythrin fluorescent dye (PE-antibody) at 4°C in the dark for 30 minutes, wash once with PBS, add 1 mL of serum-free culture medium, and resuspend the cells. The treated cells were incubated with the polypeptides for promoting epithelial cell adhesion provided in Examples 1-9 and the functional collagen material prepared with the misshapen peptide provided in Example 1 (as a control group) at 4°C in the dark for 30 minutes with rotation; after washing with PBS 2-3 times, resuspend with 300 μL of 2% paraformaldehyde (PFA0) and perform flow cytometry analysis;

[0143] The test results are shown in Table 2 below:

[0144] Table 2

[0145]

[0146]

[0147] From the test data in Table 2, it can be seen that the number of epithelial cell adhesion-promoting polypeptides prepared by the present invention binding to vaginal epithelial cells (VK2) is 9 cells / mm 2 Above, the number of retinal epithelial cells (ARPE-19) bound is 8 / mm 2 In the above, the number of peptides promoting epithelial cell adhesion provided by the best embodiment 1-2 that bind to vaginal epithelial cells (VK2) is 180 cells / mm 2 Above, the number of retinal epithelial cells (ARPE-19) bound is 160 / mm 2 Among the above, TKKTLRTGSAGAKHPAGGHPVIWS had the strongest ability to promote epithelial cell adhesion.

[0148] in, Figure 4 It is a comparative bar graph of the binding analysis between PEP1 (the polypeptide provided in Example 1) and its corresponding misstructured peptide and vaginal epithelial cells; Figure 5 The following is a comparative bar graph of the binding analysis between PEP1 (the polypeptide provided in Example 1) and its corresponding mismodeled peptide and retinal epithelial cells. Figure 4-5 As shown, the positive rate of the polypeptide provided by the present invention is significantly higher than that of its misconstructed polypeptide, indicating that the polypeptide provided by the present invention can bind to epithelial cells, thereby increasing the residence time and number of epithelial cells on the collagen material.

[0149] Test Example 3

[0150] Test samples: the functional collagen material provided in Application Example 1-19 and the functional collagen material provided in Comparative Application Example 1-2;

[0151] Test items: The ability of functional collagen materials to enrich epithelial cells;

[0152] Test method: collect epithelial cells (VK2 cells, retinal epithelial cells), wash once with PBS, resuspend in 8mL PBS containing 40μLDil dye (1mg / mL), incubate at room temperature for 15min away from light, and wash 3 times with PBS. Put the functional collagen material provided in Application Example 1-19 and the functional collagen material provided in Comparative Application Example 1-2 with a concentration of 100μM fluorescent label into EP tubes, add (at a rate of 2×10 5After 2 h of shaking incubation at 4°C in the dark, some samples were washed with PBS, fixed with paraformaldehyde for 0.5 h, and stained with DAPI for 0.5 h. Other samples were cultured for a long time (3 days, 7 days), and then immunofluorescence, scanning electron microscopy and HE staining were used to analyze the epithelial cells on the surface of collagen materials.

[0153] The specific test results are shown in Table 3 below:

[0154] Table 3

[0155]

[0156]

[0157] From the test data in Table 3, we can see that Figure 6-7 As shown, the functionalized collagen membrane material provided by the present invention is enriched with more epithelial cells, and vaginal epithelial cells are cultured in vitro. The SEM and HE results show that the functional material group has the largest number of cells, and the number of cells migrating into the functional collagen matrix is ​​the largest, and the migration distance into the material is the longest.

[0158] In particular, epithelial cells in the functional collagen material group significantly expressed cytokeratin after 7 days of culture, indicating that the cells maintained their epithelial phenotype during the culture process. Figure 8 It can also be seen that functional materials promote cell proliferation and the number of cells increases significantly.

[0159] Test Example 4

[0160] Test sample: functional collagen material provided in Application Example 1-2;

[0161] Test method:

[0162] (1) Establishment of miniature pig vaginal injury model

[0163] Miniature pig vaginal injury model: The experimental animals were Bama miniature experimental pigs, female, 4 to 6 months old, sexually mature, non-pregnant and non-parous, weighing (25±5) kg. Routine fasting for 12 hours before surgery, basic anesthesia. After waiting for about 5 minutes, the piglet was placed on the operating table, the animal was placed in a supine position, the ear vein was punctured to establish an intravenous fluid line, spontaneous breathing was maintained, oxygen was given by mask, sufentanil 0.2ug / kg was given during the operation, propofol 2mg / kg was given intermittently, no ventilator was required during the operation, and routine fluid replacement was performed.

[0164] After successful anesthesia, a urinary catheter was inserted. The abdomen, perineum and vagina were disinfected with iodine. A longitudinal incision was made in the middle of the abdomen, about 10 cm long, and the abdomen was advanced layer by layer to reveal a bicornuate uterus. The anterior and posterior leaves of the broad ligament were opened with an ultrasonic knife, and the uterine bladder was separated downward close to the uterine body. The peritoneum of the uterine bladder was opened, the bladder was pushed down, and the ureter was pushed outward at the isthmus of the uterus. The bladder cervical ligament and the uterine rectal space were further separated to expose the full length of the vagina. The length of the vagina was removed to about 4 cm. After the functionalized collagen matrix biofilm was soaked in saline, it was sutured into a cylindrical shape, lined with a mold of appropriate size, and fitted with the surrounding tissues. The upper and lower ends were end-to-end anastomosed with the vaginal stump and the external cervical opening respectively. Keep the urethra unobstructed and close the abdomen layer by layer.

[0165] After the operation, 1.6 million units of penicillin were injected intramuscularly into the gluteus maximus on both sides. The urinary catheter was removed one day after the operation, and liquid food was given within one day after the operation. 3.2 million units of penicillin were injected intramuscularly every day for 3 consecutive days to prevent infection. One week after the operation, the abdominal incision sutures were removed. Female miniature pigs were sampled under general anesthesia 12 weeks after the operation. Each female miniature pig in the experiment underwent full-thickness vaginal sampling and was killed after the sampling was completed. After the sampling, the vaginal tissue was divided into blocks and marked for subsequent analysis.

[0166] (2) HE staining of reconstructed vagina:

[0167] Tissues were taken 12 weeks after surgery after immersion in 4% paraformaldehyde solution for 24 hours, embedded in paraffin and sliced ​​to a thickness of about 5 μm for histomorphological evaluation. Tissue sections were dewaxed according to conventional procedures and stained with hematoxylin and eosin (HE), and histomorphology was observed under a microscope.

[0168] (3) Result analysis:

[0169] Normal vaginal tissue has stratified squamous epithelium, under which is a loose submucosal layer rich in collagen fibers. The submucosal layer is a smooth muscle layer with an inner circular and an outer longitudinal shape. Fig. 9 As shown, the functional collagen matrix biomembrane prepared by Application Example 1 (the amount ratio of the peptide promoting epithelial cell adhesion to the collagen material in the solution is 1×10 -3 μmol:60mm 3 ): 12 weeks after surgery, the functional collagen matrix biofilm reconstructed vagina showed several layers of epithelial cells arranged in a scale-like manner, which was a stratified squamous epithelium; the reconstructed tissue mucosa was loose connective tissue with a large number of newly formed small arteries and small veins; regularly arranged fibers were seen in the submucosal layer, and a large number of arteries and veins were seen running between the fibers. Fig.10 As shown, the functional collagen membrane prepared by Application Example 2 (the amount ratio of the peptide promoting epithelial cell adhesion to the collagen material in the solution is 1×10 -3 μmol:60mm 3 ) The results were slightly inferior to those of the functional collagen membrane repair prepared in Application Example 1.

[0170] The applicant declares that the present invention illustrates the polypeptides and functional collagen materials for promoting epithelial cell adhesion and the preparation methods and applications thereof through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A polypeptide for promoting epithelial cell adhesion, characterized in that: The polypeptide promoting epithelial cell adhesion is a collagen material binding unit, a connecting unit and an epithelial cell binding unit connected in sequence; The amino acid sequence of the collagen material binding unit is TKKTLRT; The amino acid sequence of the linking unit is GSAGA; The amino acid sequence of the epithelial cell binding unit is KHPAGGHPVIWS.

2. A method for preparing a polypeptide for promoting epithelial cell adhesion according to claim 1, characterized in that: The preparation method comprises the following steps: The collagen material binding unit and the epithelial cell binding unit are respectively assembled on both sides of the connecting unit through solid phase synthesis to obtain the polypeptide promoting epithelial cell adhesion.

3. A functional collagen material, characterized in that: The functional collagen material comprises a collagen material and a polypeptide for promoting epithelial cell adhesion as claimed in claim 1; wherein the collagen material binding unit in the polypeptide for promoting epithelial cell adhesion recognizes and connects with the collagen material.

4. The functional collagen material according to claim 3, characterized in that: The collagen material is a collagen membrane.

5. The functional collagen material according to claim 4, characterized in that: The thickness of the collagen film is 0.01-3 mm, and the standard collagen content is 20-99.5%.

6. The functional collagen material according to claim 4, characterized in that: The tensile strength of the collagen membrane is 0.001-20MPa, and the tearing force of the suture line is 0.5-20N.

7. A method for preparing a functional collagen material according to any one of claims 3 to 6, characterized in that: The preparation method of the functional collagen material comprises the following steps: The collagen material is immersed in a solution containing the polypeptide that promotes epithelial cell adhesion, and then incubated statically to obtain the functional collagen material.

8. The method for preparing the functional collagen material according to claim 7, characterized in that: The collagen material needs to be washed 1-10 times with PBS buffer before infiltration.

9. The method for preparing the functional collagen material according to claim 7, characterized in that: The soaking temperature is 30-40°C.

10. The method for preparing the functional collagen material according to claim 7, characterized in that: The ratio of the amount of the peptide promoting epithelial cell adhesion to the amount of the collagen material in the solution is (1×10 -4 -2×10 -2 )μmol:60mm 3 .

11. The method for preparing the functional collagen material according to claim 7, characterized in that: The temperature of the static incubation is 30-40° C., and the time of the static incubation is 0.5-5 h.

12. The method for preparing the functional collagen material according to claim 7, characterized in that: The static incubation further includes post-treatment: after the collagen material connected with the polypeptide is taken out, it is washed with PBS buffer for 1-10 times, each time for 1-10 minutes.

13. Use of the polypeptide for promoting epithelial cell adhesion according to claim 1 and the functional collagen material according to any one of claims 3 to 6 in preparing a functional product for repairing vaginal damage or repairing retinal damage.

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