Preparation and application of collagen gene modified intestinal stem cell extract

Abstract

The invention belongs to the field of biological medicines, and particularly discloses preparation and application of an intestinal stem cell extract modified by collagen genes. The applicant finds that stem cell supernate derived from intestinal tracts has special penetrability to skin and can deliver allogenic substances into the skin, absorption of biomacromolecules is improved, and the intestinal stem cell supernate can be used for preparing a collagen-derived stem cell extract. Therefore, the intestinal stem cells are separated from the intestinal tract, and the collagen gene is adopted to modify the intestinal stem cells, so that the intestinal stem cells generate natural collagen and a purified extract from the intestinal stem cells, the extract is applied to preparation of the skin repairing material and product application, and under the action of the skin repairing material, the skin repairing effect is improved. The biological repair of skin cells of the organism is promoted, and good whitening, wrinkle removing and oxidation resisting effects are achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically discloses the preparation and application of a collagen gene-modified intestinal stem cell extract. Background Art

[0002] Skin aging is mainly caused by natural physiological aging, abuse of cosmetics, diseases, etc., which lead to increased wrinkles, sagging, and decreased elasticity of the skin. At present, the prevention and treatment of skin aging mainly use topical antioxidant cosmetics to delay skin aging. However, most topical antioxidant products are chemical antioxidants. Frequent and excessive use will not only cause irreversible damage to the skin, but also have potential pathological effects on the body. Existing studies have shown that stem cell-derived extracts can be taken up by skin fibroblasts, promote their migration and proliferation; and by promoting the merging of type I and type III collagen, it can increase the gene expression of neural-cadherin, proliferating cell nuclear antigen, and cyclin D1 to accelerate skin repair and regeneration. The intestinal and skin barriers have many surprising common features. The intestine and skin are highly similar to each other in terms of use and function. Both organs are highly innervated and vascularized because they are essential for immune and neuroendocrine functions. Collagen is one of the most important components of the skin. It gives the skin elasticity, firmness and luster. Mung bean peptides are rich in trypsin inhibitors and are often added to cosmetics, such as creams, essences, masks, etc., to improve skin conditions. Tilapia skin peptides have the ability to enhance the activity of fibroblasts, promote the proliferation of fibroblasts, and supplement the content of elastin. Hyaluronic acid is a mucopolysaccharide that can promote the increase of epidermal cells and delay the aging of the skin. It generally has the effect of moisturizing and nourishing the skin. However, with the increase of age and the influence of the external environment, the collagen in the skin is gradually lost, resulting in wrinkles, sagging and other problems. In order to solve these problems, recombinant collagen came into being and has been widely used in the field of skin. However, since collagen is a biological macromolecule, it is very limited whether it is eaten or absorbed by application, and the repair and improvement of the skin is limited. The compliance of injected collagen is poor and it is easy to induce an immune response. The improvement and risk of the skin are disproportionate, so how to find a way to supplement collagen is crucial. Summary of the invention

[0003] In order to solve the above problems, the present invention discloses the preparation and application of a collagen gene-modified intestinal stem cell extract, which can promote the biological repair of the body's own skin cells and has good whitening, wrinkle removal and anti-oxidation effects.

[0004] The present invention includes the following technical solutions:

[0005] A method for preparing a collagen gene-modified intestinal stem cell extract, characterized in that it includes the following steps: transfecting a plasmid containing collagen into intestinal stem cells that simultaneously carry CD90+CD34+CD105+ and CD45-CD326-CD31-CD146- markers to make them express collagen; collecting the culture supernatant, and obtaining the intestinal-derived stem cell extract by high-speed centrifugation to obtain the precipitate.

[0006] Furthermore, in the method for preparing the above-mentioned collagen gene-modified intestinal stem cell extract, the collagen is type I collagen, and its amino acid sequence is shown in SEQ ID No.2.

[0007] Furthermore, the present invention discloses a collagen gene-modified intestinal stem cell extract, which is prepared by the above method.

[0008] Furthermore, the present invention discloses the use of the intestinal stem cell extract in preparing a material for repairing skin.

[0009] Furthermore, the present invention discloses a skin repair material, characterized in that the active ingredient of the material contains the intestinal stem cell extract as described in claim 2.

[0010] Furthermore, in the above-mentioned skin repair material, the content of the intestinal stem cell extract is 0.1-0.5wt%.

[0011] Furthermore, in the above-mentioned skin repair material, the active ingredients of the material also include mung bean peptide, tilapia polypeptide and hyaluronic acid.

[0012] Furthermore, in the above-mentioned skin repair material, the material is an injection.

[0013] Furthermore, the present invention discloses a method for preparing the above-mentioned skin repair material, comprising the following steps: dissolving intestinal stem cell extract, mung bean peptide, tilapia polypeptide and hyaluronic acid in a physiological saline solution and mixing them.

[0014] Furthermore, in the above preparation process, the intestinal stem cell extract is in the form of freeze-dried powder.

[0015] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0016] The present invention discloses the preparation and application of a collagen gene-modified intestinal stem cell extract. The applicant has found that the intestinal stem cell supernatant has special permeability to the skin, can deliver exogenous substances into the skin, and improve the absorption of biomacromolecules. The intestinal stem cell-derived extract can be taken up by fibroblasts, promoting their migration and proliferation, and promoting the biological repair of the body's skin cells. It has good whitening, wrinkle removal and antioxidant effects, and the intestinal stem cell extract used has high biocompatibility and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Identification of intestinal stem cells and intestinal stem cell supernatants containing COL1A1 collagen. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0019] Example 1

[0020] Preparation of intestinal stem cell supernatant containing collagen.

[0021] The donor's intestinal tissue was washed and then added with two volumes of collagenase and one volume of double antibody. It was first digested at 37°C for 30 minutes, and then culture medium was added to terminate the digestion. Finally, after centrifugation and magnetic bead sorting of cells with CD90+CD34+CD105+ and CD45-CD326-CD31-CD146- markers, intestinal stem cells were obtained, and the obtained cells were subcultured and frozen.

[0022] Synthesis and Construction of Collagen I Expression Plasmid

[0023] The nucleic acid and amino acid sequences of Collagen I were obtained from the UniProt database (as shown in the following table, the restriction sites are as follows), and the Collagen alpha-1 (abbreviated as COL1A1) molecules were synthesized by the whole gene synthesis method. Then, the obtained Collagen I nucleic acid sequences were constructed into the pCDNA3.1 molecules by the double restriction method. The transfection-positive bacteria were expanded and cultured, and the pCDNA3.1-COL1A1 plasmid was extracted using an endotoxin-free plasmid extraction kit. The obtained plasmid was sent for sequencing to confirm that the sequence was correct.

[0024] Nucleotide sequence:

[0025] As shown in SEQ ID No.1:

[0026]

[0027]

[0028]

[0029] The amino acid sequence is as shown in SEQ ID No.2:

[0030] MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDV

[0031] WKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTD

[0032] QETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGG

[0033] NFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGE

[0034] PGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLP

[0035] GMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRP

[0036] GAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSE

[0037] GPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGAR

[0038] GPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEG

[0039] KRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGP

[0040] KGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPPGAGQDGRPGPPGPP

[0041] GARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGP

[0042] PGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPS

[0043] GARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAP

[0044] GLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPA

[0045] GAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAK

[0046] GEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPG

[0047] AAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGP

[0048] AGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEP

[0049] GKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKG

[0050] DRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARG

[0051] PAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGP

[0052] AGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPG

[0053] PPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQI

[0054] ENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNM

[0055] ETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSD

[0056] PADVAIQLTFLLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEI

[0057] EIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGA

[0058] PDQEFGFDVGPVCFL

[0059] The pCDNA3.1-COL1A1 plasmid was transfected into intestinal stem cells by electroporation to make them express collagen naturally. The culture supernatant of stem cell culture was collected, and then the obtained culture medium was centrifuged at 300×g for 10 min to obtain the supernatant, 10000×g for 30 min to obtain the supernatant, and 120000×g for 90 min to obtain the precipitate to obtain the intestinal stem cell-derived extract. The obtained intestinal stem cell-derived extract containing COL1A1 was freeze-dried and stored at 4°C for use.

[0060] Specific antibodies against COL1A1 were used to perform immunofluorescence staining on intestinal stem cells that were not transfected with pCDNA3.1-COL1A1 plasmid (control group) and pCDNA3.1-COL1A1 plasmid, and western blot hybridization was performed on the supernatant of intestinal stem cells transfected with pCDNA3.1-COL1A1 plasmid. The results showed that the pCDNA3.1-COL1A1 plasmid was stably expressed in intestinal stem cells, and there was abundant COL1A1 collagen in the supernatant derived from intestinal stem cells. The specific markers of intestinal stem cells, CD90, CD34, and CD105, were all positive, and COL1A1 collagen was specifically expressed.

[0061] Example 2

[0062] Preparation of skin repair material containing collagen-modified stem cell extract.

[0063] The total volume of a single portion of skin repair material preparation is 140 ml, and skin repair materials with different formulas are prepared separately.

[0064] 1) Prepare 127 mL of physiological saline solution, 4 mL of mung bean peptide, 5 mL of tilapia polypeptide, and 4 mL of hyaluronic acid in every 140 mL of skin repair material.

[0065] 2) Prepare 127 mL of physiological saline solution, 0.3 wt% intestinal stem cell extract freeze-dried powder, 4 mL of mung bean peptide, 5 mL of tilapia polypeptide, and 4 mL of hyaluronic acid in every 140 mL of skin repair material (all the above raw materials were purchased from Guangzhou Taino Biotechnology Co., Ltd.).

[0066] 3) Prepare 132 mL of physiological saline solution, 0.3 wt% intestinal stem cell extract freeze-dried powder, 2 mL of mung bean peptide, and 2 mL of tilapia peptide in every 140 mL of skin repair material.

[0067] 4mL hyaluronic acid.

[0068] 4) 129 mL of physiological saline solution, 0.3 wt% intestinal stem cell extract freeze-dried powder, 4 mL of mung bean peptide, and 5 mL of tilapia peptide were prepared in every 140 mL of skin repair material.

[0069] 2mL hyaluronic acid.

[0070] Example 3

[0071] Clinical testing and inspection of skin repair materials

[0072] Neck skin wrinkle improvement test: 40 volunteers with neck wrinkle grade three were selected as test subjects, and the prepared skin repair material of intestinal stem cell extract was injected into the neck skin tissue. Through gentle massage, the skin repair material was squeezed and spread to the surrounding tissue. Each group of 10 volunteers was injected with skin repair materials of groups I to IV, and the skin condition of the injection site was observed 30 days after injection.

[0073] Group I skin repair material composition: composed of 127 mL of normal saline solution, 4 mL of mung bean peptide, 5 mL of tilapia polypeptide, and 4 mL of hyaluronic acid.

[0074] Group II skin repair material composition: composed of 127 mL of physiological saline solution, 0.1 wt% intestinal stem cell extract freeze-dried powder, 4 mL of mung bean peptide, 5 mL of tilapia polypeptide, and 4 mL of hyaluronic acid.

[0075] Group III skin repair material composition: 127 mL of physiological saline solution, 0.2 wt% intestinal stem cell extract freeze-dried powder, 4 mL of mung bean peptide, 5 mL of tilapia polypeptide, and 4 mL of hyaluronic acid.

[0076] The skin repair material composition of group IV is: 127 mL of physiological saline solution, 0.3 wt% intestinal stem cell extract freeze-dried powder, 4 mL of mung bean peptide, 5 mL of tilapia polypeptide, and 4 mL of hyaluronic acid.

[0077] Neck wrinkle classification: Grade 3

[0078] Neck wrinkles are classified into mild, moderate and severe grades.

[0079] Level 1: Faint transverse wrinkles appear.

[0080] Grade 2: shallow transverse wrinkles that are easily improved.

[0081] Level 3: A deep, thin depression appears on the neck.

[0082] Table 1 Record of neck skin condition after injection of different skin repair material formulas

[0083]

[0084] As can be seen from Table 1, after the skin repair material of this formula was injected into the neck, the neck wrinkles were improved, and with the increase in the amount of intestinal stem cell extract, the improvement effect on neck wrinkles became more obvious, indicating that the use of intestinal stem cell extract can promote the repair and regeneration of the body's own neck skin cells, and has good whitening, wrinkle removal and antioxidant effects.

[0085] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a collagen gene-modified intestinal stem cell extract, characterized in that: The following steps are involved: A plasmid containing collagen is transfected into intestinal stem cells carrying both CD90+CD34+CD105+ and CD45-CD326-CD31-CD146- markers to make them express collagen; the culture supernatant is collected, and the precipitate is taken out by high-speed centrifugation to obtain the intestinal stem cell extract.

2. The method for preparing a collagen gene-modified intestinal stem cell extract according to claim 1, characterized in that: The collagen is type I collagen, and its amino acid sequence is shown in SEQ ID No.

2.

3. A collagen gene-modified intestinal stem cell extract, characterized in that: Prepared by the method described in claim 1.

4. Use of the intestinal stem cell extract according to claim 3 in preparing a material for repairing skin.

5. A skin repair material, characterized in that: The active ingredients of the material contain the intestinal stem cell extract as claimed in claim 3.

6. A skin repair material according to claim 5, characterized in that: The content of the intestinal stem cell extract is 0.1-0.5wt%.

7. A skin repair material according to claim 5, characterized in that: The active ingredients of the material also include mung bean peptide, tilapia polypeptide and hyaluronic acid.

8. A skin repair material according to claim 5, characterized in that: The material is an injection.

9. The method for preparing a skin repair material according to claim 8, characterized in that: The method comprises the following steps: dissolving intestinal stem cell extract, mung bean peptide, tilapia polypeptide and hyaluronic acid in physiological saline solution and mixing them.

10. The preparation method according to claim 9, characterized in that: The intestinal stem cell extract is in the form of freeze-dried powder.

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