阅读说明：本技术 可用于激活和修复皮肤干细胞功能的生物材料的制备方法 (Preparation method of biological material for activating and repairing skin stem cell function ) 是由 冷泠 于 2020-12-28 设计创作，主要内容包括：本发明提出一种可用于激活和修复皮肤干细胞功能的生物材料的制备方法,涉及生物医学工程领域,利用脱细胞方法获得人和猪的皮肤支架；通过蛋白裂解获取能激活干细胞功能的活性蛋白成分；利用串联质谱鉴定,获得人和猪的皮肤基质蛋白成份；然后获得人和猪的皮肤特异性表达的基质蛋白；比较其成分差异,找出人特有蛋白；功能分析获得能够支持人皮肤干细胞生长的基质蛋白成分；利用上述能够支持人皮肤干细胞生长的基质蛋白成分和猪的皮肤基质蛋白成分,制备可用于激活和修复皮肤干细胞功能的生物材料。(The invention provides a preparation method of a biological material for activating and repairing the functions of skin stem cells, which relates to the field of biomedical engineering and is used for obtaining skin scaffolds of human and pigs by a cell removal method; obtaining an active protein component capable of activating stem cell function by protein lysis; identifying by tandem mass spectrometry to obtain human and pig skin matrix protein components; then obtaining the matrix protein specifically expressed by the skin of the human and the pig; comparing the component difference to find out the human specific protein; functional analysis is carried out to obtain a matrix protein component capable of supporting the growth of human skin stem cells; the matrix protein component capable of supporting the growth of the human skin stem cells and the skin matrix protein component of the pig are utilized to prepare the biological material for activating and repairing the functions of the skin stem cells.)

1. A method for preparing a biomaterial useful for activating and repairing the function of skin stem cells, comprising the steps of:

1) obtaining human and pig skin scaffolds by a decellularization method;

2) carrying out protein lysis on human and pig skin scaffolds to obtain active protein components capable of activating stem cell functions;

3) identifying the active protein components by using tandem mass spectrometry to obtain skin matrix protein components of human and pig;

4) obtaining human and porcine skin-specific expressed matrix proteins based on the human and porcine skin matrix protein components;

5) comparing the composition difference of the matrix protein specifically expressed by the skin of the human and the pig to find out the specific protein of the human;

6) carrying out functional analysis on the specific human protein by using a bioinformatics tool to obtain a matrix protein component capable of supporting the growth of human skin stem cells;

7) the matrix protein component capable of supporting the growth of the human skin stem cells and the skin matrix protein component of the pig are utilized to prepare the biological material for activating and repairing the functions of the skin stem cells.

2. The method of claim 1, wherein the decellularization method in step 1) is: cutting and collecting skin tissues of human and pig by using an electric skin collector, and removing epidermis from the skin tissues to obtain a decellularized dermis sample; the decellularized dermal sample is washed and then decellularized using an enzyme.

3. The method according to claim 1 or 2, wherein after decellularization in step 1), the integrity of the skin scaffold is tested by quantifying the amount of collagen and the collagen content is quantified using a collagen detection kit, in particular its concentration is measured by measuring the absorbance of collagen; then determining GAG content by sulfated glycosaminoglycan method, and measuring the concentration of the indicator by measuring its absorbance; when the measured concentration reaches a predetermined level, the integrity of the obtained skin scaffold is determined.

4. The method of claim 1, wherein the protein cleavage in step 2) is performed by: the decellularized human and porcine skin scaffolds were homogenized and the active protein fraction was extracted with a protein extraction reagent.

5. The method according to claim 1, wherein in step 2), after extraction of the active protein fraction, centrifugation is carried out for 30 minutes, and the supernatant is transferred to a clean tube and stored at-80 ℃.

6. The method of claim 1, wherein the peptide fragments are extracted from the cleaved protein in step 2) and the peptide fragment mixture is analyzed using an Orbitrap Q-active mass spectrometer equipped with a simple nLC nano-flow liquid chromatography system.

7. The method of claim 1, wherein the active protein components of the human and pig identified by mass spectrometry are compared in step 3) using a matrinome database to obtain the skin matrix protein components of the human and pig.

8. The method of claim 1, wherein the David tool is used in step 4) to functionally enrich the human and porcine skin-specifically expressed matrix proteins to obtain human and porcine skin-specifically expressed matrix proteins comprising: for human skin substrates, enzymes, enzyme inhibitors, growth factors, insulin factors, transforming growth factors, antimicrobial, anti-angiogenic protein components; for porcine skin substrates, metal binding, enzyme inhibitors and basement membrane associated proteins.

9. The method of claim 1, wherein the human characteristic protein of step 5) comprises: COL4A6COL28A1, COL11A1, FMOD, LUM, OGN, OMD, PRG3, PRG2, ADIPOQ, FBLN2, VTN WISP2, SPARC, ELN, EFEMP2, CTHRC1, THBS3, COMP, HMCN1, CILP, MGP, LTBP4, THBS2, LTBP1, THBS4, SRPX2, IGFALS, VWAA 5A, FB 7, PXDN, MFDN 8, MXRA 8, THSP 8, VIRN 8, ECM 8, MATN 8, MMRN 8, SN3672, HMCN 8, LAMB 8, SERPINA 8, CTSG, HTSG, HTNFNF 8, SERPINF 8, SERPINA 8, SERPISSN 8, SERPIT 8, SERPISSN 8, SER3672, SERPISSN 8, SERPISSN 36363672, SER3636363672, SER36363672, SER3636363672, SERPISSN 8, SER3672, SERPISSN 363672, SER3672, SERPISSN 8, SER363672, SER3636363636363636363672, SERPISSN 8, SER3672, SERPISSN 3636363672, SER363672, SER3672, SER363672, SERPISSN 36363636363672, SER363636363636363636363636363672, SERPISSN 3636363636363672, SERPISSN 8, SER36.

10. The method of claim 1, wherein the matrix protein component capable of supporting human skin stem cell growth in step 6) comprises: TGFBI, ANXA2, COL7a1, VTN, FBN1, HSPG2, COL5a1, LAMC1, TNC, LAMB2, SERPINF1, NID1, NID2, COL4a1, VWA1, COL18a1, EFEMP2, LAMA4, LAMA3, MATN2, HMCN1, LAMA5, TIMP3, LAMB1, THBS2, COL17a1, COL28a1, THBS4, LAMA2, COL2a1, MMRN2, HMCN2, LAMB4, LAMC3, FRAS1, LOXL1, freem 2.

Technical Field

The invention relates to the field of biomedical engineering, in particular to a preparation method of a biological material for activating and repairing functions of skin stem cells.

Background

The skin is the largest organ and is resistant to a wide variety of mechanical, chemical and biological insults. The tough, flexible structure of the skin helps to adapt to external conditions and allows rapid repair in case of injury. In mammals, different skin cell populations form the following basic layers: epidermal, dermal, subcutaneous or subcutaneous fat. The connective tissue of the dermis is rich in dermal fibroblasts and endothelial cells, which produce collagen, elastin, soluble cytokines and most other types of extracellular matrix (ECM). The extracellular matrix is a complex three-dimensional microenvironment that provides the cells with a large number of chemical and biophysical signals necessary for cellular function. ECM proteins also play a key role in wound healing. For example, cytokines (ECM-related proteins) released by platelets, such as Platelet Derived Growth Factor (PDGF), transforming growth factor b (TGF-b), Vascular Endothelial Growth Factor (VEGF), etc., can activate specific signaling pathways, stimulate immune cell responses, angiogenesis, collagen synthesis, fibrin and vitamin C support the migration of macrophages, other leukocytes, and neutrophils. Inflammatory factors such as TNF, IL-6, IL-1, and beta FGF also promote angiogenesis. In summary, the composition and organization of ECM has both physical effects and the various effects required to maintain homeostasis, and these properties make ECM an excellent candidate for the manufacture of biomimetics. For example, ECM-based biomaterials can elicit a variety of favorable cellular responses, such as angiogenesis, stem cell recruitment, and modulation of innate immune responses, all of which promote functional tissue remodeling (see Sadtler, K.; Estrellas, K.; Allen, B.W.; Wolf, M.T.; Fan, H.; Tam, A.J.; Patel, C.H.; Luber, B.S.; Wang, H.; Wagner, K.R.; Powell, J.D.; Housseau, F.; pardol D.M.; Elisseff, J.H., development a pro-regenerative biological tissue repair requirements 2cells, 2016,352, 370). Although some ECM proteins have been well studied, the overall molecular composition and regulatory function of matrix proteins is unclear, making it impossible to evaluate the activity and effectiveness of acellular biomaterials.

In addition, in order to facilitate the clinical application of tissue engineering, biomimetic materials must accurately mimic the human microenvironment. Although some structural proteins are sufficient as scaffolds for cell growth in skin tissue engineering, they are also crucial for ensuring and maintaining the biological function of cells, and the more original components are retained in the scaffold, the more they can support the function of tissues and cells. Pigs are more similar in anatomy, physiology and genome to humans and may therefore be considered the preferred source of human tissue for clinical xenotransplantation (references Sun, G.; Wojakowski, W.; Lynch, M.; Barallberge-Barreiro, J.; Yin, X.; Mayr, U.; Baig.; Lu, R.; Fava, M.; Hayward, R.; Molenaar, C.; White, S.J.; Roleder, T.; Milewski, K.P.; Gasior, P.; Buszman, P.; Jahandiri M.; Shanahan, C.M.; Hill, J.; Mayr, M.; Extralu Matrix proteins, C.M.; J.; emission, III.; C.M.; J.; Mayr, Extralu. (III.) and III.; emission of research 183. RTM.; C.D. supplement, and 183. C.. However, species differences between humans, pigs and other animals may be related to their specific skin function. Therefore, species differences between humans and other animals should be taken into full consideration in experimental studies and clinical applications of the skin extracellular matrix.

Disclosure of Invention

Aiming at the problem of insufficient function of the acellular skin scaffold of the current tissue engineering product, the invention aims to provide a preparation method of a biological material for activating and repairing the function of skin stem cells, and the biological material for activating and repairing the function of the skin stem cells is prepared.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing a biomaterial useful for activating and repairing the function of skin stem cells, comprising the steps of:

1) obtaining human and pig skin scaffolds by a decellularization method;

2) carrying out protein lysis on human and pig skin scaffolds to obtain active protein components capable of activating stem cell functions;

3) identifying the active protein components by using tandem mass spectrometry to obtain skin matrix protein components of human and pig;

4) obtaining human and porcine skin-specific expressed matrix proteins based on the human and porcine skin matrix protein components;

5) comparing the composition difference of the matrix protein specifically expressed by the skin of the human and the pig to find out the specific protein of the human;

6) carrying out functional analysis on the specific human protein by using a bioinformatics tool to obtain a matrix protein component capable of supporting the growth of human skin stem cells;

7) the matrix protein component capable of supporting the growth of the human skin stem cells and the skin matrix protein component of the pig are utilized to prepare the biological material for activating and repairing the functions of the skin stem cells.

Further, the cell removing method in the step 1) comprises the following steps: cutting and collecting skin tissues of human and pig by using an electric skin collector, and removing epidermis from the skin tissues to obtain a decellularized dermis sample; the decellularized dermal sample is washed and then decellularized using an enzyme.

Further, after the decellularization in the step 1), the integrity of the skin scaffold is tested by quantifying the amount of collagen, and the collagen content is quantified using a collagen detection kit, specifically, the concentration of collagen is measured by measuring the absorbance thereof; then determining GAG content by sulfated glycosaminoglycan method, and measuring the concentration of the indicator by measuring its absorbance; when the measured concentration reaches a predetermined level, the integrity of the obtained skin scaffold is determined.

Further, the method for protein cleavage in step 2) is as follows: skin scaffolds from decellularized human and pig were homogenized and the active protein fraction was extracted with protein extraction reagent (Sigma-Aldrich).

Further, after extracting the active protein component in step 2), centrifugation was performed for 30 minutes, and the supernatant was transferred to a clean tube and stored at-80 ℃.

Further, peptide fragments were extracted from the cleaved proteins in step 2) and the peptide fragment mixture was analyzed using an Orbitrap Q-exact mass spectrometer (Thermo Fisher Scientific) equipped with a simple nLC nano-flow liquid chromatography system (Thermo Fisher Scientific).

Further, the active protein components of the human and the pig identified by mass spectrometry are compared by using a Matrisome database in the step 3) to obtain the skin matrix protein components of the human and the pig.

Further, the David tool is used for carrying out functional enrichment on the human and pig skin-specific expressed matrix proteins in the step 4), so as to obtain the human and pig skin-specific expressed matrix proteins, wherein the matrix proteins comprise: for human skin substrates, protein components such as enzymes, enzyme inhibitors, growth factors, insulin factors, transforming growth factors, antimicrobial agents, antiangiogenesis and the like are mostly used; for porcine skin substrates, metal binding, enzyme inhibitors and basement membrane associated proteins are common.

Further, the human characteristic proteins in step 5) include: COL4A6COL28A1, COL11A1, FMOD, LUM, OGN, OMD, PRG3, PRG2, ADIPOQ, FBLN2, VTN WISP2, SPARC, ELN, EFEMP2, CTHRC1, THBS3, COMP, HMCN1, CILP, MGP, LTBP4, THBS2, LTBP1, THBS4, SRPX2, IGFALS, VWAA 5A, FB 7, PXDN, MFDN 8, MXRA 8, THSP 8, VIRN 8, ECM 8, MATN 8, MMRN 8, SN3672, HMCN 8, LAMB 8, SERPINA 8, CTSG, HTSG, HTNFNF 8, SERPINF 8, SERPINA 8, SERPISSN 8, SERPIT 8, SERPISSN 8, SER3672, SERPISSN 8, SERPISSN 36363672, SER3636363672, SER36363672, SER3636363672, SERPISSN 8, SER3672, SERPISSN 363672, SER3672, SERPISSN 8, SER363672, SER3636363636363636363672, SERPISSN 8, SER3672, SERPISSN 3636363672, SER363672, SER3672, SER363672, SERPISSN 36363636363672, SER363636363636363636363636363672, SERPISSN 3636363636363672, SERPISSN 8, SER36.

Further, the matrix protein component capable of supporting the growth of human skin stem cells in the step 6) comprises: TGFBI, ANXA2, COL7a1, VTN, FBN1, HSPG2, COL5a1, LAMC1, TNC, LAMB2, SERPINF1, NID1, NID2, COL4a1, VWA1, COL18a1, EFEMP2, LAMA4, LAMA3, MATN2, HMCN1, LAMA5, TIMP3, LAMB1, THBS2, COL17a1, COL28a1, THBS4, LAMA2, COL2a1, MMRN2, HMCN2, LAMB4, LAMC3, FRAS1, LOXL1, freem 2.

Further, matrix protein components capable of supporting the growth of the human skin stem cells are compared according to a protein molecule list in the existing data for evaluating the functionality of the acellular skin scaffold, the number of overlapped protein components is compared, the more overlapped protein components, the stronger the functionality of the scaffold product is proved, and when the number of overlapped protein components reaches a certain threshold value, the matrix protein components capable of supporting the growth of the human skin stem cells are judged to be qualified.

The invention has the following advantages:

1. the invention provides a preparation method of a novel biological material, and the biological activity and functionality identification of the biological material prepared by the invention are carried out by utilizing the system biology, so that the active ingredients of the material are completely reserved, and the functional indexes of re-epidermization of skin, hair follicle stem cell activity, wound surface angiogenesis, chemotaxis, growth and proliferation of wound surface fibroblasts and the like can be promoted, thereby being more beneficial to wound surface healing and skin aging delaying.

2. The material prepared by the invention keeps the important microenvironment of the skin stem cells and can maintain the polarity and the fate determination capability of the skin stem cells.

3. The components and the spatial composition of the material prepared by the invention provide new standards for the function and application evaluation of other biological materials. In addition, the product can be prepared into powder, liquid, gel and other different shapes, all have effective functions, and the use is more flexible, even simpler, more convenient and painless.

Drawings

FIG. 1 is a schematic representation of the biomaterial produced according to the present invention in comparison to other biomaterials.

Detailed Description

In order to make the technical solution of the present invention more comprehensible, embodiments accompanied with figures are described in detail below.

The embodiment discloses a preparation method of a biological material for activating and repairing the functions of skin stem cells, which comprises the following steps:

step 1) obtaining skin scaffolds of human and pig by a cell-removing method;

step 2) carrying out protein lysis on human and pig skin scaffolds to obtain active protein components capable of activating stem cell functions;

step 3) identifying the protein active protein components by using a tandem mass spectrum to obtain skin matrix protein components of human and pigs;

step 4) obtaining the matrix protein specifically expressed by the skin of the human and the pig based on the skin matrix protein components of the human and the pig;

step 5) comparing the component difference of the matrix protein specifically expressed by the skins of the human and the pig to obtain the human specific protein;

step 6) carrying out function analysis on the specific human protein through a bioinformatics tool to obtain a functional matrix protein component capable of supporting the growth of the skin stem cells;

and 7) preparing the biological material for activating and repairing the functions of the skin stem cells by using the matrix protein component capable of supporting the growth of the human skin stem cells.

Further, step 1) cutting and collecting skin tissues of human and pig by using an electric skin collector; removing epidermis from skin tissue, and gently scraping the surface of the skin with the back of a scalpel; after cleaning the acellular dermal sample, carrying out acellular treatment by using enzyme; after decellularization, the integrity of the bioscaffold was tested by quantifying the amount of collagen and the GAGs collagen content was quantified using Sircol dye (soluble collagen assay, Sircol Biocolor), the concentration of which was measured by measuring its absorbance at 555 nm; GAG content was determined by the Blyscan method (Blyscan method, sulfated glycosaminoglycan method, the Sircol Biocolor method), the absorbance of the indicator was determined at 625nm, and the values were normalized to the standard sodium heparin value.

Further, the method for protein cleavage in step 2) is as follows: homogenizing skin scaffolds of decellularized human and pig, and extracting active protein components with protein extraction reagent (Sigma-Aldrich); after centrifugation for 30 minutes, the supernatant was transferred to a clean tube and stored at-80 ℃.

Further, step 2) the protein was cleaved, the peptide fragments extracted, and the peptide mixture was analyzed using an Orbitrap Q-exact mass spectrometer (Thermo Fisher Scientific) equipped with a simple nLC nano-flow liquid chromatography system (Thermo Fisher Scientific).

Further, the active protein components of the human and the pig identified by mass spectrometry are compared by using a Matrisome database in the step 3) to obtain the skin matrix protein components of the human and the pig.

Further, in the step 4), the David tool is used for carrying out function enrichment on the matrix proteins of the human and the pig, which are expressed in a different way, and the skin matrix of the human is mostly protein components such as enzyme, enzyme inhibitor, growth factor, insulin factor, transforming growth factor, antimicrobial, antiangiogenesis and the like; the skin matrix of pigs is mostly metal-binding, enzyme inhibitors and basement membrane-associated proteins.

Further, step 5) the human specific proteins are: COL4A6COL28A1, COL11A1, FMOD, LUM, OGN, OMD, PRG3, PRG2, ADIPOQ, FBLN2, VTN WISP2, SPARC, ELN, EFEMP2, CTHRC1, THBS3, COMP, HMCN1, CILP, MGP, LTBP4, THBS2, LTBP1, THBS4, SRPX2, IGFALS, VWAA 5A, FB 7, PXDN, MFDN 8, MXRA 8, THSP 8, VIRN 8, ECM 8, MATN 8, MMRN 8, SN3672, HMCN 8, LAMB 8, SERPINA 8, CTSG, HTSG, HTNFNF 8, SERPINF 8, SERPINA 8, SERPISSN 8, SERPIT 8, SERPISSN 8, SER3672, SERPISSN 8, SERPISSN 36363672, SER3636363672, SER36363672, SER3636363672, SERPISSN 8, SER3672, SERPISSN 363672, SER3672, SERPISSN 8, SER363672, SER3636363636363636363672, SERPISSN 8, SER3672, SERPISSN 3636363672, SER363672, SER3672, SER363672, SERPISSN 36363636363672, SER363636363636363636363636363672, SERPISSN 3636363636363672, SERPISSN 8, SER36.

Further, step 6) obtains a matrix protein component capable of supporting the growth of human skin stem cells: TGFBI, ANXA2, COL7a1, VTN, FBN1, HSPG2, COL5a1, LAMC1, TNC, LAMB2, SERPINF1, NID1, NID2, COL4a1, VWA1, COL18a1, EFEMP2, LAMA4, LAMA3, MATN2, HMCN1, LAMA5, TIMP3, LAMB1, THBS2, COL17a1, COL28a1, THBS4, LAMA2, COL2a1, MMRN2, HMCN2, LAMB4, LAMC3, FRAS1, LOXL1, freem 2.

Further, matrix protein components capable of supporting the growth of the human skin stem cells are compared according to a protein molecule list in the existing data for evaluating the functionality of the acellular skin scaffold, the number of overlapped protein components is compared, the more overlapped protein components, the stronger the functionality of the scaffold product is proved, and when the number of overlapped protein components reaches a certain threshold value, the matrix protein components capable of supporting the growth of the human skin stem cells are judged to be qualified.

FIG. 1 is a schematic representation of the biomaterial produced according to the present invention in comparison to other biomaterials. Compared with the prior art, the biological material prepared by the invention can promote the functions of the primary isolated skin epidermal stem cells, including enhancing the proliferation activity (PCNA) and the expression of the dryness marker (CK14) of the skin epidermal stem cells.

The above embodiments are only intended to illustrate the technical solution of the present invention, but not to limit it, and a person skilled in the art can modify the technical solution of the present invention or substitute it with an equivalent, and the protection scope of the present invention is subject to the claims.