CN112485434A - Method for obtaining therapeutic target for treating keloid scar on skin - Google Patents
Method for obtaining therapeutic target for treating keloid scar on skin Download PDFInfo
- Publication number
- CN112485434A CN112485434A CN202011218151.6A CN202011218151A CN112485434A CN 112485434 A CN112485434 A CN 112485434A CN 202011218151 A CN202011218151 A CN 202011218151A CN 112485434 A CN112485434 A CN 112485434A
- Authority
- CN
- China
- Prior art keywords
- keloid
- skin
- matrix
- proteins
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- Hematology (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Library & Information Science (AREA)
- Endocrinology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for obtaining a therapeutic target spot of keloid causing skin, which relates to the fields of biomedical engineering and proteomics, and firstly, a skin matrix bracket of human keloid is obtained; identifying the matrix protein component of the skin keloid skin by using tandem mass spectrometry; then comparing the keloid with the matrix protein component of normal skin, and screening the keloid diagnosis marker; then carrying out immunofluorescence staining to confirm a specific mechanism for forming the keloid; finally, the hormone receptor which is the potential therapeutic target of the keloid is screened, and the hormone receptor which is the therapeutic target of the keloid causing skin is found out through pathological staining and animal experiment verification. The method identifies a diagnosis marker by revealing a pathological mechanism of keloid formation, finds out a hormone receptor causing the keloid of the skin, and takes the hormone receptor as a treatment target.
Description
Technical Field
The invention relates to the fields of biomedical engineering and proteomics, in particular to a biomarker combination for diagnosing skin keloid and a new potential target spot for treating the skin keloid.
Background
The skin is composed of epidermis and dermis, which are the largest organs of the human body and play an important role in protecting the body from external interference. Following skin injury, the wound healing process begins rapidly, including inflammation, proliferation, and remodeling. While the ideal result for wound healing is scar-free healing, similar in color and texture to normal skin, the best result is usually a flat, pliable scar with a slight discoloration. However, Abnormal Wound Healing can lead to pathological scars such as hypertrophic and keloid scars (1, Tredget, E.E., Levi, B., and Donelan, M.B. (2014) Biology and Principles of Scar Management and Burn Reconstruction. Surg Clin North Am 94,793 815; 2, Xue, M., and Jackson, C.J. (2015) excellular Matrix reorgation During Healing and Its Impact on normal Scarring. Adv round Care (New Rolle) 4,119 and 136). Despite advances in wound care, rehabilitation, and management over the last several decades, pathological scars remain difficult to heal with surgical methods and drugs (van der Veer, w.m., Bloemen, m.c., Ulrich, m.m., Molema, g., van Zuijlen, p.p., middlelkoop, e., and Niessen, F.B. (2009) Potential cellular and molecular computers of hypertonic scan formation burns 35, 15-29).
Keloids are fibroproliferative tumors associated with excessive accumulation of extracellular matrix (ECM) components, especially collagen deposition in The dermis and subcutaneous tissue, in response to skin injury (Andrews, j.p., Marttala, J., Macarak, e., Rosenbloom, J., and utito, J. (2016) Keloids: The param of skin fibrosis-pathomechanism and treatment. matrix Biol 51, 37-46. Keloids most commonly occur in The anterior chest, shoulder, upper back and earlobe.
ECM is a complex composed of hundreds of proteins that form the scaffold for all multicellular organisms and provide a biologically active structure that fundamentally controls the behavior of cells through chemical and mechanical signals. It is generally believed that an imbalance between ECM degradation and deposition plays an important role in scarring during pathology (Sidgwick, g.p., and baoat, a. (2012) excellent matrix emulsions formulated in hypertonic and ketoid screening. j Eur ad dermal blood vessel 26, 141-. For example, the expression of collagen, fibronectin and laminin in pathologic scars is significantly increased, while hyaluronic acid and decorin are down-regulated. Transforming growth factor-beta (TGF-beta) and fibronectin outer region A (Fn-EDA) were shown to be involved in collagen deposition during keloid formation. However, the specific role of ECM in pathological scarring is only poorly understood, resulting in an understanding of the mechanism of keloid formation, which prevents the establishment of new diagnostic and therapeutic approaches.
Disclosure of Invention
The invention aims to provide a method for obtaining a therapeutic target for causing skin keloid, which identifies a diagnosis marker by disclosing a pathological mechanism of keloid formation, finds out a hormone receptor for causing skin keloid and takes the hormone receptor as the therapeutic target.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of obtaining a target for treatment of keloid scars on skin comprising the steps of:
1) obtaining a skin matrix scaffold of human keloid by using an acellular method;
2) extracting protein of the skin matrix scaffold, and identifying the matrix protein component of the skin keloid skin by using tandem mass spectrometry;
3) comparing the matrix protein components of the keloid with the normal skin, and screening the keloid diagnosis marker, namely the matrix protein for assisting the diagnosis of the early formation of the keloid;
4) performing immunofluorescence staining on matrix protein for assisting diagnosis of early keloid formation, and confirming a specific mechanism of keloid formation from potential mechanisms of keloid formation;
5) according to the confirmed specific mechanism of keloid formation, a hormone receptor serving as a potential treatment target of the keloid is screened, and the hormone receptor serving as the treatment target of the skin keloid is found out through pathological staining and animal experiment verification.
Further, the cell removing method in the step 1) comprises the following steps: cutting and collecting skin tissue by using an electric skin collector, and removing epidermis from the skin tissue; the decellularized dermal sample is washed and then decellularized using an enzyme.
Further, after the decellularization in the step 1), the integrity of the biological 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 resulting matrix scaffold is determined.
Further, the method for extracting the protein of the skin matrix scaffold in the step 2) comprises the following steps: the decellularized skin matrix scaffold was homogenized and the proteins were extracted with protein extraction reagent (Sigma-Aldrich).
Further, after the protein extraction in step 2), after centrifugation for 30 minutes, the supernatant was transferred to a clean tube and stored at-80 ℃.
Further, in the extraction of the protein of the skin matrix scaffold in step 2), the protein was cleaved, the peptide fragments were extracted, 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, comparing the matrix protein of the keloid identified by mass spectrometry by using a Matrisome database in the step 3) to obtain the matrix protein component of the keloid acellular scaffold; then, matrix proteins for aiding in the diagnosis of the early formation of keloid scars were found, compared with normal skin.
Further, matrix proteins useful for aiding in the diagnosis of early formation of keloids include COL11A, COL26A, COL2A, CILP, CRELD, FNDC, crispd, EDIL, SPON, MMRN, IGFBP, CILP, LTBP, TNFAIP, SPARCL, TNN, SLIT, ACAN, HAPLN, P4HA, SERPINB, MMP, CTSK, ELANE, TIMP, PLOD, SERPINB, MMP, CTSA, SERPINE, ADAM, CTSS, LOXL, BMP, GPC, COLEC, S100A, SFRP, WNT, tcht, TCHH, serrp, SFRP, TGFB, MST, cthrcc, THBS, px, THBS, TNC, SPARC, COMP, FBLN, plgf, 4HA, HTRA, qtc 1, qts 11, cxnf, 100A, and gpcl.
Further, the potential mechanisms of keloid formation in step 4) include: one or more of skin microenvironment hemostasis, inflammation, angiogenesis, ECM remodeling, enzyme activity, cell adhesion, skin development, cytoskeleton development, carbohydrate and the like of the scar are biological process disorder factors, one or more of inflammatory proteins, angiogenesis promoting proteins, enzymes and fibrosis related proteins are matrix proteins of the keloid, and one or more of elastin, enzyme inhibitors and basement membrane components are components which cause the keloid to lose antiangiogenesis.
Further, representative proteins SPARC, HTRA1, CLEC11A, S100a9, MFAP4 and OGN, potentially leading to scarring, were subjected to immunofluorescence staining in step 4).
Further, the hormone receptor of step 5) as a potential therapeutic target of keloid comprises one or more of prolactin receptor, gonadotropin releasing hormone receptor, oxytocin receptor, thyroxine receptor, insulin receptor and estrogen receptor.
The invention has the following advantages:
the invention firstly clarifies the pathological formation mechanism of the skin keloid, and discloses the biological process of the micro-environment disorder of the keloid and the specific function of ECM in the formation of the skin keloid.
2. The invention provides 65 biomarkers for clinical diagnosis of keloid, and provides a diagnosis method for early prevention of keloid.
3. The potential target for treating keloid discovered by the invention takes different hormone receptors as the treatment target, and can utilize the corresponding hormone receptor inhibitor to treat keloid, thereby providing a new direction for treating keloid.
Drawings
Fig. 1 is a flowchart of one example of obtaining a target for treatment of keloid induced skin.
Figure 2 is an exemplary graph of a representative protein of a cutaneous keloid biomarker.
Figure 3 is an exemplary diagram of the hormone receptors targeted for treatment of cutaneous keloids.
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 method for obtaining a target point for treating skin keloid, which comprises the following steps:
1) obtaining a skin matrix scaffold of human keloid by using an acellular method; the cell removing method comprises the following steps: cutting and collecting skin tissue by using an electric skin collector, and removing epidermis from the skin tissue; the decellularized dermal sample is washed and then decellularized using an enzyme. After decellularization, the integrity of the bioscaffold is tested by quantifying the amount of collagen, and the collagen content is quantified using a collagen detection kit, specifically 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 resulting matrix scaffold is determined.
2) The decellularized skin matrix scaffold was homogenized, the proteins of the skin matrix scaffold were extracted with protein extraction reagent (Sigma-Aldrich), the proteins were lysed to extract the peptides when the proteins of the skin matrix scaffold were extracted, 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). Identifying the matrix protein component of the skin keloid skin by using tandem mass spectrometry; the extracted protein can be centrifuged for 30 minutes, and the supernatant transferred to a clean tube and stored at-80 ℃ for the next use.
3) Comparing matrix protein components of the keloid with normal skin, screening a keloid diagnosis marker, namely matrix protein for assisting in diagnosing early formation of the keloid, and specifically, comparing the matrix protein for identifying the keloid by mass spectrometry by using a Matrisome database to obtain the matrix protein components of the keloid acellular scaffold; then, compared with normal skin, matrix protein for assisting diagnosis of early formation of keloid is found out; these matrix proteins include COL11A, COL26A, COL2A, CILP, CRELD, FNDC, CRISPLD, EDIL, SPON, MMRN, IGFBP, CILP, LTBP, TNFAIP, SPARCL, TNN, SLIT, ACAN, HAPLN, P4HA, SERPINB, MMP, CTSK, ELANE, TIMP, PLOD, SERPINB, MMP, CTSA, SERPINE, ADAM, CTSS, LOXL, BMP, GPC, COLEC, S100A, SFRP, WNT, TCH, FGF, SFRP, FSTL, TGFB, MST, CTHRC, THBS, SRPX, THBS, TNC, SPARC, COMP, FBLN, PLOD, P4, HTRA, C1 NF, CLEC11, S100A, ANTL, CXCL.
4) Matrix proteins used to aid in the diagnosis of early formation of keloid scars are immunofluorescent stained, preferably representative proteins SPARC, HTRA1, CLEC11A, S100a9, MFAP4 and OGN that potentially cause scarring. The specific mechanism of keloid formation is identified from potential matrix of keloid formation, which includes one or more of skin microenvironment hemostasis, inflammation, angiogenesis, ECM remodeling, enzyme activity, cell adhesion, skin development, cytoskeleton development, carbohydrates, etc. of keloid as a biological process dysregulation factor, one or more of inflammatory proteins, angiogenesis promoting proteins, enzymes, and fibrosis-related proteins as matrix proteins of keloid, and one or more of elastin, enzyme inhibitors, and basement membrane components as components that cause keloid loss of antiangiogenesis.
5) According to the confirmed specific mechanism of keloid formation, hormone receptors which are potential therapeutic targets of keloids are screened, wherein the hormone receptors comprise one or more of prolactin receptors, gonadotropin releasing hormone receptors, oxytocin receptors, thyroxine receptors, insulin receptors and estrogen receptors, and then the hormone receptors which are the therapeutic targets of keloids on skin are found out through pathological staining and animal experiments.
A specific example is listed below:
the example utilizes the method of the invention to find out the treatment target of keloid, the process is shown in figure 1, which specifically comprises the following steps: and carrying out decellularization treatment on the keloid skin and the healthy skin to obtain the extracellular matrix scaffold. And carrying out matrix omics identification on the two groups of scaffolds to obtain the protein expression difference components of the two groups of scaffolds. Functional analysis and interaction network analysis of these differential components using Bioinformatics tools and databases (DAVID Bioinformatics Resources 6.8) revealed proteins that were upregulated and downregulated in keloid scars, such as the upregulated protein SPARC and the downregulated protein MFAP4, which may be responsible for keloid formation. The estrogen receptor pathway was found to be a key pathway in the mechanism of scarring and therefore inhibition of the estrogen receptors era or ER β by inhibitors may have a therapeutic effect on keloids.
Fig. 2 is an example of representative proteins of skin keloid biomarkers, showing how keloid scars are highly expressed matrix proteins (SPARC, HTRA1, CLEC11A, S100a9) and lowly expressed matrix proteins (MFAP4, OGN, AMBP) relative to normal skin. Fig. 3 is a diagram showing an example of hormone receptors as target for keloid treatment, showing that estrogen nuclear receptors ER α and ER β enter the nucleus of cells in dermal tissue, and that the cells in the dermis of keloid group enter the nucleus more than normal skin, indicating that the estrogen pathway is activated more strongly.
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.
Claims (10)
1. A method for obtaining a target for treatment of keloid scars on skin comprising the steps of:
1) obtaining a skin matrix scaffold of human keloid by using an acellular method;
2) extracting protein of the skin matrix scaffold, and identifying the matrix protein component of the skin keloid skin by using tandem mass spectrometry;
3) comparing the matrix protein components of the keloid with the normal skin, and screening the keloid diagnosis marker, namely the matrix protein for assisting the diagnosis of the early formation of the keloid;
4) performing immunofluorescence staining on matrix protein for assisting diagnosis of early keloid formation, and confirming a specific mechanism of keloid formation from potential mechanisms of keloid formation;
5) according to the confirmed specific mechanism of keloid formation, a hormone receptor serving as a potential treatment target of the keloid is screened, and the hormone receptor serving as the treatment target of the skin keloid is found out through pathological staining and animal experiment verification.
2. The method of claim 1, wherein the decellularization method in step 1) is: cutting and collecting skin tissue by using an electric skin collector, and removing epidermis from the skin tissue; 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 bioscaffold 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 resulting matrix scaffold is determined.
4. The method of claim 1 or 2, wherein the protein of the skin matrix scaffold is extracted in step 2) by: homogenizing the skin matrix scaffold after cell removal, and extracting protein with a protein extraction reagent.
5. The method of claim 4, wherein in the step 2) the proteins of the skin matrix scaffold are extracted, the proteins are cleaved, the peptides are extracted, and the peptide mixture is analyzed using an Orbitrap Q-active mass spectrometer equipped with a simple nLC nanoflow liquid chromatography system.
6. The method of claim 1, wherein matrix proteins of the mass spectrometrically identified keloid are aligned in step 3) using a matristerome database to obtain matrix protein components of the keloid decellularized scaffold; then, matrix proteins for aiding in the diagnosis of the early formation of keloid scars were found, compared with normal skin.
7. The method of claim 6, wherein the matrix protein used to aid in the diagnosis of early formation of keloids comprises COL11A, COL26A, COL2A, CILP, CRELD, FNDC, crispd, EDIL, SPON, MMRN, IGFBP, CILP, LTBP, TNFAIP, SPARCL, TNN, SLIT, ACAN, HAPLN, P4HA, SERPINB, MMP, CTSK, ELANE, TIMP, PLOD, SERPINB, MMP, CTSA, serpin, ADAM, CTSS, LOXL, BMP, GPC, COLEC, S100A, SFRP, WNT, plot, wnh, SFRP, FSTL, TGFB, MST, cthrcc, THBS, SRPX, THBS, TNC, SPARC, COMP, fbtl, plna, plp 4HA, qtra, clnf 1, htnf, 11A, cxnf, 100A, FGF 100A.
8. The method of claim 1, wherein the potential mechanism of keloid formation in step 4) comprises: one or more of skin microenvironment hemostasis, inflammation, angiogenesis, ECM remodeling, enzyme activity, cell adhesion, skin development, cytoskeleton development, carbohydrate and the like of the scar are biological process disorder factors, one or more of inflammatory proteins, angiogenesis promoting proteins, enzymes and fibrosis related proteins are matrix proteins of the keloid, and one or more of elastin, enzyme inhibitors and basement membrane components are components which cause the keloid to lose antiangiogenesis.
9. The method of claim 1, wherein representative proteins SPARC, HTRA1, CLEC11A, S100a9, MFAP4 and OGN potentially leading to scarring are immunofluorescent stained in step 4).
10. The method of claim 1, wherein the hormone receptors of step 5) that are potential therapeutic targets for keloids include one or more of prolactin receptor, gonadotropin releasing hormone receptor, oxytocin receptor, thyroxine receptor, insulin receptor, and estrogen receptor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011218151.6A CN112485434A (en) | 2020-11-04 | 2020-11-04 | Method for obtaining therapeutic target for treating keloid scar on skin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011218151.6A CN112485434A (en) | 2020-11-04 | 2020-11-04 | Method for obtaining therapeutic target for treating keloid scar on skin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112485434A true CN112485434A (en) | 2021-03-12 |
Family
ID=74928066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011218151.6A Pending CN112485434A (en) | 2020-11-04 | 2020-11-04 | Method for obtaining therapeutic target for treating keloid scar on skin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112485434A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425844A (en) * | 2021-07-09 | 2021-09-24 | 中国医学科学院北京协和医院 | Drug intervention target of keloid and application thereof |
| WO2023284660A1 (en) * | 2021-07-13 | 2023-01-19 | 上海交通大学医学院附属第九人民医院 | Scar diagnostic reagent and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552875A (en) * | 2003-05-26 | 2004-12-08 | 北京大学第三医院 | Scar and hyperplastic scar differential expression gene library and its constructing method |
| US20180042866A1 (en) * | 2016-08-11 | 2018-02-15 | Board Of Regents, The University Of Texas System | N-(2-aminoethyl) ethanolamine (aeea) and analogs to treat hypertrophic scarring and liposomal topical delivery |
-
2020
- 2020-11-04 CN CN202011218151.6A patent/CN112485434A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552875A (en) * | 2003-05-26 | 2004-12-08 | 北京大学第三医院 | Scar and hyperplastic scar differential expression gene library and its constructing method |
| US20180042866A1 (en) * | 2016-08-11 | 2018-02-15 | Board Of Regents, The University Of Texas System | N-(2-aminoethyl) ethanolamine (aeea) and analogs to treat hypertrophic scarring and liposomal topical delivery |
Non-Patent Citations (7)
| Title |
|---|
| BINGHUI LIU等: "Matrisome Provides a Supportive Microenvironment for Skin Functions of Diverse Species", 《ACS BIOMATER. SCI. ENG.》 * |
| 彭颖等: "蛋白质组学在病理性瘢痕中的研究进展", 《实用医学杂志》 * |
| 李晖: "病理性瘢痕的蛋白质组学研究", 《延安大学学报(医学科学版)》 * |
| 胡志成等: "病理性瘢痕与正常皮肤的蛋白质组学研究", 《中国病理生理杂志》 * |
| 蔡文琴等: "《实用免疫细胞化学与核酸分子杂交技术》", 31 May 1994, 四川科学技术出版社 * |
| 陆信仰等: "《外科疾病诊断与手术操作 第2版 下》", 31 March 2019, 吉林科学技术出版社 * |
| 高山等: "《R语言与Bioconductor生物信息学应用》", 31 January 2014, 天津科技翻译出版有限公司 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425844A (en) * | 2021-07-09 | 2021-09-24 | 中国医学科学院北京协和医院 | Drug intervention target of keloid and application thereof |
| WO2023284660A1 (en) * | 2021-07-13 | 2023-01-19 | 上海交通大学医学院附属第九人民医院 | Scar diagnostic reagent and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pinchi et al. | miR‐1, miR‐499 and miR‐208 are sensitive markers to diagnose sudden death due to early acute myocardial infarction | |
| Villar et al. | Myocardial and circulating levels of microRNA-21 reflect left ventricular fibrosis in aortic stenosis patients | |
| CN112485434A (en) | Method for obtaining therapeutic target for treating keloid scar on skin | |
| CN111500710A (en) | Use of circRNA730 as biomarker for the prediction, diagnosis or prognosis of sepsis | |
| KR20150036111A (en) | Bag3 as biochemical serum and tissue marker | |
| WO2018220185A1 (en) | Novel circular rna biomarkers for heart failure | |
| CN111840514A (en) | Application of CILP2 in preparation of medicine for improving heart aging and myocardial hypertrophy | |
| KR102216913B1 (en) | Minimally invasive kit evaluating skin moisturization degree including microneedle patch and biomarker for evaluating skin moisturization degree | |
| KR102216937B1 (en) | Minimally invasive kit evaluating skin oil degree including microneedle patch and biomarker for evaluating skin oil degree | |
| Astrada et al. | Concurrent validity of biofilm detection by wound blotting on hard-to-heal wounds | |
| Nie et al. | Activated SOX9+ renal epithelial cells promote kidney repair through secreting factors | |
| CN109182459A (en) | A kind of dissection of aorta FISH detection probe and kit | |
| CA2986730C (en) | Srm/mrm assay for the cyclin-dependent kinase inhibitor 2a (p16) protein | |
| EP3233084B1 (en) | Gsk3b inhibitors in the treatment of hypopigmentation disorders | |
| KR102216910B1 (en) | Minimally invasive kit diagnosing skin wrinkle or skin elasticity including microneedle patch | |
| KR102216945B1 (en) | Minimally invasive kit evaluating sensitive skin degree with sensory irritation including microneedle patch and biomarker for evaluating sensitive skin degree with sensory irritation | |
| US20170052197A1 (en) | SRM/MRM Assay for the Androgen Receptor (AR) Protein | |
| JP2017521662A (en) | SRM / MRM assay for serine / threonine protein kinase B-raf (BRAF) | |
| JP2017521663A (en) | SRM / MRM assay for GTPase KRas protein (KRas) | |
| CN110241207A (en) | A kind of unstable angina biomarker and its application and kit | |
| US20230407403A1 (en) | Method for assisting determination on suitability of esophageal cancer for endoscopic therapy, method for determining suitability of esophageal cancer for endoscopic therapy, method for collecting data for use in determination on suitability of esophageal cancer for endoscopic therapy, method for diagnosing suitability of esophageal cancer for endoscopic therapy, in vitro method for assisting determination on suitability of esophageal cancer for endoscopic therapy, ... | |
| Limbu et al. | Dandruff lesional scalp skin exhibits epidermal T cell infiltration and a weakened hair follicle immune privilege | |
| EP4086358A1 (en) | Method for the prognosis of patients suffering from atrial fibrillation | |
| CN113862348B (en) | Application of Prelp as psoriasis diagnosis mark and treatment target | |
| KR101898907B1 (en) | Composition for diagnosing adenomyosis and diagnostic kit for adenomyosis containing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210312 |