CN111856030A - Virus receptor protein for diagnosing leucoderma diseases and application thereof - Google Patents
Virus receptor protein for diagnosing leucoderma diseases and application thereof Download PDFInfo
- Publication number
- CN111856030A CN111856030A CN202010673592.9A CN202010673592A CN111856030A CN 111856030 A CN111856030 A CN 111856030A CN 202010673592 A CN202010673592 A CN 202010673592A CN 111856030 A CN111856030 A CN 111856030A
- Authority
- CN
- China
- Prior art keywords
- hsv
- leucoderma
- receptor protein
- diagnosing
- human skin
- 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
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title abstract description 14
- 201000010099 disease Diseases 0.000 title abstract description 11
- 108010066342 Virus Receptors Proteins 0.000 title abstract description 6
- 102000018265 Virus Receptors Human genes 0.000 title abstract description 6
- 206010047642 Vitiligo Diseases 0.000 claims abstract description 27
- 108010057338 simplexvirus receptor Proteins 0.000 claims abstract description 23
- 210000004511 skin melanocyte Anatomy 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 239000003147 molecular marker Substances 0.000 claims abstract description 10
- 238000001262 western blot Methods 0.000 claims abstract description 10
- 238000009007 Diagnostic Kit Methods 0.000 claims abstract description 6
- 238000003745 diagnosis Methods 0.000 claims description 13
- 108060005251 Nectin Proteins 0.000 claims description 9
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 claims description 6
- 101001129851 Homo sapiens Paired immunoglobulin-like type 2 receptor alpha Proteins 0.000 claims description 4
- 102100031651 Paired immunoglobulin-like type 2 receptor alpha Human genes 0.000 claims description 4
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 102100023064 Nectin-1 Human genes 0.000 claims 1
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 claims 1
- 210000002752 melanocyte Anatomy 0.000 abstract description 28
- 241000700588 Human alphaherpesvirus 1 Species 0.000 abstract description 27
- 230000008099 melanin synthesis Effects 0.000 abstract description 15
- 238000001514 detection method Methods 0.000 abstract description 9
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000006866 deterioration Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 101000743545 Homo sapiens Vomeronasal type-1 receptor 5 Proteins 0.000 abstract description 2
- 102100038347 Vomeronasal type-1 receptor 5 Human genes 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000002372 labelling Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 30
- 101100129922 Caenorhabditis elegans pig-1 gene Proteins 0.000 description 13
- 101100520057 Drosophila melanogaster Pig1 gene Proteins 0.000 description 13
- 210000003491 skin Anatomy 0.000 description 9
- 102000002356 Nectin Human genes 0.000 description 8
- 241000700605 Viruses Species 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 102000012220 Member 14 Tumor Necrosis Factor Receptors Human genes 0.000 description 5
- 210000003780 hair follicle Anatomy 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 102000003425 Tyrosinase Human genes 0.000 description 4
- 108060008724 Tyrosinase Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 210000002510 keratinocyte Anatomy 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 3
- 101710163881 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 3
- 102000013760 Microphthalmia-Associated Transcription Factor Human genes 0.000 description 3
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 description 3
- 101710173693 Short transient receptor potential channel 1 Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- LVTKHGUGBGNBPL-UHFFFAOYSA-N Trp-P-1 Chemical compound N1C2=CC=CC=C2C2=C1C(C)=C(N)N=C2C LVTKHGUGBGNBPL-UHFFFAOYSA-N 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000036564 melanin content Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 239000012110 Alexa Fluor 594 Substances 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 208000037952 HSV-1 infection Diseases 0.000 description 2
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 2
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 210000001787 dendrite Anatomy 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 206010040882 skin lesion Diseases 0.000 description 2
- 231100000444 skin lesion Toxicity 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- VXGRJERITKFWPL-UHFFFAOYSA-N 4',5'-Dihydropsoralen Natural products C1=C2OC(=O)C=CC2=CC2=C1OCC2 VXGRJERITKFWPL-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 101150056978 HMGS gene Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 206010019670 Hepatic function abnormal Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 206010020112 Hirsutism Diseases 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- 241001502481 Meleagrid alphaherpesvirus 1 Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 101100011891 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ERG13 gene Proteins 0.000 description 1
- 206010040799 Skin atrophy Diseases 0.000 description 1
- 206010040825 Skin depigmentation Diseases 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 239000011021 lapis lazuli Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000001722 neurochemical effect Effects 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 231100000760 phototoxic Toxicity 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000001044 sensory neuron Anatomy 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000009192 ultraviolet light therapy Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
- G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a virus receptor protein for diagnosing leucoderma diseases and application thereof. The application of a molecular marker in preparing a diagnostic kit for diagnosing leucoderma is disclosed, wherein the molecular marker is human skin melanocyte HSV-1 receptor protein. Human skin melanocyte HSV-1 receptor proteins were semi-quantitatively analyzed using a western blot assay and predicted whether protein levels were reduced. Experiments prove that after HSV-1 infects human melanocytes, the expression of melanin synthesis related protein and the melanin synthesis function are inhibited by up-regulating VN1R5, so that the vitiligo is caused. The human skin melanocyte HSV-1 receptor protein is not complex in flow, the detection result can be obtained by using an enzyme-labeling instrument, the kit manufacturing means is mature and easy to purchase, and the kit can be popularized in clinical application. The early discovery of the reduced human skin melanocyte HSV-1 receptor protein level is beneficial to diagnosing leucoderma, guiding the next treatment and reducing the risk of disease deterioration of leucoderma patients.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a virus receptor protein for diagnosing leucoderma diseases and application thereof.
Background
Vitiligo (vitiligo) is an acquired skin or mucosal depigmentation skin disease characterized by the loss of functional melanocytes and skin/mucosal melanin. The prevalence rate of the vitiligo in different regions and different ethnic groups is obviously different, and is generally about 0.1-2.9%. Vitiligo is a recurrent and multifactorial skin disease, and often brings great psychological stress to patients and families. Therefore, the early diagnosis and the active treatment of the leucoderma are very important for improving the prognosis of patients.
The etiology and pathogenesis of vitiligo are not clear so far, and various theories including an autoimmune theory, a genetic theory, a neurochemical factor theory, an oxidative stress theory, a melanocyte self-destruction theory, a keratinocyte theory and the like are proposed by different scholars, but none of the theories can completely explain the pathogenesis of vitiligo. Because the molecular mechanism of vitiligo pathogenesis is not clear, a specific treatment method is not available at present, and the existing main treatment methods comprise: chinese medicinal treatment (such as lapis pill and leucocyte-expelling granule), western medicine treatment (such as oral or topical glucocorticoid), 308nm excimer laser, narrow spectrum ultraviolet light therapy, psoralen photochemotherapy, and surgical therapy (such as autologous skin transplantation, autologous melanocyte culture transplantation, drilled skin transplantation, and skin grinding). Although some patients have expected effects, the above treatment methods have undesirable effects on some patients or are resistant to treatment after a certain period of treatment, and may cause a series of side effects, such as skin atrophy, local hirsutism, infection, nausea and vomiting, impaired liver function, phototoxic reaction and even potential carcinogenic risks. Therefore, the research of finding safer and more effective treatment means for vitiligo by clarifying the pathogenesis of vitiligo has become a hotspot. In recent years, more and more scholars have been concerned about the relationship between various viral infections and vitiligo. For example, turkey herpesvirus has shown some causal association with SL chicken vitiligo models; cytomegalovirus DNA is found in skin lesions of leucoderma patients; in addition, human immunodeficiency virus, EB virus, and hepatitis B virus are also associated with vitiligo.
Herpes simplex virus-1 (HSV-1) is a ubiquitous pathogen, spreading primarily between epithelial and neural cells. HSV-1 infects the human body through the skin or mucosa, and further induces latent infection of sensory neurons. In the case of hypoimmunity, clinical symptoms appear. It is now clear that HSV can infect keratinocytes. Keratinocytes are closely related to melanocytes, which form epidermal melanocyte units with melanocytes, which transport their synthesized melanin to surrounding keratinocytes via their dendrites. The research on the influence of HSV on melanocytes is expected to provide a new idea for the research of vitiligo.
Reports in the prior art about the relation between HSV and melanocytes all indicate that HSV can infect mouse melanocytes. In the mouse melanocyte line, HSV-1 infects cells in a dose-dependent manner, inhibits cell proliferation and induces cell apoptosis. The mouse epidermal skin sheet is used for establishing an HSV-1 in vitro infection system, and melanocytes are infected by HSV-1 and can be infected even under the condition that epidermal keratinocyte nectin-1 receptors are deficient. The hair follicle stem cells positioned at the hair follicle bulge part have important significance for leucoderma complexion by promoting the regeneration of melanocytes. In the mouse epidermal skin sheet establishment HSV-1 in-vitro infection system, HSV-1 invades hair follicle bulge stem cells in the growth phase, and the stem cells in the resting phase are not affected, so that whether HSV-1 infects the hair follicle bulge stem cells depends on the state of the hair follicle bulge stem cells. However, the results of two studies show that there is no significant difference between DNA of HSV and HSV antibody in the serum and skin tissues of patients with vitiligo and normal human. The results of the human body sample and the mouse sample are inconsistent, and the analysis may be caused by the following reasons: the virus or its antibodies in the serum or skin lesions are in a small amount and are not detected or positive enough; the effect of HSV on human melanocytes was not directly examined; HSV levels themselves are unchanged, but receptor levels are changed or other mechanisms initiated by HSV are involved in melanocyte damage; the process from the destruction of melanocytes by HSV to the formation of vitiligo is required.
HSV-1 receptor has not been reported as a molecular marker for diagnosing leucoderma. The method thoroughly achieves early diagnosis and early effective treatment in practical significance, further comprehensively and systematically studies the leucoderma action mechanism, finds out related markers for leucoderma disease diagnosis, and is a key point and a clinically urgent problem to be solved for the leucoderma disease research.
Disclosure of Invention
The invention aims to provide a virus receptor protein for diagnosing leucoderma diseases and application thereof. The research of the invention finds that HSV-1 can infect human melanocytes by combining with HSV-1 receptors, inhibit the expression of melanin synthesis related protein and the melanin synthesis function, and further cause the attack of leucoderma. The human skin melanocyte HSV-1 receptor protein is expected to become a new vitiligo disease diagnosis index. The early discovery of the decreased human skin melanocyte HSV-1 receptor protein level is favorable for diagnosing leucoderma, guiding the next treatment and reducing the risk of disease deterioration of leucoderma patients.
In order to achieve the purpose, the invention adopts the following technical scheme.
The application of a molecular marker in preparing a diagnostic kit for diagnosing leucoderma is disclosed, wherein the molecular marker is human skin melanocyte HSV-1 receptor protein.
Further, the human skin melanocyte HSV-1 receptor protein is nectin-1, HVEM and PILRA.
Further, the diagnosis is to distinguish vitiligo patients from healthy people.
Further, human skin melanocyte HSV-1 receptor proteins were semi-quantitatively analyzed using a western blot assay and predicted whether protein levels were reduced.
Compared with the prior art, the invention has the following beneficial effects.
The virus receptor protein for diagnosing the vitiligo provides a new diagnosis index or a new treatment target for the vitiligo. The research of the invention finds that after HSV-1 infects human melanocytes, the expression of melanin synthesis related protein and the melanin synthesis function are inhibited by up-regulating VN1R5, thereby causing the attack of leucoderma. The human skin melanocyte HSV-1 receptor protein is expected to become a new vitiligo disease diagnosis index. The invention provides a new diagnosis index or a new treatment target point for the vitiligo.
Human skin melanocyte HSV-1 receptor proteins were semi-quantitatively analyzed using a western blot assay and predicted whether protein levels were reduced. The diagnosis accuracy is higher, the detection flow of the human skin melanocyte HSV-1 receptor protein is not complex, the detection result can be obtained by applying an enzyme-labeling instrument, the manufacturing means of the kit is mature and easy to purchase, and the kit can be popularized in clinical application. The early discovery of the elevated human skin melanocyte HSV-1 receptor protein level is beneficial to the diagnosis of leucoderma, the guidance of the next treatment and the reduction of the risk of disease deterioration of leucoderma patients.
Drawings
FIG. 1 shows the cell morphology of HSV-1 infected melanocytes under an inverted fluorescence microscope, wherein a shows the color development of rEGFP-HSV-1 fluorescent protein, and b shows the morphological change of PIG1 cells.
FIG. 2 shows the results of melanin synthesis assay, wherein a is melanin content and b is tyrosinase activity.
FIG. 3 shows the results of immunofluorescence assay of gp100 protein.
FIG. 4 shows the results of western blot detection of proteins MITF, TYR, TRP-1, and gp 100.
FIG. 5 shows the results of immunofluorescence assay for nectin-1 protein.
FIG. 6 shows the results of western blot detection of proteins nectin-1, HVEM and PILRA.
Detailed Description
The invention is further elucidated with reference to the embodiments and the drawings.
Examples are given.
One, experimental materials and groups.
1. Materials: cell lines and HSV-1 virus.
The immortalized human epidermal melanocyte line PIG1 and the immortalized human vitiligo melanocyte line PIG3V are respectively from the dermatology department of the Beijing hospital of the fourth university of military medical science. Cultured in medium 254 (Gibco) containing the human melanocyte growth supplement hmgs (Gibco), 10% Fetal Bovine Serum (FBS) (Cellmax) and 1% penicillin-streptomycin antibiotic cocktail (Bioindustries). The STR-identified human cervical cancer cell line HeLa was purchased from iCell Bioscience Inc (shanghai, china) and cultured in dmem (hyclone) containing 10% fetal bovine serum and 1% penicillin streptomycin. All cell lines were 37 ℃ 5% CO 2Is incubated in a humidified incubator. Recombinant virus rEGFP-HSV-1 carrying green fluorescent protein was purchased from Baihao Biotechnology Inc. (Shenyang). Viral amplification was performed using HeLa cells. The specific method comprises the steps of culturing HeLa cells by using a 10cm culture dish until the confluence rate is 50% -70%, replacing a fresh culture medium DMEM10ml, and adding 1ml of virus suspension, namely a HeLa cell culture medium: the HSV-1 solution is 10: 1. collecting supernatant after 48h of amplification, and storing in a refrigerator at-80 ℃.
2. And (4) grouping the cells.
(1) Experimental groups: HSV-1 infected PIG 1; (2) control group: PIG1 without HSV-1 infection; (3) group 3V: PIG3V without HSV-1 infection.
And II, an experimental method.
1. HSV-1 infects melanocytes and is observed morphologically.
PIG1 cells were cultured in medium 254 (Gibco) + HMGS (Gibco) +10% fetal bovine serum (Cellmax) +1% diabody (Biological Industries). After the confluence rate reaches 50-70%, recombinant rEGFP-HSV-1 virus is infected. The proportion of experimental group infected viruses is respectively culture medium: virus liquid = 3: 1. 5: 1. 7: 1. 9: 1. 11: 1. the medium was replaced with fresh medium 12h after viral infection. The infection efficiency was examined by inverted fluorescence microscopy and 3 fields were randomly selected for each group. The detection time points are 0h, 6h, 12h, 24h, 36h, 48h and 72 h.
2. And (4) measuring the content of the melanin.
Cells were plated in 96-well plates and experiments were divided into 3 groups: experimental group (PIG 1 cells infected with HSV-1), negative control group (PIG 1 cells not infected with HSV-1) and PIG3V control group (PIG 3V cells not infected with HSV-1). The experimental group is infected HSV-1 and cultured together with PIG1 cells for 72 h. Melanin content was measured after 72h, and the control group and 3V group were measured at the same time point. PBS (Cellmax) 2 washes were then lysed in 200ul 1N NaOH (Bilun sky) for 30 min. The absorbance of A450 was measured using an ND-1000 spectrophotometer (Thermo Fisher).
3. And (4) determining the content of tyrosinase.
Tyrosinase activity was measured using the L-DOPA oxidation method. The cells were treated as before. PBS washing 2 times, with 1% Triton X-100 (Solebao) PBS (Solebao) 90 ul homogenate cells. 50 ul of the cell extract was mixed with 90 ul of freshly prepared PBS substrate solution containing 0.1% L-DOPA and incubated at 37 ℃ for 20 minutes. The absorbance at A475 was measured using an ND-1000 spectrophotometer (Thermo Fisher).
4、Western blot。
After cell treatment, cell proteins were collected in a ratio of 99:1 using RIPA lysate (cloudy day) and PMSF (cloudy day). Protein was quantified using BCA protein assay kit (cloudband day). Equal amounts of protein were mixed with PBS and SDS buffer (petunia) and boiled at 100 ℃ for 5 minutes. After electrophoresis and membrane conversion, 5% skim milk was sealed for 2 h. anti-MITF (1: 1000, # ab12039, Abcam), TYR (1: 1000, # ab170905, Abcam), TRP-1 (1: 1000, # ab135447, Abcam), gp100 (1: 5000, # ab137078, Abcam), nectin-1 (1: 2000, TA503469, Origene), HVEM (1: 100, #94804, R & D), PILR-1 (1: 1000, DDX 0230-0230P-100, Novus), GAPDH (1: 5000, 60004-1-Ig, Proteintetech) were incubated overnight at 4 ℃. After 1 × TBST wash, PVDF membrane (Millipore) was incubated with secondary antibody (anti-rabbit IgG, 1: 1000; anti-mouse IgG, 1: 5000) for 2h at room temperature. The bands were detected using an electrochemiluminescence western blotting substrate (Pierce) and the luminescence solution was ECL luminescence kit (petit sky).
5. Immunofluorescence.
After cell treatment, cells were washed 3 times with PBS. Cells were fixed with 4% paraformaldehyde fixing solution (bi yun day) for 20 min. 0.2% Triton X-100 (Solebao) was prepared in PBS containing 1% bovine serum albumin (Biyunyan) and permeabilized at room temperature for 20 minutes. After 3 washes with PBS, the cells were blocked with 1% bovine serum albumin in PBS for 1h at room temperature and incubated overnight at 4 ℃ with primary antibodies nectin-1 (1: 100) and gp100 (1: 250). After 3 times of PBS washing, the primary antibody was detected by using Alexa Fluor 594 labeled affinity purified goat anti-mouse IgG secondary antibody (Abcam) and Alexa Fluor 594 labeled affinity purified goat anti-rabbit IgG secondary antibody (Abcam) under the incubation condition of 37 ℃ for 1 h. After 3 washes with PBS, nuclear staining was performed with DAPI (solibao) for 10 min at room temperature. Cells were observed under a BioSpa live cell assay system (Bio Tek, USA).
And thirdly, experimental results.
1. HSV-1 infects melanocytes.
Application of 0: 1. 3: 1. 5: 1. 7: 1. 9: 1. 11:1 concentration of rEGFP-HSV-1 infects PIG1, and the cell morphology is observed under an inverted fluorescence microscope at 0h, 6h, 12h, 24h, 36h, 48h and 72h respectively. In comparison with the control group, the cells started to infect at 24h except 11:1 (48 h). Concentration 7: 1 when the cells are infected for 72h, the rEGFP-HSV-1 fluorescent protein is developed, as shown in figure 1 a; and PIG1, the cell morphology changes most significantly, as shown in fig. 1b, the number of cell dendrites decreases, shortening and rounding. It was shown that HSV-1 can infect human skin melanocytes.
2. And (4) detecting melanin synthesis.
The melanin content and the tyrosinase activity were significantly reduced in both the experimental group and the 3V group compared with the control group (p<0.05), no significant difference between the experimental group and the 3V group (p>0.05) as shown in fig. 2. HSV-1 is shown to cause the decrease of PIG1 melanin synthesis and the decrease of PIG3V melanin synthesis of leucoderma melanocytes.
3. And detecting melanin synthesis related protein.
The results of immunofluorescence gp100 protein detection are shown in FIG. 3, the experimental group is obviously lower than the control group: (p<0.05). The results of western blot detection of proteins MITF, TYR, TRP-1, and gp100 are shown in FIG. 3, andcompared with the control group, the expression level of the four proteins is obviously reduced in the experimental group and the 3V group (p<0.05); except that MITF was lower in the 3V group than in the experimental group: (p<0.05), no significant difference between the other three protein experimental groups and the 3V group: (p>0.05). The results show that HSV-1 causes the expression of protein related to PIG1 melanin synthesis to be reduced, and the expression of protein related to PIG3V melanin synthesis of leucoderma melanocytes is reduced.
4. HSV-1 receptor detection.
The results of immunofluorescence detecting the nectin-1 protein are shown in FIG. 5, and all the proteins are expressed in the experimental group, the control group and the 3V group, and the expression level of the 3V group is significantly lower than that of the control group (p<0.05), no difference between the experimental group and the control group: ( p>0.05). Results of western blot detection of proteins nectin-1, HVEM and PILRA are shown in FIG. 6, and the expression level of the three receptors is significantly lower than that of the control group no matter the experimental group or the 3V group (the expression level of the three receptors is significantly lower than that of the control group) ((thep<0.05); except that HVEM is lower than 3V group in experimental group (p<0.05), no significant difference between the other two receptor experimental groups and the 3V group: (p>0.05). The result shows that HSV-1 causes the reduction of the expression of the HSV-1 receptor of PIG1, and the expression of the HSV-1 receptor of leucoderma melanocyte PIG3V is reduced.
The research result shows that HSV-1 can infect human melanocytes by combining with HSV-1 receptors, inhibit expression of melanin synthesis related protein and melanin synthesis function, and further cause the attack of leucoderma. The reduction of human skin melanocyte HSV-1 receptors discovered by the research is expected to become a new diagnosis index of leucoderma diseases.
Claims (4)
1. The application of a molecular marker in preparing a diagnostic kit for diagnosing leucoderma is characterized in that the molecular marker is human skin melanocyte HSV-1 receptor protein.
2. The use of a molecular marker according to claim 1 for the preparation of a diagnostic kit for the diagnosis of vitiligo, wherein the human skin melanocyte HSV-1 receptor protein is nectin-1, HVEM, PILRA.
3. The use of a molecular marker according to claim 1 for the preparation of a diagnostic kit for the diagnosis of vitiligo, wherein the diagnosis is to distinguish vitiligo patients from healthy people.
4. Use of a molecular marker according to claim 1 for the preparation of a diagnostic kit for the diagnosis of vitiligo, wherein human skin melanocytes HSV-1 receptor protein is semi-quantitatively analyzed using a western blot assay and it is predicted whether the protein level is decreased.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010673592.9A CN111856030A (en) | 2020-07-14 | 2020-07-14 | Virus receptor protein for diagnosing leucoderma diseases and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010673592.9A CN111856030A (en) | 2020-07-14 | 2020-07-14 | Virus receptor protein for diagnosing leucoderma diseases and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111856030A true CN111856030A (en) | 2020-10-30 |
Family
ID=72983374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010673592.9A Pending CN111856030A (en) | 2020-07-14 | 2020-07-14 | Virus receptor protein for diagnosing leucoderma diseases and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111856030A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667738A (en) * | 2021-08-25 | 2021-11-19 | 中国医科大学附属第一医院 | Receptor protein GPR17 for diagnosing leucoderma diseases and application thereof |
-
2020
- 2020-07-14 CN CN202010673592.9A patent/CN111856030A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113667738A (en) * | 2021-08-25 | 2021-11-19 | 中国医科大学附属第一医院 | Receptor protein GPR17 for diagnosing leucoderma diseases and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10494606B2 (en) | Immortalized stem cell, compositions, preparations and uses thereof | |
| CN102631666B (en) | Preparation method for biological agent for preventing and controlling human papilloma virus infection | |
| CN107344959B (en) | Ultrashort peptide Purin-WH for promoting skin repair, and preparation method and application thereof | |
| WO2013133494A1 (en) | Adipose-derived stem cell culture fluid, preparation method for same, and hair-growth promoting composition comprising same | |
| WO2021031884A1 (en) | Method for culturing urine-derived renal stem cells and use thereof | |
| US11761002B2 (en) | Application of transgenic stem cell-derived exosome in preparing medicament or whitening cosmetic | |
| CN111879944A (en) | Molecular marker for diagnosing leucoderma diseases and application thereof | |
| CN109136174A (en) | A kind of source of human stem cell excretion body preparation of anti-aging | |
| CN108823148A (en) | A kind of method that fat mesenchymal stem cell is induced to differentiate into liver like cell | |
| CN112920989A (en) | Liver organoid model, establishment method and application thereof, and pharmaceutical composition for treating hepatocyte iron death | |
| CN111856030A (en) | Virus receptor protein for diagnosing leucoderma diseases and application thereof | |
| Zhong et al. | Expression of β-catenin and cyclin D1 in epidermal stem cells of diabetic rats | |
| CN109613254B (en) | Target marker PDIA2 for tumor treatment and diagnosis | |
| Li et al. | Protective effect and mechanisms of exogenous neutrophil gelatinase-associated lipocalin on lipopolysaccharide-induced injury of renal tubular epithelial cell | |
| WO2018155952A2 (en) | Method for isolating human brain tissue-derived neural stem cell at high efficiency | |
| CN111718898B (en) | Method and reagent for improving stress tolerance of synovial membrane mesenchymal stem cells | |
| WO2019153364A1 (en) | Stem cell preparation resisting hypoxia injury, preparation method therefor, and use thereof in preparation of medicament for treating acute myocardial infarction | |
| CN104922153A (en) | Application of NRG1 (neuregulin1)-ERBB4 complex in preparing medicaments for treating myocardial ischemia | |
| ES2322290T3 (en) | PREDICTIVE TEST OF THERAPEUTIC RESPONSE OF CANCER DE MAMA. | |
| Melnick et al. | Cervical cancer cell lines containing herpesvirus markers | |
| CN113584086B (en) | Method for directly reprogramming mouse embryo fibroblast into melanocyte | |
| Liu et al. | Stromal Vascular Fraction and Platelet-Rich Plasma Upregulate Vascular Endothelial Growth Factor Expression to Promote Hair Growth via the Wnt/β-Catenin Signaling Pathway | |
| CN114432427B (en) | Application of anti-aging active peptide and bone marrow mesenchymal stem cells in preparation of anti-aging drugs | |
| CN110368485A (en) | Antler polypeptide is preparing the purposes in the drug for treating osteosarcoma | |
| CN114452280B (en) | Application of atractylone in preparation of glioma treatment drug |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201030 |