Inhibitor capable of inhibiting killing effect of CD8<+>T cells on melanocytes, inhibitor composition and application

A technology of melanocytes and inhibitors, which is applied in the fields of biotechnology and medicine, can solve problems such as unclear structure-activity relationships, and achieve the effects of protecting melanocytes, inhibiting killing, and reducing chemotaxis

Inactive Publication Date: 2017-06-30
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the chemical components in Tripterygium wilfordii have var...

Method used

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  • Inhibitor capable of inhibiting killing effect of CD8&lt;+&gt;T cells on melanocytes, inhibitor composition and application
  • Inhibitor capable of inhibiting killing effect of CD8&lt;+&gt;T cells on melanocytes, inhibitor composition and application
  • Inhibitor capable of inhibiting killing effect of CD8&lt;+&gt;T cells on melanocytes, inhibitor composition and application

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Experimental program
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Effect test

Embodiment 1

[0035] The appropriate dose range of triptolide, triptolide, and triptolide in keratinocytes was determined by CCK-8 experiment. For the results, see figure 1 shown. Cell culture: the cell density of 6000 / well is seeded in 96-well plate; 37°C, 5% CO 2 After culturing in a constant temperature incubator for 24 hours under certain conditions, the medium was sucked off, and then 0.2uL of each test sample was added to each well (the final concentration of DMSO in each well was 0.1%). The test samples of each concentration required 5 duplicate wells, and then added 200uL of fresh medium continued to culture for 24h. Then aspirate the medium, add 100uL of CCK-8 reagent (diluted in the medium at 1:10), incubate at 37°C for 1h in the dark, and measure the OD with a microplate reader 450 absorbance value.

[0036] From figure 1 It can be seen that triptolide (LGT-4), triptolide (LGT-6) and triptolide ketone (LGT-7) in the low dose range (5-10nM) on melanocyte PIG1 and PIG3V There ...

Embodiment 2

[0038] The protective effect of triptolide, triptolide, and triptolide on melanocytes (PIG1, PIG3V) under oxidative stress model was determined by CCK-8 experiment. For the results, please refer to figure 2 . Cell culture: the cell density of 6000 / well is seeded in 96-well plate; 37°C, 5% CO 2 After culturing in a constant temperature incubator for 24 hours under certain conditions, the medium was sucked off, and then 0.2uL of each test sample was added to each well (the final concentration of DMSO in each well was 0.1%). The test samples of each concentration required 5 duplicate wells, and then added 200uL of fresh medium continued to culture for 2h before adding H 2 o 2 Make the final concentration reach 800uM, continue to culture for 24h. Then aspirate the medium, add 100uL of CCK-8 reagent (diluted in the medium at 1:10), incubate at 37°C for 1h in the dark, and measure the OD with a microplate reader 450 absorbance value.

[0039] From figure 2 It can be seen tha...

Embodiment 3

[0041] Tripterygium wilfordii series compounds acted together with IFN-γ+TNF-α on keratinocytes for 24 hours, and the cells were collected to extract proteins. See the result image 3 . Cell proteins were collected, and after SDS electrophoresis, the proteins were transferred to PVDF membranes, blocked with 5% BSA at room temperature for 1 h, and coated with primary antibodies corresponding to the proteins to be detected (among them, Anti-pSTAT1, Anti-STAT1, Anti- IRF1, Anti-SOCS3, and Anti-β-Actin were all purchased from CST Company), and incubated overnight at 4°C. The next day, the primary antibody was recovered, and the membrane was washed with TBST three times, 10 min each time, and then the secondary antibody (Goat anti-rabbit IgG-HRPG, Goat anti-mouse IgG-HRP purchased from SANTA Company) was applied, and incubated at room temperature for 1 h. Then wash with TBST 3 times, 10min each time. Afterwards, luminescent liquid was evenly applied and observed on a gel imaging...

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Abstract

The invention belongs to the field of biotechnology and medicine and relates to an inhibitor capable of inhibiting a killing effect of CD8<+>T cells on melanocytes, inhibitor composition and an application to vitiligo preventing and treating drugs. When dose of triptolide, celastrol or wilforlide A are in 5-10 nM, the inhibitor capable of inhibiting the killing effect of the CD8<+>T cells on the melanocytes can inhibit the expression level of STAT1 (signal transducer and activator of transcription 1) in keratinocytes, then the expression level of IRF1 is down-regulated, secretion of CXCL10 and CXCL16 in the keratinocytes is inhibited, and chemotaxis of CXCL10 and CXCL16 on the CD8<+>T cells is further reduced, so that the killing effect of the CD8<+>T cells on the melanocytes is inhibited; the inhibitor capable of inhibiting the killing effect of the CD8<+>T cells on the melanocytes is a tripterygium wilfordii terpenoid and particularly is triptolide, celastrol or triptonide.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, and relates to an inhibitor and its application in the prevention and treatment of vitiligo, in particular to an inhibitor capable of inhibiting CD8 + An inhibitor of T cells killing melanocytes, an inhibitor composition and its application as a medicine for preventing and treating vitiligo. Background technique [0002] Vitiligo is a common depigmentation skin disease, the prevalence of which is about 0.1%-2% in our country. Vitiligo is characterized by white spots on the skin caused by the destruction of melanocytes. The pathogenesis is still unclear, which brings great difficulties to the treatment. Previous studies have believed that the occurrence of vitiligo is related to many factors such as genetics, oxidative stress, and endocrine abnormalities. Existing treatment methods such as hormones, calcineurin inhibitors, antioxidants, Chinese patent medicines, and phototherapy have li...

Claims

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Application Information

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IPC IPC(8): A61K31/585A61K31/56C07J73/00C07J63/00A61P17/00
CPCA61K31/585A61K31/56C07J63/008C07J73/003A61K2300/00
Inventor 李春英安亚文李舒丽郭森高天文
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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