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Application of TNFR1 gene and encoded protein thereof

A gene and coding technology, applied in the field of autoimmune disease treatment, can solve the problem of unclear specific roles of inflammatory T cells and regulatory T cells, and achieve the effects of avoiding serious side effects, reducing drug dosage, and promoting differentiation

Inactive Publication Date: 2018-12-04
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific effects of TNFR1 and TNFR2 on inflammatory T cells and regulatory T cells are still unclear.

Method used

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  • Application of TNFR1 gene and encoded protein thereof
  • Application of TNFR1 gene and encoded protein thereof
  • Application of TNFR1 gene and encoded protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Extraction of naive CD4 from mouse spleen + T cell

[0071] Naive CD4 was extracted by autoMACS magnetic bead sorting method + T cells. First enrich the spleen cells of the mouse with nylon hair column for T cell enrichment, then add biotin mouse anti-CD8 antibody 6ul, biotin mouse anti-B220 antibody 8ul, biotin mouse anti-CD11b antibody 1.5ul, biotin mouse Anti-CD11c antibody 1.5ul, biotin mouse anti-CD49b 1ul, mix well, and incubate at 4°C for 20 minutes. Then add anti-biotinbeads 8ul, mix well, and incubate at 4 degrees for 15 minutes. The autoMACS instrument performs the first step of negative selection to remove non-CD4 + cell. Add mouse CD62L-beads 8ul and incubate at 4 degrees for 15 minutes. The autoMACS instrument performs the second step of positive selection. The seroselective tube is both naive CD4 + T cells. Detection of naiveCD4 by flow cytometry + T cell purity (>90%).

Embodiment 2

[0072] Example 2 naive CD4 + Induction of T cells into Th1 and Th17 cells in vitro

[0073] The extracted WT naive CD4 + T cells were planted in 96-well plates, and CD3 (1ug / ml), CD28 (1ug / ml) and anti-IL-4 monoclonal antibody (5μg / ml) were added to the culture system, with or without rmTNFα. For Th1 cell differentiation, add rmIL-12 (10ng / ml, R&D Systems); for Th17 cells, add rmIL-6 (10ng / ml) and rhTGF-β (2ng / ml), anti-IFN-γ (5μg / ml) ml), the naive CD4 + T cells were cultured in the above system for three days. Collect cells and detect IL-17A using flow cytometry + , IFNγ + expression.

[0074] The result is as figure 1 As shown in a~d, the stimulation of TNFα can not enhance WT naive CD4 +In vitro differentiation of T cells into Th1 and Th17 cells.

[0075] Will be from WT, TNFR1 - / - and TNFR2 - / - Naive CD4 extracted from mouse + T cells were planted in 96-well plate or 48-well plate, and CD3 (lug / ml), CD28 (lug / ml) and anti-IL-4 monoclonal antibody (5 μg / ml) wer...

Embodiment 3

[0076] Example 3 Naive CD4 + Induction of T cells into iTregs in vitro

[0077] Will be from WT, TNFR1 - / - and TNFR2 - / - Naive CD4 extracted from mouse + T cells are planted in 96-well plates or 96-well plates, and anti-CD3 / CD28-coated beads (5 cells correspond to one bead), rhIL-2 (50U / ml) and rhTGF-β (2ng / ml) are added to the culture system ), with or without rmTNFα (100ng / ml). Cells were collected at the same time point, and Foxp3 expression was determined by flow cytometry.

[0078] The result is as figure 2 As shown, TNFR1 - / - naive CD4 + T cells can successfully differentiate into iTregs, and when the TNFR2 gene is lost, naive CD4 + The proportion of T cells differentiated into iTregs decreased significantly. Furthermore, rmTNFα fails to promote WT and TNFR1 - / - naive CD4 + Differentiation of T cells to iTregs, response to TNFR2 - / - Cells are useless.

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Abstract

The invention discloses application of a TNFR1 (tumor necrosis factor receptor) gene, an encoded protein and a TNFR1 specific antagonist in preparation and screening of drugs treating autoimmune or inflammatory diseases. The invention for the first time puts forward that the TNFR1 antagonist can be used for treatment of autoimmune diseases; TNFalpha serves as a multidirectional functional cytokine, the receptor TNFR1 of TNFalpha is widely expressed in cells of the whole body, mainly can promote the differentiation, proliferation, anti-apoptosis and other effects of inflammatory cells, therebymediating inflammatory reaction. The TNFR1 antagonist provided by the invention can effectively treat autoimmune or inflammatory diseases, like multiple sclerosis, rheumatoid arthritis, etc. The TNFR1antagonist can target TNFR1 and block the immunoregulation function of TNFR1 signals, avoids the serious side effects caused by complete blocking of TNFalpha signals, and reduces the drug dosage.

Description

technical field [0001] The present invention relates to the technical field of autoimmune disease treatment, especially the application of TNFR1 gene and its encoded protein; more specifically, the present invention relates to TNFR1 antagonists, which can inhibit the differentiation of inflammatory T cells by acting on TNFR1 on the surface of T cells to treat autoimmune disease. Background technique [0002] As we all know, tumor necrosis factor (Tumor Necrosis Factor, TNF) α is a very important pro-inflammatory factor, involved in the activation and promotion of immune response to clear pathogens. [0003] TNFα bound to TNFR1 (recombinant human tumor necrosis factor receptor type 1) activates two different complex signaling pathways: 1) maintains cell survival and promotes the expression of inflammatory cytokines; 2) promotes apoptosis and necrosis. TNFα binds to TNFR1, and the intracellular domain binds to related proteins to form signaling complex I. Signal transduction...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17A61K45/00C12Q1/6883A61P29/00A61P37/02A61P19/02A61P25/00A61P1/00
CPCA61K38/1793A61K45/00A61K48/005A61P1/00A61P19/02A61P25/00A61P29/00A61P37/02C12Q1/6883C12Q2600/158
Inventor 郑颂国杨素娟王菊华
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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