Application of dimethylarginine and derivatives thereof in inhibition of T and B cell proliferation

A technology of dimethylarginine and derivatives, which is applied in the field of dimethylarginine and its derivatives to inhibit the proliferation of T and B cells, can solve the problem of less research and achieve the effect of inhibiting the proliferation of T cells

Inactive Publication Date: 2020-04-14
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogenesis of the above-mentioned autoimmune diseases suggests that inhibiting the abnormal activation and proliferation of T cells and B cells in the body may be a method for treating autoimmune diseases, but there are still few studies in this area in the prior art

Method used

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  • Application of dimethylarginine and derivatives thereof in inhibition of T and B cell proliferation
  • Application of dimethylarginine and derivatives thereof in inhibition of T and B cell proliferation
  • Application of dimethylarginine and derivatives thereof in inhibition of T and B cell proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 ADMA and SDMA inhibit the proliferation of mouse T cells

[0042] 1. Isolation of mouse T cells

[0043] Total splenic T cells were isolated with Stemcell cell isolation kit.

[0044] Take female 6-8 week old C57BL mice, crush the spleen, wash with biotin-linked antibody solution (including anti-B220, CD11B, CD11c, CD16 / 32, CD19, CD25, CD105, NK1.1 and TCRγδ) and add Streptavidin-coated magnetic beads, followed by a magnetic field, CD3 + T cells are enriched in suspension.

[0045] 2. Inhibitory effect of ADMA and SDMA on mouse T cell proliferation

[0046] The total T cells obtained in step 1 were stained with CellTrace and labeled with 2 × 10 5 Each well was seeded in a 96-well round-bottom cell culture plate and stimulated with anti-CD3 and CD28-coated magnetic beads (using Gibco Dynabeads mouse T-activator CD3 / CD28 for T-cell expansion and activation). The number of magnetic beads: the number of cells was 1:4. Experimental group: Add different concen...

Embodiment 2

[0048] Example 2 ADMA and SDMA inhibit the proliferation of human T cells

[0049] Isolation and in vitro proliferation experiments of human T cells: The use of peripheral blood from healthy adults was approved by the Ethics Committee of Guangzhou Medical University. Peripheral blood mononuclear cells were isolated by Sepmate peripheral blood mononuclear cell separation tube, and CD3 + Human T cell isolation kit (Miltenyi) was used to isolate human T cells. The isolated T cells were collected at a density of 1 × 10 5 Seed in a 96-well round-bottom cell plate coated with anti-human CD3 (5μg / ml) and CD28 (2μg / ml) per well. Experimental group: Add different concentrations of ADMA and SDMA respectively (ADMA and SDMA were dissolved in sterile Endotoxin-free water, stock solution concentration 0.5mM), control group: add the same volume of sterile endotoxin-free water. Cell proliferation was analyzed by flow cytometry after 4 days of culture in RPMI-1640 medium.

[0050] The exp...

Embodiment 3

[0051] Example 3 ADMA and SDMA inhibit the proliferation of mouse B cells

[0052] Isolation of total splenocytes: Mice were killed by cervical dislocation, the spleen was isolated, and the spleen was placed on a 100 μm cell mesh at the mouth of a 50 ml cytocentrifuge tube moistened with sterile endotoxin-free PBS, using a 1 ml sterile syringe The spleen tissue was crushed at the end of the piston core rod, and the cell suspension in a 50 ml tube was collected, centrifuged at 500 rcf for 5 min and resuspended in RPMI 1640 complete medium (10% FBS).

[0053] In vitro proliferation of mouse B cells: total splenocytes were isolated from mice and stained with CellTrace labeling at 5 × 10 5 Each well was seeded in a 96-well round-bottom cell culture plate with anti-IgM (10 μg / ml) and human recombinant IL-4 (10 ng / ml) to stimulate B cell proliferation. Experimental group: ADMA and SDMA (ADMA and SDMA) were added with different concentrations, respectively. SDMA was dissolved in ste...

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Abstract

The invention belongs to the technical field of immunotherapy. Firstly, dimethylarginine is found to effectively inhibit in-vitro proliferation of T and B cells, so a mechanism of ADMA and SDMA for inhibiting T and B cell proliferation in vivo is studied, and it is found that the ADMA and SDMA induced mitochondrial ROS generation is realized by activating AGXT2 enzyme, and the ADMA and SDMA are converted into a new substance DMGV through the AGXT2 enzyme, thereby the ADMA, the SDMA and the DMGV can effectively inhibit the proliferation of T and B cells; and the ADMA is converted into L-citrulline by dimethylamine hydrolase (DDAH), so the amount of ADMA that enters mitochondria, is converted into DMGV and functions is reduced, and a specific DDAH inhibitor PD404182 is the same as ADMA to suppress T cell proliferation. Therefore, the dimethylarginine, the derivatives thereof and the substances for specifically inhibiting degradation of the dimethylarginine are expected to provide a new direction for treating T and B cell mediated diseases by inhibiting abnormal proliferation of T and B cells.

Description

technical field [0001] The present invention relates to the technical field of immunotherapy, and more particularly, to the use of dimethylarginine and its derivatives for inhibiting the proliferation of T and B cells. Background technique [0002] Immunity is a physiological function of the human body. The human body relies on this function to identify "self" and "non-self" components, thereby destroying and rejecting antigenic substances entering the human body, or damaged cells and tumor cells produced by the human body itself, in order to maintain the immune system. The health of the human body, the state of resisting or preventing infection by microorganisms or parasites or the intrusion of other undesirable organisms. [0003] The immune system consists of immune organs, immune cells and immune active substances. Among them, immune cells mainly refer to cells that play an immune role, including phagocytes and lymphocytes. Lymphocytes are mainly derived from hematopoie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/198A61K45/00A61P37/08A61P35/00A61P37/02A61P29/00A61P19/02A61P17/06A61P11/06A61P3/10
CPCA61K31/198A61K45/00A61P37/08A61P35/00A61P37/02A61P29/00A61P19/02A61P17/06A61P11/06A61P3/10A61P35/04
Inventor 李钟大杨丰源沈丽亚
Owner GUANGZHOU MEDICAL UNIV
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