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Micromolecule preparation for inhibiting TH1 cell polarization, and application of micromolecule preparation

A small molecule and inhibitor technology, applied in the direction of cell culture active agent, animal cells, vertebrate cells, etc., can solve problems such as infection, and achieve the effect of simple operation, efficient transfection and cell culture

Active Publication Date: 2021-07-23
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the clinical application for the treatment of autoimmune system diseases caused by immune disorders often relies on hormone drugs or immunosuppressants, but it is often accompanied by problems such as drug resistance and infection caused by long-term immunosuppression

Method used

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  • Micromolecule preparation for inhibiting TH1 cell polarization, and application of micromolecule preparation
  • Micromolecule preparation for inhibiting TH1 cell polarization, and application of micromolecule preparation
  • Micromolecule preparation for inhibiting TH1 cell polarization, and application of micromolecule preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 mouse Isolation of CD4+ T lymphocytes

[0040] The method of isolating and extracting mouse spleen lymphocytes is as follows: take the spleen of a six-week-old C57BL / 6 mouse under aseptic conditions, place it in a 1640 medium, cut the spleen with ophthalmic scissors, and transfer it to a 200-mesh sterile screen , grind with a glass rod, drop 1640 medium while grinding, until all the splenocytes that are ground and dispersed flow out through the filter, collect the splenocytes and centrifuge at 450g for 10min, remove the supernatant and resuspend, add an equal volume of lymphocyte separation medium, and centrifuge at 450g for 30min , absorb the lymphocyte layer, add PBS to resuspend, centrifuge at 400g for 10min, add red blood cell lysate to stand for 5min after washing with PBS, centrifuge at 1500rpm for 10min, collect cells for counting, and use Miltenyi CD4 + T cell isolation kit, isolated from splenic lymphocytes CD4 + T lymphocytes.

Embodiment 2

[0041] Example 2 Induced differentiation of mouse Th1 cells

[0042] CD4 + After T cell sorting, counting, according to 1 × 10 6 / mL seeded in a 6-well plate, placed in 1640 complete medium containing 10% FBS, adding mixed factors to induce Th1 cell differentiation, the concentration of each factor is: IL12: 10ng / mL, IL2: 5ng / mL, anti CD28: 0.5 μg / mL, anti IL4: 1 μg / mL, anti CD3ε: 1 μg / ml, add NEAA and 55 μM β-mercaptoethanol, and culture for 72h to 96h. When the cells were cultured for 36 h, the medium was changed in half, 1 mL of the original medium was removed, and 1 mL of 1640 complete medium containing 10% FBS was mixed with 2 times the concentration of factor, NEAA and 55 μM β-mercaptoethanol.

Embodiment 3

[0043] Example 3 Preparation and transfection of miR-148a-3p small molecule preparation

[0044] 1. Dilute miR-148a-3p mimic at 1.00 OD260 with 125 μL DEPC water to make the final concentration 20 μM. The sequence of miR-148a-3p is shown in Table 1, and the above sequence was purchased from Shanghai Sangon Bioengineering (Shanghai) Co., Ltd.

[0045] Table 1 RNA and primer sequences

[0046]

[0047]

[0048] 2. Transfection

[0049] CD4+T cells by 1×10 6 / mL seeded in 6-well plate, after adding 1640 complete medium and Th1 inducer. Dilute 3 μL miR-148a-3p mimic into 100 μL Jet Prime Buffer, add 6 μL Jet Prime, mix gently, and let stand at room temperature for 15 minutes to form a complex. The liposome complex was added to a six-well plate, the transfection system was 100 μL, and the final miRNA concentration was 100 nM. About 36 hours half of the liquid change.

[0050] 3. Flow cytometry analysis

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PUM

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Abstract

The invention belongs to the technical field of cellular immunity regulation and control, and specifically discloses a micromolecule preparation for inhibiting TH1 cell polarization, and application of the micromolecule preparation. The micromolecule preparation comprises miR-148a-3p or a simulant thereof. By use of a regulation and control function of the miR-148a-3p for the differentiation capacity of a mouse CD4+T lymphocyte to a Th1 cell, after the miR-148a-3p is imported, the differentiation capacity of a mouse juvenile CD4+T lymphocyte to the Th1 cell is obviously lowered. Therefore, a novel micromolecule preparation for regulating and controlling the Th1 cell polarization is put forward and can be applied to regulation of differentiation of the mouse CD4+T lymphocyte to the Th1 cell and control of an immune response.

Description

technical field [0001] The invention belongs to the technical field of cellular immune regulation, and in particular relates to a small molecule preparation for inhibiting Th1 cell differentiation and application thereof. Background technique [0002] The immune system is an important physiological defense line of the organism. By activating and producing immune cells and related cytokines, it recognizes and eliminates foreign pathogens and self-produced damaged cells or tumor cells, etc., and plays the role of immune surveillance, defense, regulation, etc., and maintains the stability of the internal environment of the body. , to protect the health of the body. [0003] An uncontrolled or dysfunctional immune response originates from an abnormally activated immune system, and abnormal immune attack is also the main cause of many autoimmune diseases (such as Crohn's disease, type 1 diabetes, graft-versus-host disease, etc.). Maintaining the homeostasis of the immune system,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/715A61P37/06C12N5/0783
CPCA61K31/715A61P37/06C12N5/0636C12N2501/65
Inventor 张毅李雪张晗刘元林刘伟江王洋
Owner ACADEMY OF MILITARY MEDICAL SCI
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