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Application of miR-1165-3p in preparation or screening of drugs for inhibiting Th2 cell differentiation

A cell differentiation and drug technology, applied in the field of biomedicine, can solve problems such as Th2 cell differentiation that have not yet been reported

Inactive Publication Date: 2019-05-28
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous experiments have proved that it is highly expressed in the serum of asthmatic patients and can be used as a molecular marker of asthma, but it has not been reported whether it is involved in the differentiation of Th2 cells and whether it can improve or treat asthma

Method used

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  • Application of miR-1165-3p in preparation or screening of drugs for inhibiting Th2 cell differentiation
  • Application of miR-1165-3p in preparation or screening of drugs for inhibiting Th2 cell differentiation
  • Application of miR-1165-3p in preparation or screening of drugs for inhibiting Th2 cell differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Regulation of miR-1165-3p on Th2 cell differentiation (cellular level)

[0052] Main reagents:

[0053]Cytokines (peprotech), functional antibodies (ebioscience), serum (Gibco), primers (GenScript), sybr green (takara), miRNA reverse transcription reagents (takara), 1640 medium (Gibco), Elisa kit (Biolegend )Western antibody (Abcam, CST), flow cytometry antibody and reagent (ebioscience), Trizol and small RNA extraction reagent (Thermo), magnetic bead sorting kit

[0054] main instrument

[0055] Cell incubator, magnetic frame, flow detection machine, electrophoresis transfer membrane instrument, exposure instrument, PCR instrument and qPCR instrument, microplate reader

[0056] main method

[0057] Th1 / Th2 cells differentiated in vitro

[0058] 1. Coating: Add 100ul 5ug / ml anti-CD3e to each well, overnight at 4°C.

[0059] 2. Isolation of mouse Naive CD4+ T cells. (Day 0)

[0060] ① 6-8 week old female C57 / B6 mice were killed by cervical dislocation, s...

Embodiment 2

[0078] Example 2 miR-1165-3p improves asthma (overall level)

[0079] main reagent

[0080] House dust mite (HDM) (Greer), miR-1165-3p overexpression lentivirus (Albimon), methacholine, ELISA kit (Biolegend)), flow cytometry antibody (Thermo), PAS dye, HE dye , counting microspheres (Thermo), etc.

[0081] main instrument

[0082] Pulmonary function instrument, microplate reader, PCR instrument and qPCR instrument, microplate reader, flow detection instrument

[0083] main method

[0084] Mouse HDM acute asthma model

[0085] Thirty-two SPF grade C57 / B6 female mice were randomly divided into 4 groups, 8 mice in each group. Control group (control), HDM group (HDM), HDM+blank group (HDM+Lenti-III-mir-GFP control virus), HDM+enhancer group (HDM+LentimiRa-GFp-miR-1165-3p virus). HDM treatment group: On the 0th day, 100ug / 40ul HDM solution was administered into the trachea for sensitization, and on the 7th, 8th, 9th, 10th, and 11th days, 10ug / 40ul HDM solution was administere...

Embodiment 3

[0103] Example 3PPM1A and IL-13 are the target genes of miR-1165-3p

[0104] main reagent

[0105] Liposome (Yisheng), protein lysate (millipore), magnetic beads (Thermo), RNA extraction kit (QIAGEN), etc.

[0106] main instrument

[0107] Cell incubator, magnetic rack, 360°rotator, high-speed centrifuge

[0108] main method

[0109] Biotin-labeled miRNA-pull down experiment

[0110] Mouse embryonic fibroblasts (NIH-3T3) were used as tool cells, which were inoculated in a 10 cm cell culture dish, and when the density reached 90%, liposomes were transfected with biotin-labeled miR-1165-3p and corresponding Control (NC), 48 hours after transfection, harvest the cells, UV cross-linking, lyse the cells, incubate the lysed supernatant with magnetic beads, extract the combined RNA with the RNA extraction kit, and send it to Guangzhou Ruibo Company for sequencing .

[0111] Bioinformatics analysis of sequencing results The differential mRNA (P2) screened by Biotin-miR-1165-3p a...

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Abstract

The invention discloses application of miR-1165-3p in preparation or screening of drugs for inhibiting Th2 cell differentiation, and particularly relates to application of the miR-1165-3p as a targetspot in preparation or screening of drugs for regulating the Th2 cell differentiation, application of the miR-1165-3p as a target spot in preparation or screening of drugs for treating asthma, application of a substance capable of increasing the expression amount of the miR-1165-3p in preparation of the drugs for inhibiting the Th2 cell differentiation, and application of the substance capable ofincreasing the expression amount of the miR-1165-3p in preparation of the drugs for treating the asthma. The invention discloses application of the miR-1165-3p applied to regulating the Th2 cell differentiation in the asthma, discussion on the Th2 cell differentiation is significant for the in-depth study of a large number of Th2 diseases, and regulation of the Th2 cell differentiation by PPM1A, abrand new field, is studied for the first time.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to the application of miR-1165-3p in preparing or screening drugs for inhibiting Th2 cell differentiation. Background technique [0002] Under the stimulation of microbial pathogens, T cells activate T cell receptors, thereby differentiating into effector T cells that produce different cytokines. Under normal circumstances, the helper T lymphocyte subset Th1 / Th2 cells are in a balanced state, and the trend of Th1 / Th2 imbalance and transformation to Th1 or Th2 state is called Th1 / Th2 drift. Th1 cells mainly produce interferon gamma (Interferon gamma, IFN-γ), tumor necrosis factor beta (TNF-β), interleukin-2 (Interleukin-2, IL-2), IL-12 and other cytokines , its main transcription factor is T-bet; Th2 cells mainly produce cytokines IL-4, IL-5 and IL-13, etc., and its transcription factor is GATA3. It is customary to call the state in which Th1 and its cytokines dominate as the Th1 state, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883
Inventor 吉宁飞黄茂王正霞陈中琦马其云孙知晓张明顺
Owner JIANGSU PROVINCE HOSPITAL
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