Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of micromolecule RNA used as immunosuppressor

An immunosuppressant and small molecule technology, applied in the field of biomedicine, can solve problems such as side effects and achieve low toxicity

Inactive Publication Date: 2014-01-15
SHENZHEN UNIV
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since these drugs directly inhibit the phosphatase activity of CaN, they have serious side effects (such as nephrotoxicity, neurotoxicity, hypertension, diabetes, gastrointestinal disorders, etc.)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of micromolecule RNA used as immunosuppressor
  • Application of micromolecule RNA used as immunosuppressor
  • Application of micromolecule RNA used as immunosuppressor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of small molecule RNA miR-124a

[0027] The mature sequence of miR-124a is 5'-uaaggcacgcggugaaugcc-3' (20bp, SEQ ID NO: 1), which can be prepared in the following manner.

[0028] 1. Chemical synthesis

[0029] The mimic of miR-124a (miRNA mimic) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., which is a double-stranded RNA that can mimic the high-level expression of endogenous mature miR-124a; standard purification, stored at -20°C. Dissolve in RNase-free water to prepare a 25 μM stock solution, store at -80°C for later use, and dilute to the required concentration when used.

[0030] 2. Expression of miR-124a mediated by DNA vector or viral vector

[0031](1) Preparation of expression vector of pri-miR-124a

[0032] The mature sequence and precursor sequence (pre-miRNA) information of human miR-124a was obtained in the miRBase (http: / / www.mirbase.org / ) database. Analysis shows that the mature sequence of miR-124a has three precurs...

Embodiment 2

[0038] Verification of the target gene of embodiment 2miR-124a

[0039] Cell culture: 293A cells (purchased from ATCC, Manassas, VA) were cultured in 10% FBS-DMEM medium (FBS was purchased from Gibco, and DMEM was purchased from Hyclone). Cells in good growth state were collected, counted by centrifugation, and counted at 6×10 4 Inoculate / well in a 24-well plate at 37°C, 5% CO 2 Cultivate for 24h.

[0040] Transfection: When the cell density reaches 60-70%, cells are transfected with PEI, co-transfected with 3'-UTR reporter vector or mutated 3'-UTR reporter vector (50ng), miRNA overexpression vector pENTR / CMV-EGFP - miR-124a or pENTR / CMV-EGFP-control (500ng) and the internal reference vector phRL-TK (10ng), each experiment was done in 3 replicate wells. Two days after transfection, discard the culture medium, wash twice with PBS, and then lyse the cells with 50 μL of 1× Passive Lysis Buffer.

[0041] Luciferase detection: Use the Dual-Luciferase Reporter Assay System (E191...

Embodiment 3

[0044] Example 3 Inhibition of NFAT activity by miR-124a on human embryonic kidney epithelial cell line (293T)

[0045] Cell culture: 293T cells (purchased from ATCC, Manassas, VA) were cultured in 10% FBS-DMEM medium (FBS was purchased from Gibco, and DMEM was purchased from Hyclone). Cells in good growth state were collected, counted by centrifugation, and counted at 3×10 5 Inoculate each well in a six-well plate at 37°C, 5% CO 2 Cultivate for 24h.

[0046] Transfection: 293T cells (3×10 5 / well) inoculated in a 6-well plate, cultured overnight, and when the cell density reached 60-70%, the cells were transfected with PEI, and the plasmids pLVX-cMyc-NFATc2 (300ng) and pENTR / CMV-EGFP-miR-124a were transfected at the same time (1500ng), the control group was co-transfected with pLVX-cMyc-NFATc2 and pENTR / CMV-EGFP-control. Two days after transfection, cells were treated with Ionomycin (Sigma) at a final concentration of 1 μM and CaCl at a final concentration of 1 mM. 2 Cel...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses application of micromolecule RNA (Ribose Nucleic Acid) used as an immunosuppressor, and particularly relates to application of the micromolecule RNA which has a base sequence of SEQ ID NO:1 and is used as the immunosuppressor to preparation of a medicament for treating disease caused by abnormal activation of an NFAT (Nuclear Factor Activated T Cell) signal pathway. The micromolecule RNA in the invention uses NFAT as a target, inhibits the NFAT signal pathway by inhibiting a key factor in the NFAT signal pathway, can obviously inhibit activity of NFAT and can be used as the novel immunosuppressor for treating organ transplantation rejection reaction and autoimmune diseases. The micromolecule RNA in the invention is an endogenous RNA of a human body and has low toxicity for the human body.

Description

technical field [0001] The present invention belongs to the field of biomedicine, more specifically, the present invention relates to the application of a microRNA (miRNA), in particular to the application of miR-124a as an immunosuppressant in the preparation of drugs for diseases caused by abnormal activation of the NFAT signaling pathway . Background technique [0002] Immunosuppressant (immunosuppressant) is a class of chemical or biological substances that reduce tissue damage by inhibiting cellular and humoral immune responses. Such as rheumatoid arthritis, rheumatic fever, collagen disease, systemic lupus erythematosus, dermatophytosis, ankylosing spondylitis, membranous glomerulonephritis, idiopathic thrombocytopenic purpura, inflammatory bowel disease and autoimmune hemolytic anemia, etc.). [0003] microRNAs (miRNAs) are about 22 nucleotides (nt), non-coding small molecules of single-stranded RNA, which widely exist in animals, plants and viruses, and negatively ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K48/00A61P37/06
Inventor 苟德明康康王玉娜张小英
Owner SHENZHEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com