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Nucleic acid molecule macrophage inflammatory protein (MIP) 3 alpha antibody to nuclear antigen (ANA) 6 and application thereof to preparation of immunosuppressive medicaments

A technology of nucleic acid molecules and immunosuppression, which is applied in the field of preparation of immunosuppressive drugs, and can solve the problems of not yet finding a specific drug, poor effect, and ineffectiveness.

Inactive Publication Date: 2011-02-23
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, immune rejection is the primary reason for the failure of various tissue and organ transplants. Immune rejection works through dendritic cells and effector cells. The application of immunosuppressants is the main treatment method. Corticosteroids, cyclosporine A and tacomol Hormone therapy is a commonly used drug, which plays a role in the immune response and can only work on effector cells. Therefore, although it has a certain therapeutic effect, the effect is not good. For high-risk patients, hormone therapy is often ineffective
At the same time, other systemic immune diseases related to MIP3α: psoriasis, rheumatoid arthritis, glomerulonephritis, renal tubule nephritis, Crohn's disease and ulcerative colitis have not yet found specific drugs

Method used

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  • Nucleic acid molecule macrophage inflammatory protein (MIP) 3 alpha antibody to nuclear antigen (ANA) 6 and application thereof to preparation of immunosuppressive medicaments
  • Nucleic acid molecule macrophage inflammatory protein (MIP) 3 alpha antibody to nuclear antigen (ANA) 6 and application thereof to preparation of immunosuppressive medicaments
  • Nucleic acid molecule macrophage inflammatory protein (MIP) 3 alpha antibody to nuclear antigen (ANA) 6 and application thereof to preparation of immunosuppressive medicaments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Transfection of MIP3αANA6 short hairpin RNA into human keratinocytes inhibits the expression of MIP3α mRNA

[0033] 1. Select HaCaT cells in good growth state, press 3.0×10 4 cells / cm 2 Inoculated in 24-well plates, cultured for 24 h, used for transfection when the cell density was about 60%-80%, washed twice with PBS buffer, and added 900 μL of fresh serum-free DK medium.

[0034] 2. Take 4 μL Lipofectamine TM 2000 Reagent into the serum-free DK medium with a final volume of 50 μL, incubated at room temperature for 5 minutes, and the nucleic acid molecule MIP3αANA6 positive and antisense double strands ( 10 nmol / L -100 nmol / L) was added to 50uL serum-free DK medium. The above two solutions were mixed, allowed to stand at room temperature for 20 min, and then added to a 24-well plate covered with cells.

[0035] 3. At 37°C and 5% CO 2 After culturing for 6-8 hours under normal conditions, change the medium to fresh DK medium containing TNF-α and IL-1...

Embodiment 2

[0039] Example 2 Interfering effect of pSUPER-MIP3αANA6 on the expression of MIP3α in human embryonic kidney cells

[0040] Experimental steps:

[0041] 1. In a standard 6-well plate, place human embryonic kidney cells (293FT) at 3×10 per well 4 cells / cm 2 Inoculate and incubate for 24 hours before transfection, the cell density is 60%-80% during transfection, wash twice with PBS buffer, add 1700μL fresh DMEM medium containing 10% calf serum; 4.0μg plasmid DNA (Contains nucleic acid molecule MIP3αANA6 DNA fragment) Add 150mM NaCl to a final volume of 150μL, take another 8μL jetPEI TM Add the transfection reagent to 150mM NaCl in a final volume of 150μL, and incubate at room temperature for 5min; mix the above two solutions, let stand at room temperature for 20min, then add to a 6-well plate with cells, and incubate at 37°C and 5% CO 2 cultivated in conditions. Cells in 2 wells were co-transfected, and 3 groups of repeated experiments were set up. And the 293 cells tha...

Embodiment 3

[0048] Example 3 In vitro effect of MIP3α stabilizing interference cell clones

[0049] Experimental steps:

[0050] 1. Select human keratinocytes in good growth state, press 3.0×10 4 cells / cm 2 Seed in 24-well plates, culture for 24 hours, and use for transfection when the cell density is about 60%-80%, wash twice with PBS buffer;

[0051] 2. Infect human keratinocytes in a 24-well plate with 500 μL of the lentiviral pHSER supernatant containing the nucleic acid molecule MIP3αANA6 DNA fragment and a final concentration of 8 mg / L Ploybrene, and incubate at 37°C and 5% CO 2 Conditioned culture; after 48h, replace with fresh RMPI1640 (10%CS), at 37°C and 5%CO 2 Conditioned cultivation. On the 6th day, GFP (green fluorescence) fluorescence was observed, and the transfection efficiency was calculated.

[0052] 3. After G418 pressure culture for 5-8 weeks, the MIP3α stable interference cell clones were screened out, and the positive clones were expanded and cultured.

[0...

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Abstract

The invention belongs to the field of antisense nucleic acid medicaments, and relates to a macrophage inflammatory protein (MIP) 3 alpha antibody to nuclear antigen (ANA) 6 and preparation thereof to the preparation of immunosuppressive medicaments. The immune response of an organism plays a key role in autoimmune diseases and the exclusive reaction of the transplantation of organs and tissues. The invention provides the antisense nucleic acid molecule MIP3 alpha ANA6 used for preparing the immunosuppressive medicaments. The MIP3 alpha ANA6 is a double-stranded nucleic acid molecule. The sense strand of a DNA sequence of the MIP3 alpha ANA6 is 5'-ATATATTGTGCGTCTCCTC-3', and an antisense strand is 5'-GAGGAGACGCACAATATAT-3'. Under the condition of culture with the addition of inflammatory cytokines, the MIP3 alpha ANA6 can remarkably reduce many kinds of cell expressing MIP3 alpha mRNA and generating MIP3 alpha proteins, and functions in immunosuppression by suppressing antigen presenting cells and T cells. The invention provides the novel antisense nucleic acid medicament for treating MIP3 alpha-related immune diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine. It specifically relates to some nucleic acid molecules capable of inhibiting the expression of human MIP3α and their application in the preparation of immunosuppressive drugs. Background technique [0002] At present, immune rejection is the primary reason for the failure of various tissue and organ transplants. Immune rejection works through dendritic cells and effector cells. The application of immunosuppressants is the main treatment method. Corticosteroids, cyclosporine A and tacomol Hormone therapy is a more commonly used drug, which plays a role in the immune response and can only work on effector cells. Therefore, although it has a certain therapeutic effect, the effect is not good. For high-risk patients, hormone therapy is often ineffective. At the same time, there are no specific drugs for the treatment of other systemic immune diseases related to MIP3α: psoriasis, rheumatoid arthritis, glom...

Claims

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Application Information

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IPC IPC(8): A61P37/06A61L27/60C12N15/63A61L27/38C12N15/113C12N5/10C07H21/02A61K48/00
Inventor 彭代智王丽华董征学
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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