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LAT3 protein expressed siRNA in specific in-vitro interference RA synovial cells and application of LAT3 protein expressed siRNA

A synovial cell, specific technology, applied in the field of siRNA

Inactive Publication Date: 2018-11-02
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. The length of siRNA is generally about 22 nt, which belongs to double-stranded RNA in structure

Method used

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  • LAT3 protein expressed siRNA in specific in-vitro interference RA synovial cells and application of LAT3 protein expressed siRNA
  • LAT3 protein expressed siRNA in specific in-vitro interference RA synovial cells and application of LAT3 protein expressed siRNA
  • LAT3 protein expressed siRNA in specific in-vitro interference RA synovial cells and application of LAT3 protein expressed siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the expression of LAT3 in RA fibroblast-like synovial cells (RA FLS)) silenced by specifically synthesized siRNA;

[0061] Convert RA FLS to 2×10 5 seeded per well in a 12-well plate. Transient transfection was performed according to the HiPerFect transfection reagent instruction manual. First, siRNA and HiPerFect transfection reagent were mixed in 200 μL high-sugar DMEM medium (serum and antibiotic-free) for 10 min to form siRNA-HiPerFect complexes, and then added to containing In the 12-well plate of the cell suspension, the cells were collected after 24 hours to extract RNA and protein, and the interference efficiency was detected. The sequence of siRNA is as follows:

[0062] Sense strand: 5'-GGUACACACUGCCUCCUAUTT-3';

[0063] Antisense strand: 5'-AUAGGAGGCAGUGUGUACCTT-3';

[0064] The results are attached figure 1 As shown, 160nM siRNA was transiently transfected into RA FLS, and after 24 hours, the expression of LAT3 mRNA and protein in the inte...

Embodiment 2

[0065] Example 2: Detection of the invasion ability of RA FLS after LAT3 silencing by Transwell method

[0066] RA FLS with (5~10)×10 4 Cells / well were inoculated in the small inner chamber of Transwell, with a total volume of 100 μL, and the cells inoculated into the inner chamber were subjected to interference treatment. Add 500 μL of high-glucose DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin mixture) to the outer chamber of the small chamber, at 37°C, 5% CO 2 , cultivated for 24h. Both the inner and outer chambers were replaced with high-sugar DMEM medium (1% penicillin-streptomycin mixture) for starvation, 37°C, 5% CO 2 Cultivate for 8h. Replace the small outer chamber with high-glucose DMEN medium (20% fetal bovine serum and 1% penicillin-streptomycin mixture), 37°C, 5% CO 2 Cultivate for 24h. Use a pipette gun to suck up and discard the medium in the inner chamber of the small chamber (pay attention to pipetting carefully, and do not damage the ...

Embodiment 3

[0068] Example 3: After silencing LAT3 expression in RA FLS[ 3 H]-L-leucine absorption detection

[0069] RA FLS with 2 x 10 5 Inoculate each well in a 12-well plate, after adding drugs or interference treatment, at 37°C, 5% CO 2 Cultivate in the incubator for 24h. Remove the cell culture medium and absorb the solution with amino acid (125mM choline-Cl, 4.8mM KCl, 1.3mM CaCl 2 ,1.2mM MgSO 4 , 25mM HEPES, 1.2mM KH 2 PO 4, 5.6mM glucose, pH 7.4) washed twice, added 1mL of the above amino acid absorption solution, and starved for 10min in a 37°C incubator. Remove the absorbing solution and add a solution containing [ 3 H]-L-leucine absorption solution 1mL (concentration 1μCi / mL) was incubated in a 37°C incubator for 3min, the absorption solution was removed, and the absorption solution was washed twice with the cold absorption solution to remove unabsorbed amino acids. Add 200 μL of formic acid to each well, place in a water bath at 37°C for 1 hour, collect cells with liqu...

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Abstract

The invention discloses LAT3 protein expressed siRNA in specific in-vitro interference human RA synovial cells. The nucleotide sequence of the LAT3 protein expressed siRNA in the specific in-vitro interference human RA synovial cells is as follows: positive-sense strand: 5'-GGUACACACUGCCUCCUAUTT-3'; and antisense strand: 5'-AUAGGAGGCAGUGUGUACCTT-3'. The siRNA can effectively inhibit LAT3 expression in the human RA synovial cells in vitro. The invention further provides a method using LAT3 expression in siRNA in-vitro interference human TA cells and a kit for LAT3 expression in in-vitro interference human RA cells. The invention further proves that after transfection, human RA synovial cell invasion ability and amino acid uptake capacity are reduced, and a new research thought is provided for treatment and drug research and development of rheumatoid diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pair of siRNA specifically interfering with the expression of LAT3 (L-type amino acid transporter 3) in RA (rheumatoidarthritis) fibroblast-like synoviocytes in vitro and its use. Background technique [0002] LATs (L-type amino acid transporters, LATs) is a non-Na + The dependent amino acid transporter family exists on the surface of the cell membrane and is responsible for the transport of isoleucine, leucine, valine, and other large neutral amino acids, including branched chain and aromatic amino acids. In addition, it can also serve as an exchanger, exchanging intracellular glutamate while transporting extracellular neutral amino acids. So far, a total of four members of the L-type amino acid transporter family, LAT1, LAT2, LAT3 and LAT4, have been discovered. [0003] LAT3 was originally isolated from a human hepatoma cell line, and it is also distributed in skeletal muscle ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/87
CPCC12N15/113C12N15/87C12N2310/14
Inventor 潘继红赵瑜王林
Owner SHANDONG MEDICAL BIO TECH RES CENT
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