Application of TRAF6 inhibitor in preparation of melanoma drug
A melanoma and inhibitor technology, applied in the field of biomedicine, to achieve the effect of promoting immune function, improving effect and reducing immune tolerance
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Embodiment 1
[0027] Example 1 GEO database analysis shows that TRAF6 is a molecule that potentially regulates PD-L1
[0028]1. Search for PD-L1 in the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and the number GSE129968 is the screening data of high-throughput genetic libraries related to PD-L1. This analysis is to combine Human genome-wide CRISPR / Cas9 knockout (Genome-Scale CRISPR Knock-Out, GeCKO) lentiviral library was introduced into lung cancer cells, screened with 1 μg of puromycin for two weeks, then incubated with APC-PD-L1 flow antibody for flow cytometry Sorting, enrichment of PD-L1 high and PD-L1 low Cell. Then extract unsorted cell control group, PD-L1 low Cell experiment group, PD-L1 high The genomic DNA of the cell experimental group, two rounds of PCR amplification of the genomic DNA containing sgRNA, and finally, illumina sequencing was performed to screen out the genes corresponding to the significantly enriched sgRNA in the experimental group, so as to obtain th...
Embodiment 2
[0031] Example 2 TRAF6 can positively regulate PD-L1
[0032] 1. Construct a melanoma cell line that knocks down or overexpresses TRAF6 by means of lentiviral packaging infection. First, 2×10 6 HEK 293T cells were plated in a 6cm cell culture dish, after adding 5mL DMEM (containing 10% FBS and 1% P / S) complete medium, placed in 5% CO 2 , cultured in a cell culture incubator at 37°C. After 18-24 hours, the HEK 293T cells reached a confluence of 60%-70%, and the cell culture medium was replaced with 3 mL of 5% FBS DMEM complete medium. Add 475 μL of HBS solution into a 1.5 mL centrifuge tube, then add 5 μg of packaging plasmid, 5 μg of target plasmid (human TRAF6 empty control plasmid, TRAF6 gene cDNA lentiviral expression plasmid, human TRAF6 knockdown shRNA empty control plasmid, human TRAF6 knockdown shRNA lentiviral expression plasmids #1 and #2) were added sequentially, and finally 25μL 2.5mol / L CaCl was added dropwise 2 solution at room temperature in the dark for 20 mi...
Embodiment 3
[0037] Example 3 TRAF6 inhibitors can enhance CD8 by down-regulating PD-L1 expression + T cell activity (in vitro)
[0038] 1. The above results have proved that TRAF6 can positively regulate PD-L1, so inhibiting TRAF6 is expected to reduce endogenous PD-L1 and thus weaken tumor immune escape. Since TRAF6 also plays an important role in innate immunity and acquired immunity, it is necessary to select a suitable TRAF6 inhibitor to exert tumor killing effect without significantly affecting the normal immunity of the body. According to literature reports, parthenolide or quinine can effectively inhibit the activity of TRAF6 to inhibit the proliferation of cancer cells, and the proteasome inhibitor MG132 can slow down the proliferation of tumor cells by inhibiting the expression of TRAF6. In addition, the reversible proteasome inhibitor bortezomib can inhibit the activity of proteasome 26S subunit, reduce the degradation of NF-κB inhibitor IκB, promote the combination of IκB and ...
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