A drug for the treatment of choriocarcinoma
A choriocarcinoma and drug technology, applied in the field of biomedicine, can solve the problems of poor prognosis, enhance cell metastasis and invasion, etc.
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Embodiment 1
[0026] Example 1 Human trophoblast cell transient transfection experiment
[0027] 1. Cell culture: human trophoblast cells (JAR cells) ( HTB-144 TM ) in DMEM / F-12 medium containing 10% FBS and cultured in an incubator (37°C, 5% CO2). Change the solution every other day. After the cells grew to the logarithmic growth phase, they were digested with 0.25% trypsin.
[0028] 2. Transient transfection: JAR the cells in the logarithmic growth phase with 6×10 4 The planting density of / mL was inoculated in 6-well plates, and the transfection was performed when the cell density reached 90%. First, 4 μg of plasmids (respectively uPAR-cDNA, uPAR-mutant1, uPAR-mutant5, the vector used is pcDNA3.1his, synthesized by Takara Company) and 3 μL liposome Lipofectamine2000 (Invitrogen Company) were dissolved in 300 μL serum-free medium , let stand for 5min, then mix the two, let stand for 20min to form DNA and liposome complex. Gently wash the cells to be transfected with PBS or serum-fr...
Embodiment 2
[0031] Example 2 uPAR promotes EMT of embryonic trophoblast cells (JAR)
[0032] 1. Real-time quantitative PCR experiment
[0033] (1) Extraction of JAR cell RNA
[0034] 1) Take the transfected cells in Example 1, discard the medium, gently wash the cells 3 times with high-pressure PBS, then add 500 μL of RNAiso Plus reagent (Takara Company), fully blow the cells, and let stand on ice for 5 minutes .
[0035] 2) Transfer the cell solution to a high-pressure 1.5 mL Eppendorf tube, add 100 μL of chloroform, fully shake on a shaker for 30 s, and then stand on ice for 5 min.
[0036] 3) Centrifuge at 12000g for 10min at 4°C. After centrifugation, transfer the supernatant to a new Eppendorf tube. Be careful not to absorb the white protein layer in the middle. Then add 250μL of isopropanol, turn it upside down several times, and mix thoroughly Store overnight at -20°C.
[0037] 4) The next day, the solution was centrifuged at 12000g for 10min at 4°C, the supernatant was discard...
Embodiment 3
[0091] Example 3 Overexpression and mutation of uPAR affect its binding ability with uPA
[0092] 1. Cell Immunofluorescence
[0093] Method: The immunofluorescence experiment steps in Example 2 were followed. The primary antibody used in this experiment was: rabbit anti-human uPA IgG antibody, and the secondary antibody was: FITC-labeled anti-rabbit IgG secondary antibody.
[0094] Result: if figure 2 A. Immunofluorescence results showed that overexpression of uPAR could promote its binding ability to uPA, while the two groups of mutant group would reduce its binding ability to uPA.
[0095] 2. Protein co-immunoprecipitation experiment (co-IP)
[0096] (1) Extraction of total cell protein
[0097] 1) When the monolayer cells in the cell culture dish after transfection in Example 1 grow to 80%-90% or the number of cells reaches 2-5×10 7 First, add 1mL pre-cooled IP lysate (including protease inhibitors (1:50) and PMSF (1:100), if the protein phosphorylation level affects ...
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