Use of a pharmaceutical composition containing γδt cells in the treatment of cancer
A technology of use and cells, applied in the direction of drug combination, medical preparations containing active ingredients, animal cells, etc., to achieve the effect of good inhibitory activity, good application prospect, and growth inhibition
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Embodiment 1V
[0034] Example 1 Preparation and Identification of VEGFR2 Monoclonal Antibody
[0035] For the first immunization, 100 μg of VEGFR2 protein (recombinant human vascular endothelial growth factor receptor-2, product number: PKA-242, Amicate Technology Co., Ltd.) was subcutaneously injected on the back of the neck of the mouse. The second immunization was carried out on the 21st day after the first immunization, and each mouse was subcutaneously injected with 100 μg of VEGFR2 protein on the back of the neck. The third immunization was carried out on the 14th day after the second immunization, and each mouse was injected with 100 μg VEGFR2 protein intraperitoneally, and blood was collected from the orbital venous plexus of the mice. The fourth immunization was carried out on the 14th day after the third immunization, and mice were injected with 50 μg VEGFR2 protein in adjacent spleen and intraperitoneal cavity respectively. Seven days after the fourth immunization, blood was coll...
Embodiment 24
[0044] Example 2 Inhibition experiment of 4E09 on liver cancer cells
[0045] (1) Seed plate: take liver cancer SMMC-7721 cells in the logarithmic growth phase and digest them with trypsin, carefully pipette them with a pipette (to avoid generating air bubbles as much as possible), mix the cell suspension evenly, and count through the cell counting board, using low serum (5%) The dilution density of the complete culture medium is 1×10 5 cells / mL of cell suspension, take out the 96-well plate, add complete medium without cell suspension to 3-5 wells as zero-adjustment wells, and accurately add 100uL cell suspension to the remaining wells, and spread the plate evenly to ensure that each well The seeded cell concentration was 1×10 4 at 37°C, 5% CO 2 Incubate the suspension cells for 24 h in an incubator.
[0046] (2) Dosing: After the cells adhere to the wall, wash them with sterile PBS or normal saline for 1-2 times, add PBS solution to the 36 wells in the outer circle, and o...
Embodiment 3
[0052] Example 3 Preparation of highly active γδT cells
[0053] Take 100 mL of venous anticoagulated blood from healthy blood donors and separate peripheral blood mononuclear cells (PBMC) with lymphocyte separation medium. Add PBMCs to γδT cell RPM11640 culture medium (containing 100mL / L calf serum, IPP 2μg / L 50mL / L human AB serum and IL-2 200IU / mL), adjust the cell density to 1×10 8 / L, set 75cm 2 In cell culture flasks, at 37°C, 50mL / L CO 2 Cells were cultured in a cell culture incubator for 10 days and harvested.
[0054] After direct staining with anti-TCRγδ-FITC, anti-CD44-FITC and the cultured cells collected on day 0 and 10 of culture, flow cytometry analysis was performed, and the negative control was FITC-labeled mouse IgG. The results showed that the number of γδT cells in PBMC was 4.57% before culture, and the number of γδT cells was 71.46% after 10 days of culture. At the same time, the expression level of CD44 reached 95.1% on the 10th day, which indicated th...
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