A kind of immune cell and its preparation method and application
A technology of immune cells and cells, applied in the field of cell biology, can solve the problems of low efficiency of malignant tumors and low effective ratio of therapy, and achieve the effect of overcoming the low effective ratio
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Embodiment 1
[0067] 1. Mice and cell lines
[0068] C57BL / 6J (H-2Kb) mice were purchased from Shanghai Experimental Animal Center, Numb f / f 、Numblike f / f 、Numb&Numblike f / f Mice were donated by Professor Li Huashun of Tongji University School of Medicine, and CD11c-cre mice were purchased from Shanghai Nanfang Model Company. will Numb f / f 、Numblike f / f 、Numb&Numblike f / f The mice were crossed with CD11c-cre mice to obtain Numb KO 、Numblike KO 、Numb&Numblike KO DC mice and their respective littermate control mice Numb f / f 、Numblike f / f 、Numb&Numblike f / f . Schematic such as figure 1 shown.
[0069] 1.1 Laser confocal observation of the subcellular localization of Numb molecules
[0070] Isolate and culture mouse spleen dendritic cells, add LPS (1μl / ml) to stimulate, collect cells at designated time points, take cell pellet after centrifugation, resuspend in 100μl PBS, label with anti-mouse FITC-CD11c fluorescent antibody for 15min, PBS Wash 3 times. Fix with 2% paraformalde...
Embodiment 2
[0096] 1. Antigen-loaded DC-CTL cells
[0097] 1.1 Melanoma cell culture and antigen preparation
[0098] Melanoma cells B16F10 were cultured in DMEM medium (containing 10% FBS) at 37°C, 5% CO 2In the incubator, subculture every 2-3 days, collect the B16F10 cells in the logarithmic growth phase, and use sterile saline to adjust the cell concentration to 2×10 7 / ml, freeze-thawed three times with liquid nitrogen, and filtered at 0.45 μm to obtain the whole tumor antigen for future use.
[0099] 1.2 DC antigen loading
[0100] Adjust the cell density of the above-mentioned Numb / Numblike knockout DC cells to 1×10 6 / ml, tumor cell antigen (DC:tumor cell=1:10, number ratio) was added, and only culture medium was added to the control group. At 37°C, 5% CO 2 Incubate for 48 hours under the same conditions, add 10 μg / ml TNF-α, continue to culture for 48 hours, collect cells and supernatant for use.
[0101] 1.3 Antigen-loaded DCs induce CTL cells
[0102] The spleen cell suspe...
Embodiment 3
[0114] Construction of mouse subcutaneous melanoma model and cell therapy
[0115] Collect the B16F10 cells in the logarithmic growth phase and resuspend them with an appropriate amount of PBS to make the density 1×10 7 cells / mL.
[0116] Take 100 μL of cell suspension and inoculate C57 mice. On the 5th day after inoculation of B16F10 cells, tumor-bearing mice were injected with 1×10 6 A spleen-derived DC or bone marrow-derived DC and control DC were also set up as a negative control group injected with PBS. Mice were sacrificed on day 27 after inoculation with B16F10, and subsequent experimental analysis was performed, including tumor weight measurement and tumor infiltrating lymphocyte content detection. The result is as Figure 14 and 15 shown.
[0117] Figure 14 Among them, dendritic cells knocked out of Numb and Numblike proteins can effectively inhibit tumor growth.
[0118] Figure 15 Among them, the content of tumor-infiltrating lymphocytes increased in mice ...
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