Method for efficiently amplifying NK cells
A technology of NK cells and cells, applied in the field of cell culture, can solve the problem of weak killing ability of macrophages and B cells, and achieve the effect of enhancing killing function and promoting proliferation
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Embodiment 1
[0026] According to the method of the present invention, expand and cultivate NK cells with the following specific steps:
[0027] (1) Use the lentiviral transfection system to simultaneously transfect the genes of CD8α-interleukin 21, CD19, CD137L, CD86, and CD64 into K562 cells. CD8α is a membrane protein expressed on the cell membrane, and the CD8α gene is linked to the interleukin-21 gene so that Interleukin 21 is expressed on the cell membrane and becomes a transmembrane protein; CD19, CD137L, CD86, and CD64 are membrane proteins, and the vector contains a viral promoter and a selection marker gene; when the foreign expression vector enters the host cell, the promoter is activated, After being cultured for a period of time, transmembrane IL-21, CD19, CD137L, CD86, and CD64 can be expressed on the K562 cell membrane. The above cells were sorted by flow cytometry to obtain K562 cells stably expressing CD8αIL-21, CD19, CD137L, CD86, and CD64 as trophoblast cells of NK cells;...
Embodiment 2
[0036] The NK cells prepared in Example 1 were tested by flow cytometry, and the NK cells amplified by traditional IL-2 and K562 engineered cells were used as controls. Analyze the proportion of NK cells to the total cells (such as image 3 shown), the expression of Fc receptor CD16 on the surface of NK cells (such as Figure 4 shown), the expression of NK cell surface activation receptor NKG2D (such as Figure 5 shown) and the expression of the inhibitory receptor CD158α on the surface of NK cells (such as Figure 6 shown). image 3 , Figure 4 , Figure 5 and Figure 6 Show, utilize the NK cell that the method for the present invention prepares, the purity of NK cell (being CD3 - CD56 + ratio) was 97.8%, significantly higher than 86.1% of the control group; and the expression of CD16 on the surface of NK cells prepared by the method of the present invention was 88.7%, which was also significantly higher than 76.3% of the control group; in addition, using the method of...
Embodiment 3
[0038] The determination of NK cell killing rate was carried out according to the following operations:
[0039] (1) Target cell preparation: take K562 and MCF-7 cells in good growth state, centrifuge at 1000rpm for 5min, and count the cells; use RPMI-1640 complete medium to adjust the cell density to 2×10 5 / mL, spare;
[0040] (2) Preparation of effector cells: take NK cells in good growth state, centrifuge at 1500rpm for 5min, count the cells and adjust the cell density to 2×10 with RPMI-1640 complete medium 6 / mL, spare;
[0041] (3) Co-cultivation of target cells and effector cells: Add 10,000 target cells (ie 50uL) in a 96-well cell culture plate, and add effector cells (ie NK cells) at a ratio of 1:1, 5:1 and 10:1 , to a final volume of 100uL / well; and set blank control wells, target cell control wells and corresponding effector cell control wells, set 3 replicate wells for each well, and place the cell culture plate at 37°C, 5% CO 2 Cultivate in the incubator for 8 ...
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