A kind of preparation method of autologous trophoblast and the culture method of SNK cell
A technology of trophoblasts and culture methods, which is applied in the fields of medicine, immunology, cell biology and molecular biology, and can solve the problems of low NK activity, low killing activity of solid tumors, and low proportion
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[0023] The present invention provides a preparation method of autotrophic cells, comprising the following steps:
[0024] 1) connecting the 41BBL-MICA fusion gene to a lentiviral expression vector, and transferring it into competent cells to obtain a lentivirus containing the 41BBL-MICA fusion gene;
[0025] 2) Infect the peripheral blood mononuclear cells with the lentivirus containing the 41BBL-MICA fusion gene obtained in step 1), and culture them for more than 14 days, and the obtained CD3-41BBL+MICA+ cells are autotrophoblast cells.
[0026] In the invention, the 41BBL-MICA fusion gene is connected to a lentiviral expression carrier, and transformed into a competent cell to obtain a lentivirus containing the 41BBL-MICA fusion gene. In the present invention, the 41BBL-MICA fusion gene has the effect of activating NK cells and stimulating the expansion of NK cells. In the present invention, the nucleotide sequence of the 41BBL-MICA fusion gene is shown in SEQ ID No.1, deta...
Embodiment 1
[0040] 1. Fusion gene 41BBL-MICA (SEQ ID No.1).
[0041] 2. Lentiviral packaging
[0042] The MICA-41BBL fusion gene was synthesized for the whole gene, and the virus supernatant was harvested through lentiviral packaging, concentrated and then transfected into PBMC. The specific operation process is as follows:
[0043] 1) Use gene synthesis technology to synthesize the fusion gene of MICA+41BBL (Jinweizhi Biotechnology Co., Ltd.), clone it into the pCDH vector, extract the plasmid through transformation and amplification, and obtain a sufficient amount of the target gene plasmid. The specific operation is as follows:
[0044] a) Take out the competent cells from the refrigerator at -80°C, place them on ice for 10 minutes to slowly dissolve them, take 1 μl of the plasmid and add them to 100 μl E. coli competent cells DH5α, and place them on ice for 30 minutes.
[0045] b) Insert the above-mentioned centrifuge tube on the float and place it in a 42°C water bath for heat shock...
Embodiment 2
[0074] 1. Culture of SNK cells:
[0075] a) Separate human peripheral blood mononuclear cells, centrifuge and count, and resuspend with 1640+10% FBS+200IU / mL IL-2;
[0076] b) According to the ratio of 200:1, add the above-mentioned autologous trophoblast cells; 37°C, CO 2 incubator cultivation;
[0077] c) According to the actual volume, add 500IU / mL of IL-2 every day, and change the medium in half when the medium turns yellow according to the growth of the cells;
[0078] d) SNK cells are obtained when cultured to 14-28 days.
[0079] 2. Flow cytometric detection of SNK phenotype
[0080] After the cells were cultured for 14 days, the expressions of CD3, CD4, CD8, CD56, and CD16 were detected by flow cytometry, and the phenotype of SNK was analyzed. Specific steps are as follows:
[0081] a) Take the SNK cells cultured for 14 days, centrifuge at 1000rpm for 5min, discard the supernatant, add 10ml PBS to wash once, and discard the washing solution.
[0082] b) The cells...
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