nk92 cells modified by il7 and il21, preparation method and application thereof
A technology of NK cells and cells, applied in the field of biomedicine, can solve the problems of irreversible and poor cell status.
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Embodiment 1
[0228] Example 1 Construction of NK92MI / IL21 and NK92MI / IL7&21 cell lines
[0229] Molecular structure design of the first fusion protein: GM-CSF signal peptide-IL7 sequence-hinge region-Fc segment-CD4 transmembrane region
[0230] Molecular structure design of the second fusion protein: GM-CSF signal peptide-IL21 sequence-hinge region-Fc segment-CD4 transmembrane region
[0231]The first fusion protein coding sequence and the second fusion protein coding sequence are synthesized by gene synthesis, and the present invention synthesizes the first fusion protein coding sequence (SEQ ID NO.:9) and the second fusion protein coding sequence (SEQ ID NO. : 10) were respectively constructed on the pHULK piggyBac N-Comet GFP vector (BioVector NTCC Plasmid Vector Strain Cell Gene Collection Center), and the IL7-pHULK piggyBac N-Comet GFP plasmid and the IL21-pHULK piggyBac N-Comet GFP plasmid were successfully constructed. First, the IL21-pHULK piggyBac N-Comet GFP plasmid was electrop...
Embodiment 2
[0233] Example 2 Comparison of cytotoxicity and in vitro proliferation of NK92MI, NK92MI / IL21 and NK92MI / IL7&21 cells
[0234] In the in vitro killing test, CFSE / 7-AAD flow detection method was used for the detection of killing results. Specifically, the target cells were taken in a 15ml centrifuge tube, resuspended in PBS, added a small amount of CFSE and incubated at 37°C for 30 minutes, and the target cells were taken. Cell 4×10 5 each in a 24-well plate, then add the corresponding number of effector cells, mix well, and the final system is 1.5ml. Effector cells and target cells were co-cultured, then the supernatant was removed, washed once with PBS, and after resuspended in PBS, 2ul 7-AAD was added to each group, and then detected on the FACS Calibur machine. The CFSE positive cell population is the target cell, and the proportion of the 7-AAD positive cell population in this cell population is the death rate of the target cell.
[0235] NK92MI, NK92MI / IL21 and NK92MI / I...
Embodiment 3
[0245] Example 3 Phenotype analysis of PBMC cells co-cultured with NK92MI, NK92MI / IL7, NK92MI / IL21 and NK92MI / IL7&21 cells
[0246] In order to understand whether NK92MI, NK92MI / IL7, NK92MI / IL21 and NK92MI / IL7&21 cells have influence on the activation of T cells in normal people after entering the human body, the present invention has carried out a simulation test in vitro. The present invention extracts the peripheral blood of three normal people, extracts PBMC cells respectively, and divides the obtained PBMC cell counts into 5 groups (placed in 24-well plates for culture, and the medium used is RPMI1640). The first group puts 1×10 6 PBMC cells, without any treatment, as a negative control for flow cytometry analysis; put 1×10 in the second group 6 PBMC cells, add IL2 and 2012 cells to activate and expand T cells, as a positive control for flow analysis; put 1×10 in the third group 6 PBMC cells, then add 1×10 5 NK92MI cells were co-cultured; the fourth group put 1×10 6 P...
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