Study Of PSMA-CAR Transfected NK Cells And Their Efficiency In Killing Prostate Cancer Cell Lines

Objective:Worldwide,cancer remains a public health problem.Conducting immune cell research is one of the most important approaches to fight cancer today,and it is of great practical importance to conduct research about NK(natural killer cells)for malignancy treatment in order to effectively control the impact of prostate cancer on human life and health.Natural killer(NK)cells,named for their ability to kill target cells autonomously,are the main effector cells of cancer in innate immunity.Most current therapeutic regimens that exploit the tumor microenvironment focus on T-cell immunity,either by promoting activating signals or inhibiting suppressive signals.the limited success of T-cell immunotherapy highlights the importance of developing new generations of immunotherapies,such as the use of previously neglected NK cells.Although tumors have also evolved to resist NK cell-induced cytotoxicity,cytokine supplementation,blockade of inhibitory molecules and genetic engineering of NK cells can overcome this resistance and hold great promise in both solid and hematologic malignancies.In this study,the study on CAR-NK(Chimeric antigen receptor NK cells)against prostate malignancies was carried out especially for PSMA(Prostate specific membrane antigen).In this study,the PEI lentiviral packaging method was used to package PSMA-CAR lentiviral vectors into lentiviruses,which were infected with natural killer(NK)cells to form PSMA-CAR-NK cells,and further in vitro experiments were conducted to test the role of these cells in cytotoxicity against prostate cancer cell lines.In order to obtain more targeted and accurate tumor killing data,in the study,we first explored for how to improve the efficiency of lentiviral infection of NK cells,and then based on the optimal ratio of effector and target cells for killing prostate cancer cell lines by transgenic cells.Methods:Chimeric antigen receptor NK cells(CAR-NK)infected with NK cells by chimeric antigen receptor packaging lentivirus can make them express chimeric antigen receptor more precisely to further specifically target recognition of PSMA and become transgenic cells to kill prostate cancer cell lines.1.After drawing healthy human peripheral blood,PBMC(Peripheral blood The ratio of NK cells was measured by flow cytometry.2.Lentivirus was prepared by transfecting 293 FT cell packaging with three vector system,and then Lentivirus was concentrated by PEG-8000 method.3.PSMA-CAR-NK cells were prepared.By comparing the transfection efficiency of NK cells induced by Lentivirus infected PBMC for 3 days with that induced by PBMC for 14 days,the best time for Lentivirus to infect NK cells was explored.4.Three prostate cancer cell lines PC3,LNCaP,DU145,transgenic cells,and prostate cancer cell lines were cultured with a target to effect ratio of 0.1:1,0.5:1,1:1,2:1,5:1,10:1,15:1,and 20:1,respectively.The killing efficiency was compared under these target to effect ratios.Results:1.The percentage of NK cells detected by flow cytometry increased from(6.68 % ±1.57 %)on day 0 to(80.58 % ± 7.51 %)on day 14.2.The 293 FT cells were observed to emit green fluorescence by inverted fluorescence microscope,so the three carrier system packaging method for preparing Lentivirus and PEG-8000 Lentivirus was successful.3.PSMA-CAR-NK cells were successfully prepared.The transfection efficiency of NK cells induced by PBMC culture for 3 days was higher than that of 14 days.Lentivirus infection of NK cells should be carried out at the early stage of NK cell culture.4.PSMA-CAR-NK cells were killed with the highest efficiency at a 20:1 ratio of effector to target cells(prostate cancer cell line).Conclusion:The results of this experimental study show that a variety of cytokine induction methods have high purity and activity in NK cells cultured in vitro,Lentivirus has high transfection efficiency for NK cells in early culture,and PSMA-CAR-NK cells have high killing efficiency for prostate cancer cell lines.As a new type of anti prostate cancer immunotherapy platform,it has great prospects,providing a research basis for the next in vivo killing experiment.