Study On Effect Of ??T Cells Expanded In Vitro To Kill Hepatocellular Carcinoma Cells

Objective: To investigate stimulation of expanded ??T cells in vitro to kill hepatocellular carcinoma cells and its mechanism and validation in vivo.Methods: 1.Peripheral blood mononuclear cells(PBMC)were isolated from heparin anticoagulated venous blood by density gradient centrifugation.The stimulation was amplified in five groups in vitro,and the negative control group was divided into two groups.One group only was added interleukin-2(IL-2),the other group plus phytohaemagglutinin(PHA),experimental group plus phosphorylated antigen 1-hydroxy-2-methyl-2-buten-4-yl-4-diphosphate(HDMAPP),zoledronic acid(ZOL)and mycobacterium tuberculosis heat resistant antigen(Mtb-HAg),in which PHA stimulates activation of ??T cells,and HDMAPP,ZOL,and Mtb-HAg stimulate activation of ??T cells.Subsequently,each group was separately added with IL-2 to maintain the proliferation.Morphological changes were observed under real-time microscope.2.The proportion of ??T cells in T cells in the control group and the experimental group after 12 days of culture in vitro was determined by flow cytometry.??T cells were selected as effector cells and HepG2 cells as target cells in the cytotoxicity experiment.After stimulation and expansion in vitro,the killing ratios at 0:1,10:1,and 40:1 were determined and compared by flow cytometry.Live cell workstation was used to observe the picture of the killing effect.3.To consider investigating the killing mechanism,the natural killer cell(NK)activating receptor NKG2D(natural-killer group 2,memberD,NKG2D)blocker was used to block effector cells from identifying target cells and a specific inhibitor of extracellular signal regulatedkinase(ERK)1/2 PD98059 was used to block intracellular signaling pathways.4.The nude mice tumor model was established in two batches,the tumor growth curve was drawn and the tumor formation effect was tested by small animal imager to verify the killing effect of ??T cells.Results: The ??T cells accounted for about 1-10% in T cells,and the proportion of ??T cells in T cells in PBMC was(4.64±0.37)%,which was within the normal range.When ??T cells were cultured in vitro for 12 days,the ??T cells in the three experimental groups showed a large amount of amplification(P<0.01),and the amplification efficiency reached 90%.The experimental group and the control group showed significant differences(P<0.01),in which the proportion of ??T amplified by HDMAPP and ZOL stimulation was significantly higher than that of Mtb-HAg group(P<0.05).In the killing experiment,the killing effect increased with the increase of the effective target ratio.The killing rate of ??T cells stimulated by ZOL in the experimental group was significantly higher than that in the HDMAPP group and the Mtb-HAg group(P<0.05).A dynamic killing process was observed under the living cell workstation,and the target cells showed obvious atrophy and necrosis in the image.In the blocking experiment,the blocking effect of NKG2 D blocker and ERK1/2 signaling pathway blocker was verified.When PD98059 was used to treat ??T cells and the target ratio was 40:1,the killing effect was observed in each experimental group.It can be seen that the blocking effect of PD98059 is stronger than that of NKG2 D blocker(P<0.05).Among them,in the three experimental groups,in the HDMAPP group,when the target ratio was 40:1,the NKG2 D blocker showed a significant blocking effect(P<0.05).In the ZOL group,when the effect ratio was 10:1,the effector cells were significantly blocked after treatment with PD98059(P<0.05),further illustrating the major blocking effect of PD98059.It can also be said that the intracellular signaling pathway plays an important role when effector cells kill liver cancer cells.Experiments in vivo basically verified the killing effect of ??T cells.According to the tumor growth curve,there was a difference between the experimental group and the control group after cell treatment(P<0.05).Tumors appeared in the control group of the small animal imager,but no tumor signal was observed in the experimental group.Conclusion: HDMAPP,Mtb-HAg and ZOL can achieve the purpose of stimulating the amplification of ??T cells in vitro,and the amplification efficiency of HDMAPP and ZOL can reach over 90%.HDMAPP,Mtb-HAg and ZOL stimulated the amplification of ??T cells to kill HepG2.The killing rate of ??T cells stimulated by ZOL and Mtb-HAg was higher than that of HDMAPP group.In comparison,ZOL has high amplification efficiency and good effect on killing tumor cells.