The Effects Of BCG-PPD In Γ△T Cells On Lung Cancer A549 Cells

Objective:To research the effect of Purified Protein Derivative of BCG(BCG-PPD) on the expression of perforin and granzyme B of y8T cells, and also to study the synergistic action of BCG-PPD on γδT cells killing lung cancer A549 cells.Methods:(1)To prepare y8T cells:Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of healthy volunteers with Ficoll density gradient centrifugation method, and y5T cells were amplified by Zoledronic acid(ZA,5nmol/ml) and Interleukin-2(IL-2,200IU/ml).(2)To determine the best E:T ratio:The proliferation capacity of γδT cells was assessed by flow cytometry(FCM) after 9-14 days cultivated.γδT cells and lung cancer A549 cells were co-cultured with different effector-target ratio(E:T).After 48 hours cultivated,we observed the growth status of lung cancer A549 cells by inverted microscope and measured the cytotoxicity of γδT cells against lung cancer A549 cells by LDH Cytotoxicity Assay Kit,then we would get the best E:T ratio.(3)To test the related indicators after treated by BCG-PPD.γδT cells and lung cancer A549 cells were co-cultured with the best E:T ratio, and BCG-PPD at various concentrations was uesed to change the effect of γδT cells on lung cancer A549 cells.We observed the dynamic changes of lung cancer A549 cells by inverted microscope.After 48 hours cultivated,we measured the cytotoxicity of γδT cells against A549 cells by LDH Cytotoxicity Assay Kit and the expression of perforin and granzyme B of γδT cells by flow cytometry,and observed the change of cellular morphology of lung cancer A549 cells by transmission electron microscope(TEM).Results:(1)The percentage of γδT cells in PBMC increased from (4.65±4.36)% to (83.82±2.18)% after 9-14 days cultivated in vitro.(2)The cytotoxicity of γδT cells against lung cancer A549 cells increased with E:T ratio.We chose 20:1 as the best E:T ratio.(3)BCG-PPD at concentration of 20IU/ml could significantly enhance the cytotoxicity of γδT cells against lung cancer A549 cells and the expression of perform and granzyme B of γδT cells,and the growth status of lung cancer A549 cells showed different.The results of TEM showed that the integrality of A549 cell membrane was destroyed, nuclear membranes disappeared and chromatin occured pyknosis and chromatolysis.Conclusion:γδT cells expanded in vitro had cytotoxic effect on lung cancer A549 cells;BCG-PPD could enhance the expression of perform and granzyme B of γδT cells,accelerate the cellular morphology change of lung cancer A549 cells,and increase the killing effect of γδT cells on lung cancer A549 cells.