The Study On The Apoptosis Of Stomach Adenocarcinoma Cells Induced By Tumor Infiltrating Lymphocytes

【OBJECTIVE】To investigate the effect of tumor infiltrating lymphocytes( TIL) against stomach adenocarcinoma cells(SGC-7901) via inducing cancer cells apoptosis as well the variation of cytokines secreted by TIL.【METHODS】Tumor infiltrating lymphocytes were extracted from fresh stomach adenocarcinoma tissue,and cultivated at least for 14 days in 2500IU/ml IL-2 RPMI1640 medium with 10% new cattle serum. TIL and SGC-7901 were Mixed according to effector-target ratios( E:T )10:1,50:1 for 2h, 6h,12h,24h. The apoptotic rate were detected for all by Flow cytometry (FCM) at one same time after SGC-7901 were isolated and labeled with Annexin V and PI . SGC-7901 which were treated for 48h by TIL was also detected with TUNEL(terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling)for the internucleosomal DNA strand breaks. Agarose Gel electrophoresis of DNA from control and treated group cells was performed to detect chromatin cleavage in apoptotic tumor cells . ELISA assay was performed to detect the Concentration of TNF-α,IFN-γin the supernatant of TIL(2×106/ml×2ml) getting in 4th,6th,8th,10th,12th,14th day.【RESULT】 TUNEL assay and DNA ladder agarose Gel electrophoresis both demonstrate the occurrence of apoptosis in SGC-7901 which were treated by TIL. Statistical analysis with Flow cytometry detection result indicates that significant statistic difference is exist between disposal groups and negative control groups(p<0.01) about the apoptotic rate,and the difference is also exist within disposal groups(p<0.05). 2h group show remarkably higher apoptotic rate, but 6h group has lower rate,and the apoptotic rate of 12h group recovers even surpasses 2h group,rate.In generally, the apoptotic rates of disposal groups take on a look of wave,and 12h group has the highest apoptotic rate.Another ,the apoptosis rate dependent on the E:T. The Concentration of TNF-alpha intendes to step-up with the elongation of cultivated time.Data shows the12th day of cultivated time possibly is the turn point of acute increasing. In generally,IFN-gamma intends to step-up with the elongation of cultivated time ,and its dose changes more remarkably after 10-12th day of cultivated time.【CONCLUSION】 TIL posses the apoptotic killing-effect agaist adenocarcinoma with its effect occuring in acute and chrinic patterns,and up to its summit in about 12h. The killing-effect of TIL also dependent on the E:T. TNF-α,IFN-γconcentration turn on almost the same trend to step-up with the elongation of cultivated time, which imply some degree restore of the antitumor activity of TIL.