| ObjectiveTo study the specific cytotoxicity and mechanism of cytotoxic T lymphocyte(CTL) in vitro induced and activated by dendritic cells loading with freeze-thrawing antigen from heat-shocked renal tumor cells,and maybe it will provide a new simple effect way to treat advanced renal cell carcinoma.Methods1.Cultivation of renal carcinoma cells786-0 cells were cultured and collected in exponential phase of growth to prepare for the experiment.2.Cultivation of dendritic cells(DC),morphological identification and detection of surface marker on DC(1) Peripheral blood monouclear cells(PBMC) were separated from peripheral blood of healthy human by density gradient centrifugation.After a short peroid of cultivation,the adherent cells were cultured in three culture flasks with mixture of GM-CSF and IL-4 for 4 days and TNF-αfrom the 5th day.(2) Freeze-thrawing antigen from heat-shocked renal tumor cells(named HSAg) were put to the first culture flask in the 6th day,and DC in which were named HSAg-DC group.Freeze-thrawing antigen(named Ag)were put to the secend culture flask in the 6th day,and DC in which were named Ag-DC group.PBS were put to the last culture flask in the 6th day,and DC in which were named non-Ag-DC group.(3) DC were taken pictures with light microscope(LM) in the sencond day,the 5th to 6th day and the 7th to 8th day.DC were taken pictures with scanning electron microscope(SEM) in the 3th to 4th day and the 7th to 8th day.(4) 4 kinds of surface marker(CD1a,CD83,CD80,CD86) of DC in HSAg-DC group and Ag-DC group were detected with flow cytometer(FCM) in the 7th to 8th day of cultivation of DC.3.Cultivation of T lymphocyte cells and mixed lymphocyte reaction(MRL)(1) Float cells that from PBMC after a short peroid of cultivation are filled into nylon piller and then T lymphocyte cells were separated.T lymphocyte were cultured with IL-2 for days.(2) Three groups of DC in the 8th day were mixed with T lymphocyte by a ratio of 20 to 1 and then were induced as 3 groups of CTL,named HSAg-DC-CTL group,Ag-DC-CTL group,non-DC-CTL group.4.Cytotoxic experiment and detection of killing activity of CTLThree groups of CTL were mixed with renal carcinoma cells by different ratio(10: 1,20:1,50:1).Optical density of each cultural hole was detected with MTT method and finally the killing ratio of 3 groups of CTL were calculated with the formula.Results1.Morphological identification DCMature DC were induced from naive DC with combination of GM-CSF,IL-4,TNF-αand tumor antigen and characterized as typical dendrite-like.2.Detection of surface marker on DCFour kinds of surface marker(CD1a,CD83,CD80,CD86) of DC in HSAg-DC group and Ag-DC group were detected with flow cytometer(FCM) in the 7th to 8th day of cultivation of DC,the result of which demonstrated that express of surface maker of HSAg-DC group was high than that of Ag-DC group and statistical significance exist among express of CD1a,CD83 and CD86(P＜0.05).3.Detection of killing activity of CTLAll groups of CTL could kill renal carcinoma cells,and the killing effect increase as the ratio of effective cells to target cells increase.At the same ratio,HSAg-DC-CTL group has the strongest killing effect,Ag-DC-CTL group less,and non-Ag-DC group least.Statistical significance exist among killing effect of each group(P＜0.05). Conclusion1.The rusults of LM,SEM,FCM show that DC can get mature by using TNF-αand renal carcinoma antigen.2.The rusults of detection of surface marker on DC show that the presentational ability of DC can become stonger obviously after loading with freeze-thrawing antigen from heat-shocked renal tumor cells.3.The rusults of detection of killing activity of CTL with MTT method show that DC loading with freeze-thrawing antigen from heat-shocked renal tumor cells can induce CTL with stong killing effect.4.Our study shows that DC loading with freeze-thrawing antigen from heat-shocked renal tumor cells have stong anti-carcinoma immune effect,and maybe it will provide a new simple effect way to treat advanced renal cell carcinoma.
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