The Study Of The Sensitivity Of Cervical Carcinoma Cell Towards Trail And The Expression Pattern Of Trail Receptors

[Objective] : To study the role of TRAIL in the treatment of cervical carcinoma, we analyzed the sensitivity of cervical carcinoma cell to TRAIL killing in the context of the expression of different TRAIL receptors, and we studied the relative expression level of death receptors (DR4, DR5) and decoy receptors (DcR1, DcR2) of TRAIL in cervical carcinoma cell line Hela. We hope to investigate the degree of TRAIL-induced apoptosis in cervical carcinoma, which may offer useful suggestions to the clinic physicians to treat the cervical carcinoma patients.[Methods]: The cervical carcinoma cell line Hela is cultivated in culture medium of RPMI-1640 +10% calf serum . (1) Hela cells are seeded into 96-well plate, and there are 2×10~4 cells each well. Different final concentration of TRAIL protein (10,20,100,200μg.L~-1) are added into Hela. After 24 hours cultivation, 10μL MTT (0.5g.L~-1) are added into every well, then incubate for hours. Discard the supernant and add dimethylsulfoxide into every well, mix together sufficiently. Under 490nm wavelength measure the value of Absorbance. (2) Extract total RNA from Hela and immediately do the RT-PCR reaction. The amplification products are runned on 2% agorse gel, and stained with ethidium bromide, photographed and analyzed by imageanalysis software. (3) Pellets of Hela is dissolved in 200ul PBS. luL monoclonal anti-human antibody (little rat resist human DR4, DR5, DcRl, DcR2) is added into different tube, incubated at 4°C for 30 minutes, then washed twice with cold PBS. Cell pallets are suspended into PBS solution in which contain FITC-labeled antibody, incubated at 4°C for 30 minutes. After washing, measure average fluorescent intensity of each sample.[Results] : Cervical carcinoma cell Hela has high sensitivity towards TRAIL-induced apoptosis. Under the lower concentration (lOOng / ml) of TRAIL, near to 50% cell were killed. The expression of TRAIL decoy receptors mRNA in Hela is weaker than that of death receptors, and there is no remarkably difference between the decoy receptors and between the death receptors. Expressions of receptor protein in Hela were DcRl 17.7%^ DcR2 5.3% ^ DR4 99.9%, DR5 97.8%.[Conclusion] : In the study, we add TRAIL into Hela, measure the rate of apoptosis in MTT; and extract total RNA from Hela, RT-PCR reaction is done immediately,then get the mRNA of TRAIL receptors; and use FCM to measure the protein of TRAIL death receptors(DR4^DR5 )and decoy receptors (DcRl >DcR2) on the surface of Hela. The human cervical carcinoma cell line Hela has high sensitivity to TRAIL and the expressions of decoy receptors (DcRl ^ DcR2) in Hela are much lower than that of death receptors (DR4n DR5) , and there are not remarkably difference between the decoy receptors and the death receptors. We conclude that application of TRAIL in molecular therapy of human cervical carcinoma may have a widely applied foreground.