Study On The γTNFβ , TNF P And IFN Y In Murine Cell Line L929 And Human Cell Line Hela

The cytotoxicities of human recombinant γTNFβ (IFNγ ITNFβ hybrid protein)^ TNF P and IFN Y were measured on murine cell line L929 and human cell line Hela. L929 Cell is sensitive to TNF 3 and Hela is resistant to it. The results revealed that the cytotoxicity of Y TNF- P on cell L929(64%) was obviously higher than that of TNF P (53.9%) (pO.Ol) when at the same dose( 100u/ml)and treated time (48h), while IFN Y had no obvious effect on the L929cell. For Hela cell, the cytotoxicity effect of Y TNF P and IFN Y was very obvious but the effect of TNF P was not found.. The examination of appotosis suggested that Y TNF P and TNF P induce the appotosis of L929 cell by TUNEL method and by measuring FACS and fluoresent dying, while Y TNF P and IFN Y mainly inhibited the proliferation of Hela cell.To furrher explore the mechanism of apoptosis induced by γTNFβ or TNF P ,the activity of the caspase 8 and caspaseS were measured when cells were induced by cytokines Y TNF P. TNF- P or IFN . The results showed that the activity of caspase 8 and caspaseS obviously elevated inside the induced L929 cell by Y TNF P or TNF P while the activity had not changed when induced by IFN Y .As for Hela, caspaseS was dectected easily when induced by the three cytokines ,while the activation of caspaseS was invisible. Whether caspase 8 takes part in the process of antiproliferation need to be further investigated.To investigated the relationship of the signal transduction pathway and other signal molucules, three signal transduction inhibitors were tested for their effects on L929 cell and the change of caspase activation. The inhitors include Staurosporine (PkC inhibitor) Quinacrine (PLA2 inhibitor) andVerapamil (Ca2+ channel blocker). The results indicated that Staurosporine can increase the cytotoxities of Y TNF P or TNF P on L929 cell. On the cantrary Quinacrine or Verapamil can reduce the cytotoxicities. The activity of caspaseS and caspase3. became higher when cell was treated by Staurosporine while they are lower when treated by Quinacrine or Verapamil.To identify when the cytoxic signal transducts into the plasma and the time efffects of various inhibitor after cytokines combinds with their receptors. We tested the pretreatment of inhibitor and the pretreatment of y TNF P or TNF P . The pretreatment of inhibitor was divided into two groups: discard pretreatment liquid group and reserve pretreatment liquid group. At the same time we also tested the activity of caspase3 when cell was pretreated by VerapamiK YTNF3 or TNF-3 . The results indicated that the blocking effect on Y TNF P or TNF P induced L929 cytolysis was significantly lower in inhibitor pretreatment discarding group than in pretreatment reserved group. Pretreatment time of inhibitor was also important .The time was longer, the effect of inhibition was weaker. The results of Y TNF P or TNF P pretreatment suggested that the longer the pretreatment, the weaker the blocking effect of the inhibitor. The same conclusion was drawn from the activity of caspaseS that the stronger the blocking effect of the inhibitor the lower the caspase3 activation. There were obvious apoptosis or blocking effect when cell was pretreated with cytokins or inhibitor for 1 hour. That indicate the signal transduct is a very rapid process. That the blocking effect of discarding group is obviously lower than reserved group is suggests the remain of the cytokines or inhibitor is very important for the signal transduct.