Research Of Oral Squamous Cell On The Endoplasmic Reticulum Apoptosis Pathway Induced By The Component ? From Agkistrodon Acutus Venom

Objective:To investigate the effects of endoplasmic reticulum apoptosis pathway induced by AAVC-I(Agkistrodon acutus venom component-I,AAVC-I)on oral squamous cell carcinoma Tca8113.Methods: The Tca8113 cells was chosen as the research object and were cultured in vitro conditions.1.Using DTT(dithiothreitol)as endoplasmic reticulum stress inducers referring to demands for the experimental,observing cell morphology changed by inverted microscope after different concentrations of DTT and AAVC-I in different time periods,to select the optimum concentration range and action time of AAVC-I induced human oral squamous cell carcinoma Tca8113 cells may occur endoplasmic reticulum stress;2.MTT assay detect the proliferation inhibition rate change of Tca8113 cell in different concentrations and different time periods after AAVC-I and DTT;3.Experimental grouping,set the normal control group,the experimental group(Different concentrations AAVC-I 3.125?g/m L,6.25?g/m L,12.5?g/m L acting successively on the Tca8113),after normal cultured 12 h,24h using RT-PCR technique,Western blotting to detect the expression of ERS specific indicators CHOP,GRP78,Caspase-4 at the RNA level and protein level in different periods;4.By immunocytochemistry detect qualitativly the expression of CHOP,GRP78,Caspase-4 in cells under the action of different concentrations AAVC-I;5.By RT-PCR,Flow Cytometr to detect the change of expression of CHOP,GRP78,Caspase-4 and apoptosis rate when the occurrence of AAVC-I induced endoplasmic reticulum stress response after using CHOP inhibitor.Results:1.Observing through the inverted microscope normal Tca8113 cells grow adhering to the wall,polygonal or spindle and the cytoplasm density is uniform,DTT group,with the increasing of DTT(1mmol/L,2mmol/L,4mmol/L,8mmol/L)concentration and duration of action(2h,4h)extended,the number of cells semi-attached to the wall gradually increased,cells rounding and shrinkaged,the cell gap becomes larger,the density of the cytoplasm increasing,AAVC-I group(3.125?g/m L,6.25?g/m L,12.5?g/m L,25?g/m L),with the increasing of AAVC-I concentration and reaction time(12h,24h)extended,most Tca8113 cells rounded,smaller,semi-adherent and suspension cells increased,cells fusion hold together,intercellular boundaries were unclear.2.MTT assay showed that the two experimental groups DTT group(1mmol/L,2mmol/L,4mmol/L,8mmol/L,2h,4h)and AAVC-I group(3.125?g/m L,6.25?g/m L,12.5?g/m L,25?g/m L 12 h,24h)with drug concentration increasing and the role of time prolonged the rate of cells proliferation inhibition showed an upward trend(P <0.05).3.The detect results of RT-PCR technology and Western blotting results indicate that low concentration AAVC-I group endoplasmic reticulum stress index CHOP,GRP78,Caspase-4 expression increased compared with normal control group,medium and high concentrations AAVC-I group CHOP,GRP78,Caspase 4 expression were significantly higher than the normal control group(P <0.05).4.Immunocytochemistry staining the expression of CHOP,GRP78,Caspase-4 was up-regulated with AAVC-I concentration increases and the extension of time.5.After using CHOP inhibitor epirubicin,CHOP,GRP78,Caspase-4 expression decreased by RT-PCRand the apoptosis rate also decreased by flow cytometry.Conclusions:1.Successfully screened out DTT concentration induced ER stress response and reaction time;2.Using DTT as a positive control,AAVC-?can induce endoplasmic reticulum stress occurs;3.Low concentrations AAVC-? can induced Tca8113 cells adapt or occur mild endoplasmic reticulum stress response,middle and high concentrations AAVC-? can induced Tca8113 cells occur sustained severe endoplasmic reticulum stress response;4.Epirubicin can inhibit the expression of CHOP,CHOP pathway may be one of apoptosis ways through endoplasmic reticulum road induced by AAVC-?for Tca8113 cell lines.