Detection Of The Apoptosis Changes And Effects On Bcl-2 And Bax Of The Adult Normal Hepatocyte In Vitro Exposure To Bilirubin

Objective: To explore the effects of bilirubin indues apoptosis of human liver cell line L-02 and the expression of Bcl-2 and Bax in liver cells treated by different doses of bilirubin .Methods : The growth inhibition of bilirubin in different concentrations by MTT assay.L-02 cells were treated with bilirubin at different concentrations.The cell growth and proliferation was assessed by ,DNA ladder,apoptosis rate and changes of cell nuclei were studied by agarose electrophresis and flow cytometry,respectively.Liver cells of human being(L-02) was planted in 6-well plates,then being treated with bilirubin for 12h.The cells were digested and beated upon by trypsin,then were centrifuged in refrigerated centrifuge and suspended in cold PBS solution twice then suspend the cells in 200μl binding buffer.Add 5μl Annexin-V and 5μl PI,then incubate in the dark for 15min.Add 400μl Bingding buffer and decect the cells in Flow cytometer.L-02 cells were approximately l×106cell/m1and planted in 6-well plates,after one night,treated with bilirubin at different concentrations(0 mg/L,100 mg/L,150 mg/L,200 mg/L,250mg/L,300 mg/L).Results: When the concentration of bilirubin solution was increased from 0mg/L to 300mg/L,the cell surviving rates significantly decreased in a concentration-and time-dependent manner as evidenced by MTT assay(P<0.01).The OD number in 0.2251±0.0324,0.2745±0.0466,0.6412±0.073,0.7295±0.0337,0.7629±0.0327,0.8322±0.0336.Compared with the control group,the bilibubin solution at a concentration of 150mg/L,200mg/L,250mg/L,300mg/L solution significantly inhibited 1the proliferation of L-02 cells(P<0.01).A ladder pattern of DNA fragments appeared after L-02 cells exposed to 200mg/L,250mg/L,300mg/L bilirubin for 24h.In cells treated with 0 mg/L,100 mg/L,150 mg/L,200 mg/L,250mg/L,300 mg/L bilirubin for 12h,the apoptotic rate was 57.3% respectively,significantly higher than that in the negative control group 1.16%.L-02 cells treated with different dosages of bilirubin for 12h, the rates of apoptotic is significant difference among different groups(p<0.01).The apoptotic rate added from 1.24% to 57.3%,when bilirubin increased from 0mmol/L to 300mmol/L.The apoptotic rate is significantly higller in the group of 300mmol/L than the group of 0mmol/L(p<0.01).Conclusions:Different dosages of bilirubin could early induec the apoptosis of L-02cells and demonstrated that was result in the express of Bcl-2 and Bax.