To Study The Effects Of HBV X Gene On The Biological Characteristics Of The L02 Hepacyte And Study The Role Of The ADM In The Regulations Of HBx That Influences On The Hepacytic Apoptosis

Objective(1)To establish the gene-transfected cell model L02/HBx,which will lay the foundation for us to do further research to find out the mechanisms of malignant transformation and hepatocellular carcinoma(HCC) induced by HBx.(2)To observe the changes of the morpholophy ,proliferation,apoptosis ,cell cycles of the L02/HBx and get further recognition about the influences of HBx on the L02 so that we can disclose the mechanisms of the hepatocellular carcinoma thoroughly.(3)To observe the growth trends,apoptosis and cell cycles of L02/HBx expose to the Adriamycin (ADM)and acquire their changes so as to stand the chance of breaking through the traditional theories on the mechanisms of HCC and get new ones.We have been hoping that the experiments could supply the valuable data to direct the chemotherapy of hepacellular carcinoma.Methods(1)To obtain the lowest concentration of G418 that kill the L02 cytes by using the G418 dosage-reaction test.(2)To transfect the HBx gene into the L02 cytes by the Effectene Transfection Reagent,next,We obtained the positive clones in two weeks by the G418 selection base on its lowest killing concentration,followup,we detected the HBx protein by RT-PCR and western blot respectively,which will assure that we establish the L02/HBx cell model successfully.(3)To culture the L02/HBx,L02/pcDNA3.1 and L02 respectively,after that,we observed the morphology of the L02/HBx,detected all groups about the proliferation with MTT arrays ,in the meanwhile,we detected the apoptosis and cell cycles by FCM.Base on these results, We compared the L02/HBx with the other two groups about the apoptosis and cell cycles.(4)To culture the L02/HBx ,L02/pcDNA3.1 and L02 cytes by the serum obtaining ADM and observe its effects on the growth of all groups,following,we detected all the groups about the changes of apoptosis and cell cycles by FCM.Results(1)G418 dosage-reaction test showed that the concentration of G418 from 500 to 1000 mg/L can kill all the cells in two weeks,but the range from 0 to 400 mg/L can't have the same effects,there also had the cells surviving the selection,which determine that 500 mg/L was the lowest killing concentration.(2)We obtained the positive clones by G418 selection in two weeks,the concentration for G418 selection was 500 mg/L. the RT-PCR and western detection revealed that there had HBx mRNA and HBx protein in the transcriptional level and HBx protein expression in the protein level respectively.(3)Inversion phase contrast microscope showed that the morphologic characteristic of L02/HBx had been changed more obviously than that of the L02,that is,the cells like to gather and pile up together,the figure become unregular and bigger,the borderline and the surface become obscure. The MTT arrays showed that L02/HBx proliferated more quickly than the control groups,the proliferating double time of the L02/HBx was 24 hours,but the control groups were two times of L02/HBx. FCM showed that ,in contrast with the control groups,the apotosis rates of L02/HBx were at a low level (0.09±0.13% vs 3.74±1.29%,p<0.05)and the propotion of L02/HBx falled in G1 phase(61.35±0.82% vs 67.80±6.84%,p<0.05) but rose in S phase(36.59±2.54% vs 22.37±2.17% , p<0.05 ) , the diploid cells decreased ( 87.54±0.82% vs 97.94±1.77% ,p<0.05) but the aneuploid cells reversed(12.46±0.82% vs 2.06±1.77% ,p<0.05)。(4)After used the ADM,MTT arrays showed that the apoptosis speeds of the L02/HBx had been promoted more obviously than that of the L02/HBx without disposing to the the ADM,FCM also showed that the apoptosis rates of HBx rose more obviously(34.91±5.85% vs 0.09±0.13%,p<0.05),the propotion of L02/HBx still falled in G1 phase(82.81±6.48% vs 87.19±1.92%,p<0.05) and rose in S phase compared with control groups(13.84±6.16% vs 2.22±1.26%,p<0.05),the diploid cells decreased(75.33±9.47% vs 89.42±5.16%) and the aneuploid cells increased obviously(24.67±9.47%vs10.58±5.16%).Conclusions(1)The L02/ HBx cell strain that stably expressed the protein had been established successfully,it means that we have the ideal cell model to study the mechanisms of malignant transformation and HCC induced by HBx.(2)All the data revealed that HBx can accelerate the cell cycle and improve the growth instead of facilitating the apoptosis with the expulsion of the proapoptotic factors,which will induce the uncontrollable proliferation that can result in maltransformation.But if HBx is under the conditions that induce apoptosis,it will prevent the damaged DNA from repairing so as to lead the cell to a higher apoptotic rates,afterall,it possibly involve in the cell abberance and cause maltransformation,all the truth told us that the circumstances that the HBx was in would affect its biological functions.