| Objective:To find appropriate experimental model for in vitro study of the role of c-erbB-2 and Bcl-2 oncogene in neoplasia and development of tumor cells,by detectecting the expression of c-erbB-2 and Bcl-2 oncogene in Tca8113 cell. Method:Total RNA was extracted from Tca8113 cell and mRNA expression of c-erbB-2 and Bcl-2 oncogene in Tca8113 cell was quantitatively detected by real-time RT-PCR(real-time reverse transcriptase-polymerase chain reaction). Result:There were three transparent strap(5s 18s 28s) on agarose gel electrophoresis photo ,which indicated that RNA extracted was qualified for the experiment.The mRNA purity was tested by spectrophotometer(A260/A280)and the tites of both c-erbB-2 and Bcl-2 gene in Tca8113 cell were between 1.8 to 2.0.Coefficient of product-moment correlation reached 1.0, and it is thus evident that the linearity of standard curves of c-erbB-2 and Bcl-2 gene was well. Correcting curves of gene expression and gene amplification with house-keeping gene(GAPDH),we got the relative content of c-erbB-2 and Bcl-2 gene.The relative content of c-erbB-2 gene was 4.97E+03 copies/μl,house-keeping gene2.09E+06 copies/μl,each a million house-keeping gene content2.38E+03,while the relative content of Bcl-2 gene was 5.47E+0.4 copies/μl , house-keeping gene2.09E+06 copies/μl , each a million house-keeping gene content2.62E+0.4. Conclusion:c-erbB-2 and Bcl-2 genes significantly overexpressed in human tongue squamous carcinoma(Tca8113)cell line,so this cell line can be used as the experimental model for in vitro study of the role of c-erbB-2 and Bcl-2 oncogene in the progress of neoplasia and development of tumor. PART TWO SILENCE THE EXPRESSION OF C-ERBB-2 AND BCL-2 GENE IN TCA8113 CELL LINE BY RNAI TECHNIQUE Objective: To investigate the interfering efficiency of RNAi technique on the expression of c-erbB-2 and Bcl-2 oncogene in human tonguesquamous carcinoma (Tca8113). Methods: The recombined RNAi plasmids for c-erbB-2 and Bcl-2 oncogene were constituted by the four succussive steps-designing of Oligo DNAs,synthesis of Oligo DNAs, transfection of Oligo DNAs into pSUPER.neo+gfp vectors and selection of positive plasmids.In order to silence the expression of c-erbB-2 and Bcl-2 oncogenes, the recombined RNAi plasmids were transfected into Tca8113 cells by culturing together for about 10 hours,and the interfering efficiency of RNAi for the two oncogenes was evaluated by fluorescence-quantitative RT-PCR. Results:The interfering efficiencies for c-erbB-2 oncogene were 0,62.68% and 40.61%, respectively in psiC1,psiC2,psiC3,while the interfering efficiencies for Bcl-2 oncogene were 0,66.20% and 0, respectively in psiB1,siB2,psiB3. Conclusion:The recombined RNAi plasmids of psiC2 (gAgTCCCAACCATgTCAAA)for c-erbB-2 oncogene and of psiB2(CCgggAgATAgTgATgAA)for Bcl-2 oncogene can effectively silence the expression of c-erbB-2 and Bcl-2 oncogenes in Tca8113 cell. PART THREE THE EFFECT ON PROLIFERATION AND APOPTOSIS OFObjective:To investigate the effect of the silence of c-erbB-2 and Bcl-2 genes on the proliferation and the apoptosis of Tca8113.Methods There were four groups:Control group,c-erbB-2 gene-interfered group, Bcl-2 gene-interfered group,combined-interfered group (both c-erbB-2 and Bcl-2 genes being interfered).The recombined RNAi plasmids for c-erbB-2 and Bcl-2 oncogene were constituted by the four succussive steps-designing of Oligo DNAs,synthesis of Oligo DNAs,transfection of Oligo DNAs into pSUPER.neo+gfp vectors and selection of positive plasmids.In order to silence the expression of c-erbB-2 and Bcl-2 oncogenes, the recombined RNAi plasmids were transfected into Tca8113 cells by culturing together. Proliferation, apoptosis, cell cycle, expression of mRNA and proteins in each group were tesed by MTT test, clone-forming test,cell cycle test,electron microscopy,fluorescent microscopy,and RT-PCR,respectively. Result:The OD value at 72h after transfection for combined-interferd group,c-erbB-2 gene-interfered group,and Bcl-2 gene-interfered group were 0.084(p<0.005),0.1...
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