c-erbB-2、Bcl-2共沉默对人舌鳞癌细胞株（Tca8113）细胞作用的实验研究

【作者】 季平；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 肿瘤药物， 2005， 博士

【摘要】 目的:检测人舌鳞癌细胞株(Tca8113)细胞中 c-erbB-2、Bcl-2基因的表达水平,为进一步探讨 c-erbB-2、Bcl-2 基因在人舌鳞癌形成、发展过程中的作用机制,建立体外人舌鳞癌细胞株研究模型。方 法 采用实时荧光定量 PCR(real-time RT-PCR)实验技术检测人舌鳞癌Tca8113 细胞株中 c-erbB-2、Bcl-2 基因 mRNA 表达水平。 结果:从 TCA8113 细胞中抽提总 RNA,1%琼脂糖凝胶电泳图片上可见 5s,18s,28s 三条明显的条带,说明所提 RNA 质量较好。分光光度计(A260/A280)检测 Tca8113 细胞株细胞中 c-erbB-2、Bcl-2 基因mRNA 纯度均在 1.8～2.0,c-erbB-2、Bcl-2 基因标准曲线图可见线性良好,相关系数达到 1.0。通过基因表达扩增曲线图与看家基因 GAPDH校正,得出目的基因的相对含量,c-erbB-2 基因相对含量:4.97E+03copies/μl、看家基因 2.09E+06copies/μl、每百万看家基因含量 2.38E+03;Bcl-2 基因相对含量:5.47E+04 copies/μl、看家基因 2.09E+06copies/μl、每百万看家基因含量 2.62E+04。 结论:Bcl-2 与 c-erbB-2 基因的表达水平在人舌鳞癌 Tca8113 细胞株细胞中显著升高,可作为探讨 c-erbB-2、Bcl-2 基因在肿瘤形成、发展过程中的作用机制的体外肿瘤细胞株研究模型。 目的:探讨表达重组干扰质粒(RNAi 技术)对人舌鳞癌细胞株(Tca8113)细胞中 c-erbB-2、Bcl-2 基因表达的干扰效率。方 法 按siRNA 设计原则设计针对 c-erbB-2、Bcl-2 基因的 Oligo DNA,体外化学合成 Oligo DNA,Oligo DNA 退火、连接、转化、筛选克隆,构建针对 c-erbB-2 基因的表达重组 RNA 干扰质粒(psiC1、psiC2、psiC3)和针对 Bcl-2 基因的表达重组 RNA 干扰质粒(psiB1、psiB2、psiB3),细胞脂质体转染 Tca8113 细胞,Real-time RT-PCR 的标准曲线、扩增曲线和熔解曲线的数据收集处理,采用荧光定量 RT-PCR 方法检测重组干扰质粒对 Tca8113 细胞中 c-erbB-2、 Bcl-2 基因表达的干扰效率。 结果:实时荧光定量 RT-PCR 方法检测 psiC1、siC2、psiC3 质粒脂质体转染后 c-erbB-2 基因的表达情况结果显示:psiC2、psiC3 相对于对照组均有不同程度的干扰效果,psiC1 相对于对照没有干扰效果。更多还原

【Abstract】 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更多还原