重组干扰质粒对多药抗性Acc-3细胞Bcl-2基因表达作用实验研究

Silence the Expression of Bcl-2 Gene in Cisplatin-Resistant Cell Line Acc-3/Cddp

目的探讨重组干扰质粒对口腔腺样囊性癌多药抗性细胞株(Acc-3/CDDP)细胞中Bcl-2基因表达的干扰效率。方法按siRNA设计原则设计针对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Bcl-2基因的表达重组RNA干扰质粒(psiB1、psiB2、psiB3),细胞脂质体转染多药抗性Acc-3/CDDP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多药抗性Acc-3/CDDP细胞中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psiB1、psiB2、psiB3质粒脂质体转染后Bcl-2基因的表达情况结果显示:psiB1、psiB3相对于对照组无明显干扰效果,psiB2的干扰程度达66.20%。结论针对Bcl-2基因的表达重组RNA干扰质粒psiB2(CCgggAgATAgTgATgAA)能有效沉默多药抗性Acc-3/CDDP细胞中Bcl-2基因的表达。

Objective To investigate the interfering efficiency of RNAi technique on the expression of Bcl - 2 oncogene in human oral adenoid cystic carcinoma （Acc- 3/CDDP）. Methods The recombined RNAi plasmids for Bcl -2 oncogene were constituted by the four successive 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 Bcl - 2 oncogenes, the recombined RNAi plasmids were transfected into Acc- 3/CDDP ceils 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 Bcl - 2 oncogene were 0,66.20% and 0, respectively in psiB1 .siB2.psiB3. Conclusions The recombined RNAi plasmids of psiB2 （CCgggAgATAgTgATgAA） for Bcl - 2 oncogene can effectively silence the expression of Bcl -2 oncogenes in Acc- 3/CDDP cell line.