CAJ | 학술논문

目的构建表达人膀胱癌bcl-2基因的小分子RNA(siRNA)的慢病毒载体并建立稳定转染细胞株.方法根据GenBank提供的bcl-2 cDNA序列,设计3条针对bcl-2 siRNA序列,分别构建pSIH1-H1-copGFP重组质粒.筛选出最有效沉默靶基因的siRNA后,将携带目的序列的慢病毒载体转染到293T细胞中,感染膀胱癌细胞株T24,建立稳定转染细胞株.结果设计的针对bcl-2的序列中第3条的抑制效果最好,下调59.0%.以此目的序列构建稳定转染细胞株,对bcl-2 mRNA抑制率达65.6%,对bcl-2蛋白的抑制率高达74.5%.结论成功构建表达人膀胱癌bcl-2-siRNA的慢病毒载体,它能有效沉默bcl-2基因在膀胱癌细胞株T24中的表达并建立了稳定转染细胞株.
목적 구건표체인방광암bcl-2기인적소분자RNA(siRNA)적만병독재체병건립은정전염세포주.방법 근거GenBank제공적bcl-2 cDNA서렬,설계3조침대bcl-2 siRNA서렬,분별구건pSIH1-H1-copGFP중조질립.사선출최유효침묵파기인적siRNA후,장휴대목적 서렬적만병독재체전염도293T세포중,감염방광암세포주T24,건립은정전염세포주.결과 설계적침대bcl-2적서렬중제3조적억제효과최호,하조59.0%.이차목적 서렬구건은정전염세포주,대bcl-2 mRNA억제솔체65.6%,대bcl-2단백적억제솔고체74.5%.결론 성공구건표체인방광암bcl-2-siRNA적만병독재체,타능유효침묵bcl-2기인재방광암세포주T24중적표체병건립료은정전염세포주.
Objective To obtain small interfering RNA (siRNA) sequences that can stably block the expression of bcl-2 in the bladder cancer cells, construct the bcl-2-siRNA lentiviral vector and establish a stably transfected cell line. Methods According to genetic information, 3 siRNA targeting bcl-2 were designed. The corresponding pSIH1-H1-copGFP recombinant plasmids were constructed. After the most effective siRNA was achieved, the shuttle plasmid pSIH-bcl-2-siRNA with lentiviral packaging plasmid was mixed, and 293T cells were transfected. Virus solution was collected to infect T24 cells and a stably transfected cell line was established. Results The third siRNA sequence, located in bcl-2 1210-1228, showed the best gene silencing effect as 59. 0%. The lentiviral vector was constructed successfully and the inhibition ratio was 65. 6% for bcl-2 mRNA and 74. 5% for bcl-2 protein. Conclusion It is successful to obtain bcl-2-siRNA lentiviral vector that can stably block the expression of bcl-2 in the bladder cancer cell line T24 and establish a stably transfected cell line which provides foundation for further experimental studies on the function of bcl-2.