CAJ | 학술논문

目的构建携带骨桥蛋白双链小干扰RNA(OPN-siRNA)的质粒表达载体,筛选出基因沉默效果最明显的短发卡样RNA(shRNA)质粒表达载体.方法设计并合成2个针对OPN的shRNA寡核苷酸片段(shRNA1,shRNA2),退火形成双链并分别克隆进入载体pGenesil-1.酶切鉴定后,采用LipofectamineTM2000介导的转染方法将重组的shRNA质粒转入大鼠血管平滑肌细胞(VSMC),筛选出稳定转染株.采用RT-PCR及Western免疫印迹检测其对OPN表达的抑制效果.结果 shRNA质粒成功转染入VSMC且效率达50%以上.转染含OPN shRN A1的VSMC的OPN mRNA和蛋白的表达分别为0.16±0.04和0.30±0.09,含OPN shRNA2分别为0.23±0.06和0.44±0.06,两组之间及分别与正常细胞组比较差异均有统计学意义(P<0.01).空载体组和阴性对照组与正常细胞组比较,差异均无统计学意义(P>0.05).结论成功构建了靶向OPN基因的shRNA质粒表达载体,其中抑制效果最为明显的shRNA质粒表达载体为shRN A1质粒.
목적 구건휴대골교단백쌍련소간우RNA(OPN-siRNA)적질립표체재체,사선출기인침묵효과최명현적단발잡양RNA(shRNA)질립표체재체.방법 설계병합성2개침대OPN적shRNA과핵감산편단(shRNA1,shRNA2),퇴화형성쌍련병분별극륭진입재체pGenesil-1.매절감정후,채용LipofectamineTM2000개도적전염방법장중조적shRNA질립전입대서혈관평활기세포(VSMC),사선출은정전염주.채용RT-PCR급Western면역인적검측기대OPN표체적억제효과.결과 shRNA질립성공전염입VSMC차효솔체50%이상.전염함OPN shRN A1적VSMC적OPN mRNA화단백적표체분별위0.16±0.04화0.30±0.09,함OPN shRNA2분별위0.23±0.06화0.44±0.06,량조지간급분별여정상세포조비교차이균유통계학의의(P<0.01).공재체조화음성대조조여정상세포조비교,차이균무통계학의의(P>0.05).결론 성공구건료파향OPN기인적shRNA질립표체재체,기중억제효과최위명현적shRNA질립표체재체위shRN A1질립.
Objective To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting osteopontin (OPN) gene (OPN-siRNA),and to screen for OPN-siRNA that may be most effective in gene-silencing. Methods Two pairs of oligonucleotides for short hairpin expression targeting OPN mRNA (shRNA1 and shRNA2) were inserted into pGenesil-1 vector. Recombinant expression vector was identified by enzyme cutting and sequencing analysis. OPN shRNAs were transfected into rat blood vessel smooth muscle cells (VSMC) via LipofectamineTM 2000. The transfected cells were visualized by using inverted fluorescent microscope. Forty-eight hours later,the VSMCs transfected by optimal recombined plasmid were selected by culturing in G418. Nude cells and cells transfected by PGH were used as control.The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. Results VSMCs were stably transfected by pshRNA1-OPN in more than 50% of total cells. The expression levels of OPN mRNA and proteins were 0.16±0.04 and 0.30±0.09 in shRNA1 transfected VSMCs,0.23±0.06 and 0.44±0.06 in shRNA2 transfected VSMCs,which were significantly different between the two groups and with compared normal cells (P<0.01). The blank vector group and negative control group did not show any difference from normal controls (P>0.05). Conclusions The plasmid expression vectors coding for shRNA targeting OPN mRNA are constructed successfully. shRNA 1 appears to be most effective for gene-silencing.