骨桥蛋白短发卡样RNA质粒载体的构建与筛选

Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting osteopontin gene

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.

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.