目的 观察RNA干扰技术对肝癌细胞株内骨桥蛋白(osteopontin,OPN)不同片段的抑制效果,以期为针对OPN的靶向治疗提供更准确、有效的位点.方法 应用合成OPN序列特异性双链RNA(OPNi-A、OPNi-B、OPNi-C),转染人HEP G2肝癌细胞株,用荧光定量PCR和免疫组化检测比较OPN的mRNA和蛋白表达情况.结果 OPNi-A、OPNi-B、OPNi-C转染HEP-G2后,A片段的荧光强度大于B片段和C片段.RNA干扰HEP-G2细胞株48 h后,A、B、C、三个片段的OPNmRNA的△CT值分别从干扰前的8.31±1.58、8.78±1.49、8.25±1.51上升至12.14±1.43、10.22±1.97、10.48±1.88 (P＜0.05)；三个片段的OPN蛋白水平免疫组化评分也从干扰前的6.44±1.67、5.43±2.05、5.45±2.52下降到2.84±1.52、4.43±1.65、3.95±1.43.结论 RNA干扰对肝癌细胞株内骨桥蛋白不同片段有不同的抑制作用.合成更具针对性的siRNA可能对抑制肝癌侵袭转移具有重要的科学和卫生经济学意义.%Objective Within human hepatoma cell lines,we aimed to investigate the effects of the down-regulation by RNAi on different fragments of osteopontin (OPN) in order to discover more effective and accurate sites for OPN.Methods Specific small interfering RNA of OPN (OPNi-1) were synthesized and transfected into human hepatoma cell line (HEP-G2).Fluorescent quantitative PCR and immunohistochemical methods were used to test*the OPN expression levels of mRNA and protein before and after RNAi.Results After transfection,the △CT value of the A fragment was greater than B and C fragments of OPN mRNA in HEP-G2.Before RNAi was added to HEP-G2 cells,the three fragments A,B,C had OPN mRNA CT values of 8.31±1.58,8.78±1.49,8.25±1.51 respectively.Once the RNAi were added,the CT values were measured 48h after for the fragments A,B,and C which were 12.14±1.43,10.22±1.97,10.48±1.88 (P＜0.05) respectively.The immunohis tochemical values of A,B,C were down from 6.44±1.67,5.43±2.05,5.45±2.52 to 2.84±1.52,4.43± 1.65,3.95± 1.43 respectively after interference.Conclusions RNAi can inhibit the expression of OPN gene selectively.siRNA targets different segments of OPN,which may have more effects on invasion and metastasis of liver cancer for a more important significance in science and health economics.
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