靶向血小板衍生生长因子-B siRNA对喉癌Hep-2细胞增殖的影响

Effect of targeted PDGF-B siRNA on the proliferation of Hep-2 cells

目的探讨血小板衍生生长因子(platelet derived growth factor,PDGF)B链mRNA的siRNA质粒转染喉癌细胞株Hep-2细胞,沉默Hep-2细胞中PDGF-B mRNA并检测细胞增殖。方法以脂质体lip2 0 0 0为载体,设计4组针对PDGF-B的siRNA片段瞬时转染喉癌Hep-2细胞,实时荧光定量PCR、Westernblot分别检测各组PDGF-B mRNA、PDGF-B蛋白的表达及干扰效果,噻唑蓝(MTT)法检测各组细胞增殖抑制率,并与阴性对照组和空脂质体组进行比较。结果实时荧光定量PCR检测显示:SiRNA2、SiRNA3、SiRNA4组PDGF-B mRNA表达均有明显抑制,其中SiRNA2抑制率最高;与SiRNA1、阴性对照组及空脂质体组进行比较,差异具有统计学意义(P<0.05)。Westernblot检测显示:SiRNA-1、SiRNA-2、SiRNA-3组PDGF-B mRNA蛋白表达均有下降,其中SiRNA-2下降最明显,而SiRNA-4、阴性对照组及空脂质体组对PDGF-B蛋白表达无明显抑制作用。MTT法检测显示:SiRNA-1、SiRNA-2、SiRNA-3均明显抑制Hep-2细胞增殖,其中SiRNA-2对Hep-2细胞的抑制作用最明显;且与SiRNA-4、阴性对照组及空脂质体组比较,差异具有统计学意义(P<0.05)。结论成功运用siRNA干扰喉癌Hep-2细胞PDGF-B mR-NA,靶向沉默PDGF-B mRNA能有效抑制细胞增殖。

Objective To transfect siRNA into Hep-2 cells for knocking down platelet-derived growth factor（PDGF-B） mRNA,and to detect its effect on the proliferation of Hep-2 cells.Methods Four groups of PDGF-B targeted siRNA were transfected into Hep-2 cells with lipofectamine 2000 as vector.The expression of PDGF-B mRNA and PDGF-B protein was detected by real-time PCR and Western Blot respectively.MTT was used for detecting the inhibitory rates of proliferation.Results Real-time PCR showed that the expression of PDGF-B mRNAs in siRNA2,siRNA3,and siRNA4 groups was inhibited with the highest inhibitory rate in siRNA2 group.The differences of the expression levels of PDGF-B mRNAs in siRNA2,siRNA3,and siRNA4 groups and those of the siRNA2,the negative control and the empty liposome groups were statistically significant（P0.05）.Western Blot showed that the expression of PDGF-B protein in siRNA1,siRNA2,and siRNA3 groups declined with the lowest expression in siRNA2 group.The expression of PDGF-B protein in siRNA4,the negative control and the empty liposome groups were not inhibited.The MTT assay showed that siRNA1,siRNA2,and siRNA3 could inhibit cell proliferation with the most obvious inhibitory effect on siRNA2.The differences of the inhibitory rates of cell proliferation in siRNA1,siRNA2,and siRNA3 groups and those of the siRNA4,the negative control and the empty liposome groups were statistically significant（P0.05）.Conclusion siRNAs targeting PDGF-B can down-regulate the expression of PDGF-B gene and the knockdown of PDGF-B mRNA can inhibit the proliferation of Hep2 cells effectually.