β2-spectrin的siRNA对AML-12肝细胞增殖特性的影响  

Effect of siRNA against β_2-spectrin on Proliferation of AML-12 Cells

作　　者：汪志军[1] 涂炜[2] 刘芳[3] 

机构地区：[1]华中科技大学同济医学院附属协和医院消化内科,武汉430022 [2]华中科技大学同济医学院附属同济医院消化内科,武汉430030 [3]华中科技大学同济医学院附属协和医院血液内科,武汉430022

出　　处：《华中科技大学学报（医学版）》2013年第1期8-11,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30600277)

摘　　要：目的应用特异性siRNA干扰小鼠肝细胞株AML-12中β2-spectrin表达,检测AML-12细胞增殖凋亡以及细胞周期的改变。方法化学合成β2-spectrin特异性siRNA,分别转染AML-12细胞,荧光定量PCR和Western blot确定其抑制效果。AML-12细胞经TGF-β细胞因子处理4h后,转染β2-spectrin特异性siRNA,48h后,BrdU标记法以及流式细胞术检测β2-spectrin沉默前后AML-12细胞增殖、凋亡以及周期变化;共聚焦技术检测干扰β2-spectrin表达对TGF-β/Smad信号通路中Smad3、Smad4核定位的影响。结果 siRNA可以高效特异沉默β2-spectrin的表达。流式细胞术检测显示,β2-spectrin沉默促进AML-12细胞增殖,抑制凋亡,S期细胞比例显著升高。免疫荧光结果显示,TGF-β处理后,Smad3、Smad4表达定位于细胞核,沉默β2-spectrin干扰Smad3、Smad4细胞核内定位。结论沉默β2-spectrin的表达促进AML-12增殖,抑制凋亡,促进细胞向S期转化。其机制可能是通过干扰Smad3、Smad4核定位而抑制TGF-β/Smad信号通路,进而发挥诱导凋亡效应。Objective To investigate the effect of siRNA against β2-spectrin on proliferation,apoptosis and cell cycle of AML-12 cells （a murine hepatic cell line）. Methods AML-12 cells were pretreated with TGF-β1 for 4 h, and subsequently transfected with β2-spectrin siRNA. BrdU assay and flow cytometry were used to evaluate the proliferation,apoptosis and cell cycle of siRNA-transfeeted AML-12 cells. The nuclear location of Smad3/4 was detected by using double-immunofloresence staining. Results β2-spectrin gene was effectively silenced by specific synthesized siRNAs as demonstrated by the flow cytometric analysis. The cell proliferation and the cell percentage of S phase were conspicuously increased after siRNA transfection. Moreover,β2-spectrin silencing also resulted in the nuclear translocalization of Smad3/4. Conclusion Silencing β2- spectrin expression may increase the proliferation of AML-12 ceils by the nuclear translocalization of Smad3/4, blockade of TGF-β/Smad signaling, increase in S phase cells and inhibition of cell apoptosis.

关 键 词：β2-spectrin AML-12细胞株 细胞增殖 细胞周期 细胞凋亡 

分 类 号：R322.4[医药卫生—人体解剖和组织胚胎学]