摘要
目的研究siRNA干扰β-Catenin表达,阻断Wnt/β-Catenin信号通路对肝星状细胞(HSC)增殖和凋亡的影响。方法将化学合成的siRNAβ-Catenin以Lipofectamine包裹,转染HSC-T6细胞,设阴性对照和空白对照;采用逆转录-聚合酶链反应和Western blotting检测细胞β-Catenin表达;采用Western blotting法检测细胞Ⅰ、Ⅲ型胶原表达;采用MTT法和流式细胞仪检测细胞增殖和细胞凋亡。结果以不同浓度的β-Catenin SiRNA转染HSC-T6细胞后,β-Catenin mRNA水平比阴性对照组分别下调了51.3±3.6%、85.7±6.8%和94.5±7.5%(P均<0.01),比空白对照组分别下调了50.5±3.4%、86.1±6.2%和93.7±7.2%(P均<0.01);β-Catenin蛋白表达被抑制了56.43±2.88%(P<0.01);Ⅰ和Ⅲ型胶原表达分别被抑制了46.58±3.46%(P<0.01)和50.48±3.72%(P<0.01);细胞增殖明显被抑制,最大抑制率达到44.8±2.8%(P<0.01);细胞凋亡增加达28.6%(P<0.05)。结论 siRNAβ-Catenin能高效抑制HSC-T6细胞β-Catenin表达,阻断Wnt/β-Catenin信号通路,从而抑制HSC-T6细胞增殖,促进凋亡,显著减少Ⅰ、Ⅲ型胶原的合成和分泌,具有预防及治疗肝纤维化的潜力。
Objective To investigate the effect of synthetic small interfering RNA（siRNA）targeting β-Catenin on the biological characters of hepatic stellate cells（HSCs）in vitro.Methods Synthetic siRNA targeting β-Catenin was transfected into HSC-T6 cells by lipofectamine package.Negative siRNA transfection and no transfection were used as negative and blank control,respectively.After incubation with siRNA,total RNA and protein of HSC-T6 cells were extracted.The expression of β-Catenin gene and protein was detected by reverse transcription-polymerase chain reaction and Western blotting.The proliferation of HSC-T6 was determined using MTT assay.HSC-T6 proliferation and apoptosis were measured by flow cytometry.Content of collagen type Ⅰand Ⅲin the supernatants were determined by Western blotting.Results After HSC-T6 cells transfected with different concentrations of β-Catenin SiRNA,the level of β-Catenin mRNA decreased significantly as compared to the negative and blank control;β-Catenin protein expression was inhibited by 56.43±2.88%（P0.01）,and Ⅰ and Ⅲ collagen expression were also inhibited by 46.58±3.46%（P0.01）and 50.48±3.72%（P0.01）;the cell proliferation was significantly inhibited（P0.01）,while cell apoptosis rate was significantly higher than in the negative control（P0.05）.Conclusion Inhibition of β-Catenin by siRNA may have a potential effect on prevention and treatment of hepatic fibrosis by inhibiting proliferation of and promoting apoptosis of stellate cells.
出处
《实用肝脏病杂志》
CAS
2011年第2期91-94,共4页
Journal of Practical Hepatology
基金
上海市教委选拔培养优秀青年教师科研专项基金(编号j:dy08086)
