核因子κBp65反义寡核苷酸对大鼠肝星状细胞增殖和Ⅰ型胶原表达的影响  

Effect of nuclear factor-κB p65 ASOND on type Ⅰ collagen expression and rat hepatic stellate cells proliferation

作　　者：周宇[1] 陈科全[1] 王皓[1] 唐志凌[1] 魏国丽[1] 叶文桃[1] 叶石才[1] 刘荣火[1] 冯晓[1] 

机构地区：[1]广东医学院附属医院消化内科,湛江524001

出　　处：《中华生物医学工程杂志》2008年第6期405-408,共4页Chinese Journal of Biomedical Engineering

基　　金：广东省自然科学基金（04011392）

摘　　要：目的探讨核因子κB（NF-κB）p65反义寡核苷酸（ASOND）对大鼠肝星状细胞（HSC）增殖和Ⅰ型胶原表达的影响。方法Ⅳ型胶原酶消化密度梯度离心法分离培养大鼠HSC。脂质体介导不同浓度的NF—κB p65 ASOND（0．001、0．01、0．1和μmol／L）转入HSC。锥虫蓝染色排斥法检测NF—κB p65 ASOND对HSC的毒性并检测各组乳酸脱氢酶（LDH）活性。MTT法测定NF—κB p65 ASOND对1mg／LTNF-α刺激后HSC增殖影响。RT—PCR法和ELISA法检测不同浓度NF—κB p65 ASOND对1mg／LTNF—α刺激后HSCI型胶原表达的影响。结果转染NF—κB p65 ASODN后，HSC细胞NF—κB蛋白的表达下降。不同浓度（0．001、0．01、0．1和1μmol／L）的NF-κB p65 ASOND对于体外培养HSC的存活率和LDH活性无明显影响（P〉0．05）。0．01～1μmol／L的NF—κB p6 5ASOND抑制1mg／LTNF-α刺激后的HSC的增殖和Ⅰ型胶原蛋白和mRNA的表达，且随浓度的增加作用增强（P〈0．05）。结论NF—κB p65 ASOND可通过抑制NF-κB活性减少HSC活化增殖及I型胶原生成，从而减少细胞外基质的产生。Objective To study the effect of nuclear factor-κB （NF-κB）antisense oligonucleoties （ASOND） on type Ⅰcollagen expression and rat hepatic stellate cells （HSCs） proliferation. Methods Rat HSCs were separated by affusing and digesting of type Ⅳ collagen enzyme and density acentric method. Lipidmediated NF-κB p65 ASOND（0.O01,0.01,0.1,1 μmol/L） was transferred into rat HSCs. Toxicity of HSCs caused by NF-K B p65 ASOND and activity of LDH were determined by trypan blue staining. Affection of transferring NF-κB p65 ASOND on proliferation of HSCs induced by 1 mg/L TNF-α was detected by MTT. In different concentrations of NF-κB p65 ASOND, expression of type I collagen stimulated by l mg/L TNF-α was examined by RT-PCR and ELISA. Results After transfeetion of NF-κB p65 ASODN, expression of NF- κB protein in HSCs was down-regulated. Toxicity experiment indicated that NF-κB p65 ASOND of different concentrations （0.001,0.01,0.1 and 1.0 μmol/L） had no effect on HSCs survival and LDH activity（P〉0.05）. Different concentrations of NF-κB p65 ASOND（0.01-1μmol/L） could restrain HSCs proliferation stimulated by 1 mg/L TNF-α as well as the expression of type I collagen protein and mRNA, and associated with concentration （P〈0.05）. Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and type Ⅰcollagen expression, thus may reduce extracellular matrix.

关 键 词：寡核苷酸类 反义 核因子ΚB 转染 肝星状细胞 Ⅰ型胶原 增殖 肝 纤维化 

分 类 号：R575.2[医药卫生—消化系统]