核因子κB p65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

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;脂质体介导的不同浓度的NF-κB p65 ASOND(0.001、0.01、0.1和1μmol/L)进入HSC;台盼蓝染色排斥法检测NF-κBp65 ASOND对HSC的毒性实验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HSC I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1 μmol/L)的NF-κB p65 ASOND对于体外培养HSC的存活率和LDH无明显影响(P>0.05),0.01～μmol/L的NF-κB p65 ASOND抑制HSC的增殖,lmg/L TNF-α刺激HSC的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.

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

Objective Sstudy effect of nuclear factor-κB ASOND on I type collagen expression and rat hepatic stellate cells(HSC)proliferation.Methods Rat HSCs were separated by affusing and digestingof Ⅳ type collagenenzyme and density acentric method.Lipid-mediated NF-κB p65 ASOND(0.001,0.01,0.1,1μmol/L)Was transferred into rat HSCs.Toxicity of HSCs caused by NF-κB p65 ASOND and activity of LDH were determined by trypan blue staining.Proliferation affection of transferring NF-κB p65 ASOND into HSCs was determined by MTT.In different concentration NF-κB p65 ASOND.expression of Ⅰ type collagen stimulated by 1mg/L TNF-αwas determined by RT-PCR and ELISA.Results After transfection of NF-κB p65 ASODN,expression of NF-κB protein in HSCs was decreasing.Toxicity experiment indicated that NF-κB p65 ASOND of different concentration(0.001,0.01,0.1 and 1.0 μmol/L)had no effect on HSCs livability and LDH activity(P<0.05).Four different concentration NF-κ B p65 ASOND could restrain HSCs proliferation stimulated by 1 mg/L TNF-α.The expression of I type collagen and mRNA stimulated by 1mg/LTNF-αwas increased,and had a positive correlation with concentration(P>0.05).Conclusion NF-κB p65 ASOND may depress NF-κB activity to restrain HSCs proliferation and Ⅰ type collagen expression,and reduce extracellular matrix.