中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2009年
3期
131-134
,共4页
陈海龙%李海龙%张桂信%李宏%刘佳%贺雪梅
陳海龍%李海龍%張桂信%李宏%劉佳%賀雪梅
진해룡%리해룡%장계신%리굉%류가%하설매
基因沉默%RNA干扰%核转录因子-κB%核转录因子-κB抑制蛋白激酶%脂多糖%巨噬细胞
基因沉默%RNA榦擾%覈轉錄因子-κB%覈轉錄因子-κB抑製蛋白激酶%脂多糖%巨噬細胞
기인침묵%RNA간우%핵전록인자-κB%핵전록인자-κB억제단백격매%지다당%거서세포
gene silencing%RNA interference%nuclear factor-κB%inhibitory κB kinase%lipopolysaccharide%macrophage
目的 观察RNA干扰介导的核转录因子-κB抑制蛋白(IκB)激酶(IKK)α和γ基因沉默对核转录因子-κB(NF-κB)信号通路的调节作用,进一步阐明其基因调控机制.方法 设计IKKα及IKKγ靶向的小分子干扰RNA(siRNA),合成互补的寡核苷酸链,转染到RAW264.7小鼠巨噬细胞.观察经脂多糖(LPS)刺激后NF-κB的活化、IKKα及IKKγ基因表达的变化、NF-κB p65及p50核移位的变化以及前体蛋白NF-κB p105的表达情况.结果 IKKα及IKKγ基因沉默后,可导致IKKα及IKKγ基因表达出现明显下调,同时NF-κB p65及p50的核移位受到抑制,胞质、胞核中NF-κB p65、p50及p105的表达显著下调,而且能抑制NF-κB p65、p50及p105蛋白的核移位.结论 IKKα及IKKγ两种蛋白激酶参与NF-κB信号通路的调节,不仅能分别在抑制蛋白的泛肽化、组蛋白磷酸化等环节发挥作用,而且还能够通过相互之间的协同作用,弥补其中某种蛋白受到过度抑制时所引起的炎症信号通路的功能障碍.
目的 觀察RNA榦擾介導的覈轉錄因子-κB抑製蛋白(IκB)激酶(IKK)α和γ基因沉默對覈轉錄因子-κB(NF-κB)信號通路的調節作用,進一步闡明其基因調控機製.方法 設計IKKα及IKKγ靶嚮的小分子榦擾RNA(siRNA),閤成互補的寡覈苷痠鏈,轉染到RAW264.7小鼠巨噬細胞.觀察經脂多糖(LPS)刺激後NF-κB的活化、IKKα及IKKγ基因錶達的變化、NF-κB p65及p50覈移位的變化以及前體蛋白NF-κB p105的錶達情況.結果 IKKα及IKKγ基因沉默後,可導緻IKKα及IKKγ基因錶達齣現明顯下調,同時NF-κB p65及p50的覈移位受到抑製,胞質、胞覈中NF-κB p65、p50及p105的錶達顯著下調,而且能抑製NF-κB p65、p50及p105蛋白的覈移位.結論 IKKα及IKKγ兩種蛋白激酶參與NF-κB信號通路的調節,不僅能分彆在抑製蛋白的汎肽化、組蛋白燐痠化等環節髮揮作用,而且還能夠通過相互之間的協同作用,瀰補其中某種蛋白受到過度抑製時所引起的炎癥信號通路的功能障礙.
목적 관찰RNA간우개도적핵전록인자-κB억제단백(IκB)격매(IKK)α화γ기인침묵대핵전록인자-κB(NF-κB)신호통로적조절작용,진일보천명기기인조공궤제.방법 설계IKKα급IKKγ파향적소분자간우RNA(siRNA),합성호보적과핵감산련,전염도RAW264.7소서거서세포.관찰경지다당(LPS)자격후NF-κB적활화、IKKα급IKKγ기인표체적변화、NF-κB p65급p50핵이위적변화이급전체단백NF-κB p105적표체정황.결과 IKKα급IKKγ기인침묵후,가도치IKKα급IKKγ기인표체출현명현하조,동시NF-κB p65급p50적핵이위수도억제,포질、포핵중NF-κB p65、p50급p105적표체현저하조,이차능억제NF-κB p65、p50급p105단백적핵이위.결론 IKKα급IKKγ량충단백격매삼여NF-κB신호통로적조절,불부능분별재억제단백적범태화、조단백린산화등배절발휘작용,이차환능구통과상호지간적협동작용,미보기중모충단백수도과도억제시소인기적염증신호통로적공능장애.
Objective To investigate the role of inhibitory κB (IκB) protein kinase (IKK) α and γ genes silencing induced by RNA interference (RNAi) in modulation of nuclear factor-κB (NF-κB) signal pathway,to elucidate further the regulatory mechanism of NF-κB gene. Methods IKKα and IKKγ targeting small interference RNA (siRNA) were designed to synthesize complementary oligonucleotide chains,then it was transfected into mouse macrophage cell line RAW264.7. The transfected cells were treated with lipopolysaccharide (LPS,10 μg/ml),then the expression of IKKα and IKKγ genes,the nuclear translocation changes in NF-κB p65 and p50 proteins,and the expression of precursor protein NF-κB p105 were detected at selected time points. Results It was shown that RNAi targeting IKKα and IKKγ could down-regulate the expression of IKKα and IKKγ genes,and at the same time,down-regulate the expression of NF-κB p65,p50 and p105 proteins both in cytoplasm and nucleus,and inhibit the nuclear translocation of NF-κB p65,p50 and p105 proteins. Conclusion These findings provide evidence for the involvement of the two protein kinases IKKα and IKKγ in the modulation of NF-κB signal pathway,not only they can inhibit the ubiquitin of protein and histone phosphorylation respectively,but also can redeem the functional impairment of the inflammation signal pathway induced by over inhibition of some proteins,through synergistic action of the two kinases.
