NNK联合LPS孵育对小鼠巨噬细胞增殖及相关基因表达影响  

EFFECT OF COMBINED INCUBATION OF NNK AND LPS ON PROLIFERATION OF MACROPHAGES AND ITS RELATED GENE EXPRESSION IN RATS

作　　者：陈琛[1] 卞晶晶[1] 刘晓萍[1] 张静[1] 于忠杰[1] 杨童茜 

机构地区：[1]青岛大学医学院组织胚胎学教研室,山东青岛266021

出　　处：《齐鲁医学杂志》2015年第5期505-507,511,共4页Medical Journal of Qilu

基　　金：山东省自然科学基金资助项目(ZR2012CM008)

摘　　要：目的观察脂多糖（LPS）、4-（甲基亚硝氨基）-3-吡啶-1-丁酮（NNK）对小鼠巨噬细胞增殖及相关基因表达的影响。方法体外培养小鼠巨噬细胞Raw264.7细胞,检测不同浓度的LPS及NNK刺激后Raw264.7细胞C-myc mRNA的表达情况,选择药物最佳浓度。用所选浓度的LPS、NNK及LPS＋NNK孵育Raw264.7细胞不同时间,利用细胞计数仪检测细胞增殖能力,采用实时定量PCR方法检测C-myc、核转录因子-κB（NF-κB）、精氨酸酶（Arginase）、一氧化氮合酶（iNOS）、肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1α及IL-6mRNA表达,应用酶联免疫试剂盒检测炎性递质TNF-α及IL-6的含量。结果选择浓度为10μmol/L NNK与1mg/L LPS进一步实验。NNK孵育Raw264.7细胞24h,其细胞增殖和各基因表达及IL-6、TNF-α蛋白表达与对照组比较差异无显著性。LPS和LPS＋NNK孵育Raw264.7细胞24h,其细胞增殖活性显著降低;C-myc、NF-κB、Arginase、iNOS、IL-1α、IL-6mRNA及IL-6、TNF-α蛋白表达显著升高（F=19.4～5 895.0,t=4.04～97.53,P〈0.05）。NNK对LPS刺激的Raw264.7细胞C-myc mRNA、TNF-α蛋白的表达升高具有拮抗作用;对NF-κB、IL-1αmRNA的表达升高具有协同作用。结论 LPS可抑制Raw264.7细胞的增殖,并促进Raw264.7细胞活化;NNK单独孵育对巨噬细胞的增殖和相关基因表达无明显影响,但可以强化LPS诱导的NF-κB、IL-1α基因高表达,促使巨噬细胞向M1型活化。Objective To observe the effect of LPS and NNK on the proliferation of macrophages and expression of its related gene. Methods Raw 264.7cells were cultured,in vitro,in rats.The expression of C-myc mRNA in Raw264.7cells was detected after stimulation with different-concentration LPS and NNK,so as to choose the optimal concentration of the drug.Employing selected concentration of LPS,NNK and combined LPS＋NNK to incubate Raw264.7cells for different time,the expressions of C-myc,NF-kB,Arginase,iNOS,TNF-α,IL-1αand IL-6were detected,and employing Real time-PCR to quantitatively measure the content of TNF-αand IL-6. Results The concentration of 10μmol/L of NNK and 1mg/L of LPS was selected for further study.The Raw264.7cells were cultured with NNK for 24 hours,the cell multiplication and gene expression,as well as expressions of IL-6mRNA and IL-6were not significantly different versus the control group,when Raw264.7cells were incubated with LPS and LPS＋NNK for 24 hours,its cell proliferation activity notably reduced,and expressions of C-myc,NF-κB,Arginase,iNOS,IL-1α,IL-6mRNA,IL-6and TNF-αobviously elevated（F =19.4-5 895.0,t=4.04-97.53,P〈0.05）.NNK showed antagonistic action to the expressions of C-myc mRNA and TNF-αprotein in LPS-stimulant Raw264.7cells,and showed joint action on the elevation of expressions of NF-κB and IL-1α mRNA. Conclusion LPS can inhibit the proliferation of Raw264.7cells and enhance its activation.Incubation with NNK alone does not impact the proliferation of macrophages and its related genes,but can strengthen high expressions of NF-κB and IL-1αthat induced by LPS,and promote the shift of Raw264.7cells to M1.

关 键 词：脂多糖类 丁酮类 巨噬细胞 细胞增殖 基因表达 

分 类 号：R392.12[医药卫生—免疫学]