沉默Nogo-A对脂多糖诱导的PC12细胞TNF-α、IL-6分泌及TH下调的影响  

Effect of Silencing Nogo-A on Secretion of TNF- alpha, IL-6 and Downregulation of TH in PC12 Cells Induced by Lipopolysaccharide

作　　者：钟建斌[1,2] 范胜诺[3] 肖颂华[3] 万利梅[1,2] 陈炽邦 钟思敏[1,2] 刘璐[1,2] 刘军[3] 

机构地区：[1]增城市人民医院 [2]中山大学附属博济医院神经内科,广东广州511300 [3]中山大学孙逸仙纪念医院神经内科,广东广州510120

出　　处：《中山大学学报（医学科学版）》2015年第3期391-395,402,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金(S2012010010242)

摘　　要：【目的】构建髓磷脂相关抑制物A(Nogo-A)基因sh RNA表达质粒,探讨沉默Nogo-A对脂多糖(LPS)诱导大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)释放炎症因子肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的调节作用,以及对PC12细胞表达酪氨酸羟化酶(TH)的影响。【方法】构建Nogo-A基因sh RNA表达质粒,荧光定量PCR法、Western Bolt法检测细胞中Nogo-A蛋白及m RNA的表达;用不同浓度LPS处理PC12细胞24 h,CCK8法检测细胞活力,选取合适浓度LPS做后续试验;将PC12细胞分为对照组、p Genesil-1.1+LPS组以及p Genesil-nogo A-sh RNA+LPS组,各组细胞相应处理24 h后,用ELISA法检测各组上清液中的TNF-α和IL-6的含量,Western blot检测TH的表达。【结果】构建出Nogo-A基因sh RNA表达质粒,p Genesil-nogo A-sh RNA组PC12细胞的m RNA及蛋白水平均下调,具有明显的统计学差异(P<0.05);随着LPS浓度的增加,PC12细胞的活力逐渐下降;与对照组比,LPS组上清液中TNF-α和IL-6的含量明显增多,TH的表达明显降低;而与LPS对照组比较,p Genesil-nogo A-sh RNA+LPS组的上清液中TNF-α和IL-6的含量明显减少,TH的表达明显升高,差异均具有统计学意义(P<0.05)。【结论】LPS可激活PC12细胞分泌炎症因子及降低TH表达,沉默Nogo-A基因对此现象有调节作用。[Objective] To construct the small hairpin RNA （shRNA） expression plasmid targeting myelin associated inhibitor A （Nogo-A） and study the effect of Nogo-A gene shRNA on lipopolysaccharide （LPS） induced the release of inflammatory cytokines tumor necrosis factor alpha （TNF-alpha） and interleukin-6 （IL-6） in PC12 cells,and the expression of tyrosine hydroxylase （TH）.[Methods] Nogo-A gene shRNA plasmid was constructed.Fluorescence Quantitative Real-Time PCR and Western blot were used to detect the expression of Nogo-A.PC12 cells were treated with different concentration of LPS for 24 hours and detected with cell viability assay CCK8.An appropriate concentration of LPS was chosen for the following experiment.PC12 cells were divided into control group,pGenesil-1.1 ＋LPS group and pGenesil-nogoA-shRNA＋LPS group.The content of the supernatant TNF-alpha and IL-6 of each group was determined by ELISA assay.Western blot was used for detection of TH.[Results] shRNA expression plasmid targeting Nogo-A was successfully constructed.Both mRNA and protein level of Nogo-A in pGenesil-nogoA-shRNA group were decreased,the difference is statistically significant （P ＜ 0.05）.The viability of PC12 cells decreased when LPS concentration increased.Compared with the control group,the content of TNF-alpha and IL-6 in the supernatant of LPS group was increased,while the TH level decreased.Compared with the LPS group,the content of TNF-alpha and IL-6 in the supernatant of pGenesilnogoA-shRNA＋LPS group decreased significantly,while the TH level increased,the difference of both comparisons were statistically significant （P ＜ 0.05）.[Conclusion] LPS induced PC12 cells to release inflammatory cytokines and lower the TH expression,while these effects can be regulated by silencing of the Nogo-A gene.

关 键 词：髓磷脂相关抑制物A PC12细胞 SHRNA 脂多糖 肿瘤坏死因子-α 白介素-6 酪氨酸羟化酶 

分 类 号：Q7[生物学—分子生物学]