摘要目的 探讨雷公藤内酯醇(TP)对人胰腺癌PANC1细胞株侵袭能力的影响及其与Toll样受体4/核因子-KB(TLR4/NF-KB)信号通路的关系.方法 将PANC1细胞分为亲本细胞组、TP组、脂多糖(LPS)组和TP+ LPS组.TP组培养液中加入50 ng/ml的TP,LPS组加入1μg/ml的LPS,TP+ LPS组先用50 ng/ml的TP处理2h,再加入1 μg/ml的LPS.各组细胞均常规培养24 h.采用实时定量PCR和蛋白质印迹法检测TLR4、基质金属蛋白酶-9(MMP-9)mRNA和蛋白的表达,双荧光素酶报告基因系统检测NF-KB活性,Transwell小室检测细胞侵袭能力.结果 亲本组、LPS组、TP组、TP+ LPS组的TLR4mRNA表达量分别为0.41 ±0.06、0.46±0.10、0.20±0.04和0.25±0.06,TLR4蛋白表达量分别为0.55±0.06、0.60±0.03、0.18±0.04和0.13±0.00；NF-KB活性分别为13.0±3.0、31.6±4.3、7.3±1.5和10.8±2.1；穿膜细胞数分别为(56.8±8.6)、(104.5±12.8)、(32.0±5.7)和(46.8±7.0)个；MMP-9mRNA表达量分别为0.36±0.05、0.58 ±0.07、0.18±0.03和0.30±0.004,MMP-9蛋白表达量分别为0.31±0.04、0.53±0.08、0.11±0.02和0.15±0.00.LPS组TLR4 mRNA和蛋白表达量与亲本组差异无统计学意义,但NF-KB活性、穿膜细胞数、MMP-9 mRNA和蛋白表达量显著高于亲本组(t值分别为8.654、7.593、6.655、4.982,P值均＜0.01).TP组TLR4 mRNA和蛋白表达量、NF-KB活性、穿膜细胞数、MMP-9 mRNA和蛋白表达量均显著低于亲本组(t值分别为-7.609、-9.948、-4.176、-5.915、-8.179、-9.948,P值均＜0.01).TP+ LPS组TLR4 mRNA和蛋白表达量、NF-KB活性、穿膜细胞数、MMP-9 mRNA和蛋白表达量均显著低于LPS组(t值分别为-4.437、-14.805、-10.506、-9.700、-9.055、-8.932,P值均＜0.01).结论 TP具有抑制胰腺癌细胞侵袭的作用,其机制与抑制TLR4/NF-kB信号通路、下调MMP-9的表达有关.

abstractsObjective To investigate the role of TLR4/NF-kB signaling pathway in inhibited invasion ability of pancreatic cancer cells caused by triptolide (TP).Methods PANC1 cells were divided into parental cells group,TP group,lipopolysaccharide (LPS) group and TP + LPS group.50 ng/ml of TP was added in culture medium in TP group,and 1 μg/ml of LPS was added in culture medium in LPS group,while 50 ng/ml of TP was pretreated for 2 h and 1 μg/ml of LPS was added in culture medium in TP + LPS group.All the ceils were cultured for 24 h.The TLR4 and matrix metalloproteinase-9 (MMP-9) mRNA and protein expression were evaluated by real-time PCR and Western blot.The NF-kB activity was determined by dual-luciferase reporter assay system.The invasion ability of pancreatic cancer cells was evaluated by transwell invasion chamberassay.Results The TLR4 mRNA expressions in parental cells group,TP group,LPS group and TP + LPS group were 0.41 ± 0.06,0.46 ± 0.10,0.20 ± 0.04,0.25 ± 0.06 ; the TLR4 protein expressions were 0.55 ±0.06,0.55 ±0.06,0.18 ±0.04,0.13 ±0.00; the activities of NF-kB were 13.0 ±3.0,31.6 ±4.3,7.3 ±1.5 and 10.8 ± 2.1,and the numbers of invasion cell were (56.8 ± 8.6),(104.5 ± 12.8),(32.0 ± 5.7) and (46.8 ± 7.0) ; the MMP-9 mRNA expressions were 0.36 ± 0.05,0.58 ± 0.07,0.18 ± 0.03,0.30 ± 0.004 ;the MMP-9 protein expressions were 0.31 ± 0.04,0.53 ± 0.08,0.11 ± 0.02,0.15 ± 0.00.In LPS group,TLR4 mRNA and protein expressions were not statistic significant when compared with those in parental cells group,but the activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions were statistically increased when compared with those in parental cells group (t =8.654,7.593,6.655,4.982,P ＜0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP group were significantly lower than those in parental cells group (t =-7.609,-9.948,-4.176,-5.915,-8.179,-9.948,P＜ 0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP +LPS group were significantly lower than those in LPS group (t =-4.437,-14.805,-10.506,-9.700,-9.055,-8.932,P＜ 0.01).Conclusions TP can inhibit pancreatic cancer cell invasion,and the mechanism is related to the inhibition of TLR4/NF-kB signaling pathway and down-regulation of MMP-9 expression.