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[目的]研究Toll样受体4(toll-like receptor 4,TLR4)促进低氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)的高活性在宫颈癌中的作用及机制。[方法 ]体外培养Siha细胞,分为空白对照组(control组)、脂多糖组(LPS组)、NF-κB信号通路干预组[吡咯烷二硫氨基甲酸(PDTC)+LPS组]和TLR4信号干预组(si TLR4+LPS组)共4组,用MTT法、软琼脂形成实验观察各组细胞的活性增殖、克隆情况;用免疫细胞化学染色法和Western blot法检测细胞内HIF-1α含量变化;DCFH-DA法检测活性氧(ROS)含量变化;光泽精化学发光法检测NADPH氧化酶含量变化。[结果 ]MTT和软琼脂形成实验结果显示:与control组相比,LPS组细胞活性和增殖能力增强,PDTC+LPS组无明显变化,si TLR4+LPS组降低(P<0.05);与LPS组相比,PDTC+LPS组和si TLR4+LPS组细胞活性和增殖能力均降低(P<0.05)。与PDTC组+LPS组相比,si TLR4+LPS组细胞活性显著减弱(P<0.01)。免疫细胞化学染色和Western blot法结果显示:与control组相比,LPS组HIF-1α活性增强(P<0.05),si TLR4+LPS组活性降低(P<0.05);与LPS组相比,PDTC+LPS组HIF-1α表达降低(P<0.05),si TLR4+LPS组HIF-1α表达明显降低(P<0.01);与PDTC+LPS组相比,si TLR4+LPS组细胞HIF-1α活性减弱(P<0.05)。NADPH氧化酶和ROS活性检测结果显示:与control组相比,LPS组NADPH氧化酶和ROS活性增强(P<0.05),si TLR4+LPS组降低(P<0.05),LPS组与PDTC+LPS组无明显差异。与PDTC+LPS组相比,si TLR4+LPS组NADPH氧化酶和ROS活性均降低(P<0.05)。[结论]TLR4可通过核因子-κB(NF-κB)信号途径促进宫颈癌的发生和发展,NADPH氧化酶参与TLR4维持细胞内HIF-1α高活性,而此过程与NF-κB信号无关,这为研究宫颈癌的发生发展机制提供新的思路。
[Objective] To investigate the effect and mechanism of toll-like receptor 4 (TLR4) on the high activity of hypoxia inducible factor-1α (HIF-1α) in cervical cancer. [Methods] Siha cells were cultured in vitro and divided into control group, lipopolysaccharide group (LPS group), NF-κB signal pathway intervention group [pyrrolidine dithiocarbamate (PDTC) + LPS group) and TLR4 signal The intervention group (si TLR4 + LPS group) were divided into 4 groups. MTT assay and soft agar assay were used to observe the proliferation and cloning of cells in each group. The changes of intracellular HIF-1α contents were detected by immunocytochemistry and Western blot ; DCFH-DA method was used to detect the content of reactive oxygen species (ROS); glossary chemiluminescence method was used to detect the content of NADPH oxidase. [Result] The results of MTT assay and soft agar assay showed that compared with the control group, the cell viability and proliferation ability of LPS group were enhanced. There was no significant change in PDTC + LPS group and si TLR4 + LPS group (P <0.05) The cell viability and proliferation ability of PDTC + LPS group and si TLR4 + LPS group were decreased (P <0.05). Compared with PDTC + LPS group, the cell viability of si TLR4 + LPS group was significantly decreased (P <0.01). Immunocytochemical staining and Western blot showed that compared with the control group, the activity of HIF-1α in LPS group was increased (P <0.05) and the activity of si TLR4 + LPS group was decreased (P <0.05). Compared with LPS group, PDTC (P <0.05), while the expression of HIF-1α in si TLR4 + LPS group was significantly lower than that in the PDTC + LPS group (P <0.01). Compared with the PDTC + LPS group, the HIF- (P <0.05). The results of NADPH oxidase and ROS assay showed that the activity of NADPH oxidase and ROS in LPS group were significantly increased (P <0.05), and decreased in si TLR4 + LPS group (P <0.05). Compared with control group, LPS group and PDTC + LPS group No significant difference. Compared with PDTC + LPS group, NADPH oxidase and ROS activity in si TLR4 + LPS group decreased (P <0.05). [Conclusion] TLR4 can promote the occurrence and development of cervical cancer through NF-κB signaling pathway. NADPH oxidase participates in TLR4 to maintain high intracellular HIF-1α activity, which has nothing to do with NF-κB signaling To provide a new idea for studying the mechanism of cervical cancer occurrence and development.