四环素对人牙龈成纤维细胞增殖及凋亡的影响

Effects of tetracycline on proliferation and apoptosis of human gingival fibroblasts

目的:探讨四环素(TET)对人牙龈成纤维细胞(HGFs)增殖和凋亡的影响及其可能的作用机制。方法:体外培养HGFs,采用不同浓度(0、5、10、50、100、500、1000、2000、2500μg·ml^(-1))的TET干预HGFs,细胞计数法(CCK-8)检测TET对HGFs增殖的影响。0、10、100和2000μg·ml^(-1)TET培养细胞,分别作为对照组、低浓度组、中浓度组、高浓度组;倒置显微镜下观察TET对HGFs生长状态的影响,流式细胞术检测细胞周期的变化,Annexin V-FITC/PI双染色法检测细胞凋亡情况,酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、细胞间黏附因子-1(ICAM-1)和白细胞介素-6(IL-6)的分泌情况,实时荧光定量PCR(qPCR)和蛋白质免疫印迹法(Western blotting)分析NF-κB信号通路相关蛋白的表达水平。结果:低浓度(10~50μg·ml^(-1))TET能够促进HGFs的增殖,中浓度(100~1000μg·ml^(-1))、高浓度(2000~2500μg·ml^(-1))TET均可抑制HGFs的增殖。低浓度TET对HGFs生长状态无明显影响,而中、高浓度TET组细胞呈现不同程度的不规则形状。低浓度TET组G;/G;期细胞比例增多,S期细胞比例减少,而中、高浓度TET组G;/G;期细胞比例减少,S期细胞比例增多。中、高浓度TET可促进HGFs凋亡。低浓度TET可抑制TNF-α、ICAM-1和IL-6的分泌,而中、高浓度TET可促进TNF-α、ICAM-1和IL-6的分泌。低浓度TET可促进NF-κB p65和IκBα蛋白磷酸化,而中、高浓度TET可抑制NF-κB p65和IκBα蛋白磷酸化。结论:中、高浓度TET可抑制HGFs增殖和细胞周期进程,并诱导细胞凋亡,但是低浓度TET对细胞增殖无明显影响,其作用机制可能与NF-κB信号通路的活化有关。

Objective:To investigate the effect and possible mechanism of tetracycline(TET)on the proliferation and apoptosis of human gingival fibroblasts(HGFs).Methods:HGFs were cultured in vitro,and different concentrations(0,5,10,50,100,500,1000,2000,2500μg·ml^(-1))of TET were used to intervene in HGFs,and the effect of TET on the proliferation of HGFs was detected by cytometry(CCK-8).The cells were cultured with 0,10,100 and 2000μg·ml^(-1)TET and divided into control group,low concentration group,medium concentration group and high concentration group;the effect of TET on the growth state of HGFs was observed under an inverted microscope,and the cell cycle and apoptosis were detected by flow cytometry;enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of tumor necrosis factor-α(TNF-α),intercellular adhesion factor-1(ICAM-1)and interleukin-6(IL-6).Real-time quantitative PCR(qPCR)and Western blotting were used to detect the expression levels of NF-κB signaling pathway related protein.Results:Low concentration(10~50μg·ml^(-1))of TET could promote the proliferation of HGFs,and medium(100~1000μg·ml^(-1))and high(2000~2500μg·ml^(-1))concentration of TET could inhibit the proliferation of HGFs.Low concentrations of TET had no significant effect on the growth state of HGFs,while cells in medium and high concentrations of TET showed irregular shapes to varying degrees.The proportion of cells in G;/G;phase increased and the proportion of cells in S phase decreased in the low concentration TET group,while the proportion of cells in G;/G;phase decreased and the proportion of cells in S phase increased in the middle and high concentration TET groups.Medium and high concentrations of TET could promote the apoptosis of HGFs.Low concentration of TET could inhibit the secretion of TNF-α,ICAM-1 and IL-6,while medium and high concentration of TET could promote the secretion of TNF-α,ICAM-1 and IL-6.Low concentration of TET could promote the phosphorylation of NF-κB p65 and IκBαproteins,while medium and high concentration of TET could inhibit the phosphorylation of NF-κB p65 and IκBαproteins.Conclusion:TET can inhibit the proliferation and cell cycle progression of HGFs,and induce cell apoptosis,but low concentrations of TET has no significant effect on cell proliferation,and its mechanism of action may related to the activation of NF-κB signaling pathway.