Doxycycline抑制人肝癌细胞系HepG2生长实验探究.docVIP

Doxycycline抑制人肝癌细胞系HepG2生长实验探究 　 作者：张强波　李杰　时昌文　李捷　孙京杰　曹莉莉 【摘要】 目的：观察多西环素（Doxycycline）对人肝细胞性肝癌细胞系HepG2生长抑制和凋亡的作用，并初步探讨其作用机制。方法：不同浓度的Doxycycline（5、10、20、30和50 mg/L）对体外培养人肝癌细胞系HepG2进行不同的时间干预（24、48和72 h），MTT法测定细胞生长抑制率，TUNEL法检测细胞凋亡率，免疫细胞化学检测Fas、FasL及MMP-2蛋白表达的变化。结果：Doxycycline作用HepG2细胞48 h和72 h后，细胞生长被明显抑制，与无药对照组比较差异有统计学意义（Plt;0.001），且呈时间、浓度依赖趋势。其细胞凋亡率较无药对照组也明显升高（Plt;0.01）；20 mg/L Doxycycline作用于癌细胞48 h后，实验组FasL蛋白阳性表达较无药对照组明显升高，MMP-2蛋白阳性表达较无药对照组明显降低，两组差异均有统计学意义（Plt;0.01），而Fas蛋白表达则差异无统计学意义（Pgt;0.05）。结论：Doxycycline对人肝癌细胞系HepG2具有明显的生长抑制和促凋亡作用，其机制可能与下调MMP-2、上调FasL从而启动Fas/FasL膜受体途径有关。 【关键词】 肝肿瘤·Doxycycline·细胞凋亡·FasL·MMP-2 【ABSTRACT】Objective: To observe the effect and mechanism of doxycycline on cell growth inhibition and the apoptosis of human hepatoma cell HepG2 in vitro. Methods: HepG2 cells were treated with doxycycline at different concentrations and time in vitro. Cell growth inhibition rate was detected by MTT assay, and apoptosis rate was identified by TUNEL assay, the expression of Fas, FasL and MMP-2 proteins were examined by immunocytochemical staining. Results: Treated with doxycycline at different concentrations （5，10，20，30 and 50 mg·L-1） for 48 and 72 hours, the growth of HepG2 cells decreased significantly （Plt;0.001） and there was a tendency of dose-time dependent manner. Incubated HepG2 cells with doxycycline at 20 mg·L-1 for 48 hours, the expression of FasL were up-regulated and MMP-2 were down-regulated compared with those of control group （Plt;0.01）, while the difference of Fas expression was insignificant （Pgt;0.05）. Conclusion: Doxycycline might inhibit cell growth and induce apoptosis of HepG2 significantly. The mechanism of doxycycline induced apoptosis might be associ 　　多西环素（Doxycycline）为四环素类抗生素的一种，已有研究表明其对前列腺癌、乳腺癌、胰腺癌等多种肿瘤生长有明显的抑制作用［1-3］，而其对人肝癌细胞的生长抑制作用国内外未见报道。本研究观察了Doxycycline对人肝癌细胞系HepG2的生长抑制和凋亡的影响，并初步探讨其作用机制。 1　材料与方法 1.1　实验材料　人肝细胞性肝癌细胞系HepG2购自美国典型物保藏中心细胞库，用含10%胎牛血清的RPMI1640培养基，37℃、5%CO2饱和湿度的孵箱内常规培养，每日倒置显微镜观察细胞生长情况，3d换液，适时0.25%胰酶消化传代。四甲基偶氮唑盐（MT