【作者】 李立祥；

【导师】 熊奇如； 耿小平； 朱立新；

【作者基本信息】 安徽医科大学 ， 外科学， 2008， 硕士

【摘要】 目的探讨脂氧合酶（lipoxygenase）抑制剂对肝癌细胞株HepG2增殖与凋亡的调节作用及其对凋亡相关基因表达的影响。进一步揭示脂氧合酶抑制剂的抗肿瘤机制,为药物实验及临床应用提供理论依据,以拓宽肝癌的发病机制及药物治疗学的研究视野。方法①选用人肝癌细胞株HepG2作为研究对象,加入脂氧合酶抑制剂去甲二氢愈创木酸（NDGA）（浓度设为25、50、100、200μmol/L）进行体外培养,采用对数生长期的细胞进行实验。②应用MTT比色法观察NDGA对HepG2增殖的影响。③荧光染色法、透射电镜观察细胞凋亡形态。④流式细胞仪进行细胞周期及凋亡检测。⑤RT-PCR检测NDGA对端粒酶hTERTmRNA、bcl-2及bax凋亡相关基因表达水平的影响。结果①NDGA（25～200μmol/L）对肝癌细胞HepG2增殖有抑制作用,其体外增殖呈量效和时效关系,生长抑制率由5.23%增至61.20%。药物浓度越高,抑制作用越强,呈明显的剂量效应关系;系列浓度NDGA作用于细胞24h、48h、72h,作用时间越长,抑制作用越强,呈时间依赖性。高浓度NDGA可诱导HepG2凋亡,各组之间差异有显著性（P＜0.01）。根据中效方程式计算出72h的IC₅₀值为128.37μmol/L。以给药浓度为自变量,抑制率为因变量对作用不同时间的各组进行回归分析,显示给药浓度与抑制率呈明显正相关,r24=0.965,P＜0.01,r48=0.902,P＜0.01,r72=0.981,P＜0.01。②荧光染色法显示NDGA作用HepG2细胞48h后染色,细胞体积缩小、形态变圆、胞膜皱缩呈毛刺状、细胞内含高度浓缩的染色质胞核,部分呈核碎片状,可见到核裂解成双核。有较多的细胞脱落,悬浮于细胞液中。③透射电镜可见细胞变圆缩小,细胞膜凹陷,伪足断裂,微绒毛卷曲缩短、消失,典型的细胞核浓缩致密、凋亡小体形成的细胞形态学改变。④流式细胞仪显示,随药物浓度增加,细胞凋亡率显著增加,经NDGA 200μmol/L作用48h后早期凋亡率达41.6±2.3。NDGA能引起肝癌细胞阻滞于S期,G₀/G₁期细胞比例下降。⑤RT-PCR检测显示NDGA明显下调端粒酶5-LOX、hTERTmRNA和bcl-2及上调bax的表达,存在一定的剂量依赖关系,各处理组与对照组相比,有统计学意义。结论①脂氧合酶抑制剂NDGA对肝癌细胞HepG2的体外生长增殖具有抑制作用,呈剂量和时间依赖性效应,同时可以阻滞细胞周期于S期。其作用机理可能式通过抑制细胞增殖和诱导细胞凋亡的途径来实现的。②NDGA对HepG2细胞的形态学产生明显影响,对细胞的粘附侵袭有一定影响。③NDGA通过抑制5-LOX mRNA的表达,可能是其抑制细胞增殖和诱导细胞凋亡的作用机制之一。④端粒酶hTERT mRNA、bcl-2 mRNA及bax mRNA等相关凋亡基因表达水平可能与细胞凋亡过程有关。LOX可能是肝癌生物化学治疗的新靶点。更多还原

【Abstract】 Objective To investigate the effects of lipoxygenase inhibitor,on proliferation and apoptosis in hepatocellular carcinoma cell line HepG2 and its apoptosis related genes in vitro,which we can elucidate the antineoplastic mechanisms of lipoxygenase inhibitors for the drug experiment and clinical application.Widening the research field of hepatocellular carcinoma in the pathogenesis and pharmacotherapeutics.Methods①A human hepatocellular carcinoma cell line,HepG2,was cultured in vitro and nordihydroguaiaretic acid（NDGA） was divided into 25、50、100、200μmol/L at concentration in the different times.Cells in logarithmic growth phase were selected and examined.②The proliferation inhibition was analysed by alive cell count,MTT assay.③Cells apoptosis was observed with inverted microscope,Hoechst33258 labeling and transmission electron microscope.④Cell cycle analysis,apoptosis rate were evaluated by Annexin V/PI and flow cytometry（FCW）method.⑤The mRNA expression of apoptosis related genes telomerse,bcl-2 and bax were examined by RT-PCR.Results①NDGA can inhibit HepG2 cells proliferation viability within a certain range （25～200μmol/L） of treating time and dose,in which the inhibition increased from 5.23%to 61.20%.The higher the concentration of NDGA was,the stronger the cytotoxic effect reached,which suggested obvious dose-dependent manner of NDGA;The inhibitory effects were augmented with the prolongation of culture（24h、48h、72h）,which had time-dependence.The r values of dose-effect curves for NDGA on HepG2 cell line were 0.965、0.902 and 0.981 respectively.The IC₅₀ values were 128.37μmol/L（72h）.High concentration NDGA can induce the apoptosis of HepG2.②Under the Hoechst33258 labeling,the cell morphology obviously changed treated with different concentrations of NDGA for 48h.Some cells became much more round and brighter,shrinking with the membrane crimpled like burrs.The cytoplasm contained highly concentrated chromosomes which attached to the nuclear membrane and came into the shape of clump,crescent or fragment.Many cells fell off the wall of medium and were suspensive in the culture medium.③Under the scanning electron microscope,HepG2 Cell showed typical apoptotic morphological changes after treatment of NDGA,characterized by cellular roundness,volume shrinkage and membranous crinkle.The changes of microvillus and filopodium were very distinct.The filopodia were broken,and the microvilla were curled and shortened.Some cells nuclear condensated and apoptosis bodies were formed.④With the increase in the concentrations of NDGA,the HepG2 cells apoposis increased gradually After 48h treatment（200μmol/L）,the early apoptosis reached 41.6±2.3.NDGA can induced cell cycle arrest at the S phase in HepG2 cell lines,along with the decrease in the population of G₀/G₁ phase cells.⑤The expression of bax mRNA was up-regulated and down-regulated of 5-LOX mRNA,bcl-2 mRNA and hTERT mRNA in HepG2 cells by RT-PCR after NDGA treatment for 48h,all the expressions of mRNA with dose-related effect and were compared significantly（P＜0.001）.Conclusion①NDGA can inhibited HepG2 cells proliferation in a time- and dose-dependent manner,and cells proliferation was blocked in the S phase.The mechanism may be implemented though inducing apoptosis and preventing proliferention.②Because NDGA has a dramatical effect on the morphology of HepG2 cells,especially on the filopodia and microvilla,we can conclude NDGA might prevent the adhesion and invasion of the ceils.③The expression of 5-LOX mRNA was strongly positive in HepG2 cells.One of the mechanisms of NDGA inhibition proliferation and inducing apoptosis is likely to connect with the down-regulation of 5-LOX mRNA.④The effect of NGDA on cells proliferation and induced the apoptosis which may be associated with the upregulating expression of bax mRNA and downregulating expression of hTERT mRNA,bcl-2 mRNA.Lipoxygenase might be a novel therapeutic target for the hepatocellular carcinoma.更多还原