【作者】 王梦华；

【导师】 张正茂； 单保恩；

【作者基本信息】 河北医科大学 ， 妇产科学， 2009， 硕士

【摘要】 目的:研究格尔德霉素（Geldanamycin,GA）及其与抗癌药物顺铂（DDP）联合应用对人卵巢癌细胞SKOV3的体外抑制作用,分析格尔德霉素抑制SKOV3细胞的增殖及诱导其凋亡的可能机制,探讨格尔德霉素用于卵巢癌临床治疗的可行性。方法:1体外培养人卵巢癌细胞SKOV3,采用四甲基偶氮唑蓝（tetrzolium-based colorimetric assay,MTT）比色法检测格尔德霉素对该细胞增殖的影响。2格尔德霉素作用48后,采用Giemsa染色显微镜观察SKOV3细胞形态学改变及流式细胞术（flow cytometry,FCM）检测细胞周期及凋亡率的变化。3免疫细胞化学法（immunocytochemistory,ICC）检测格尔德霉素处理48小时后SKOV3细胞内Akt及raf-1蛋白的表达情况。4流式细胞术分析格尔德霉素处理SKOV3细胞48后Akt、raf-1蛋白的表达水平。5 MTT比色法检测格尔德霉素与顺铂联合应用对SKOV3细胞增殖的抑制作用,采用金氏公式分析相互作用指数（Interaction Index）,评价两药联合应用后的作用,如相互作用指数＞0.85表示相加作用,相互作用指数＜0.85为拮抗作用。结果:1不同浓度的格尔德霉素（20、40、200、400、2000nmol/L）对SKOV3细胞的增殖均可产生抑制作用,且格尔德霉素对SKOV3细胞增殖的抑制作用呈明显的时间—剂量依赖关系,最大抑制率可达（74.72±3.16）%。GA作用48h后的IC50为496.57nmol/L。2经20、40、200、400、2000nmol/L的格尔德霉素作用48h后SKOV3细胞,随着药物浓度的增大,G₂/M期细胞逐渐增多,该作用呈剂量—效应依赖关系。不同浓度组格尔德霉素均诱导细胞凋亡,其凋亡率与对照组相比差异有统计学意义（P＜0.05）。3光学显微镜下观察未经格尔德霉素作用的对照组SKOV3细胞呈长梭形或菱形,形态饱满:而经格尔德霉素作用48h后的SKOV3细胞皱缩,破裂,呈不规则形,有的细胞两端出现细长的伪足,细胞胞浆中出现空泡,脱落细胞增多。经Giemsa染色光学显微镜下显示,对照组SKOV3细胞呈长梭形或菱形,形态饱满,细胞核染色均匀,核仁清晰可见,未见凋亡细胞出现;而经格尔德霉素作用48h后的SKOV3细胞变圆,胞质浓缩,核着色不均匀,出现核分裂现象,呈现凋亡细胞的形态学改变。4免疫细胞化学法检测显示,经0、20、200、2000nmol/L格尔德霉素处理48小时后,SKOV3细胞中Akt、raf-1蛋白的表达随药物浓度增大而逐渐降低。流式细胞术检测结果同样显示Akt、raf-1蛋白表达显著降低（P＜0.05）。5 MTT结果显示格尔德霉素与顺铂联合应用对SKOV3细胞的增殖有明显的抑制作用。20、40、200、400nmol/L的格尔德霉素联合顺铂对SKOV3细胞增殖的抑制作用随浓度的增加逐渐增强。金氏公式分析相互作用指数,计算结果显示不同浓度的顺铂联合不同浓度的格尔德霉素相互作用指数均＞0.85。结论:1格尔德霉素对人卵巢癌细胞SKOV3的增殖具有明显的抑制作用,为卵巢癌的临床治疗提供了一种新的手段。2格尔德霉素抑制SKOV3细胞增殖的作用呈明显的时间一剂量依赖关系。3格尔德霉素可使SKOV3细胞阻滞于G₂/M期,并可诱导其凋亡。4格尔德霉素可通过下调Akt、raf-1蛋白的表达而发挥抑制SKOV3细胞增殖、诱导该细胞凋亡的作用。5格尔德霉素与顺铂联合应用可增强顺铂对SKOV3细胞增殖的抑制作用。更多还原

【Abstract】 Objective:To investigate the cytotoxic effects of geldanamycin on the proliferation of human ovarian cancer SKOV3 cells.Moreover,and to check the cytotoxic effects of geldanamycin combined with cisplatin（DDP） on human ovarian cancer SKOV3 cells.Furthermore,to analyze the possible mechanisms of GA inhibiting the proliferation and inducing apoptosis to SKOV3 cells.Methods:1 Human ovarian cancer SKOV3 cells were incubated with different concentrations of geldanamycin in vitro.The effect of geldanamycin on the proliferation of SKOV3 cells was measured by MTT colorimetric method.2 Microscopy was used to observe the cells changes in morphology and flow cytometry was used to detected cell cycle and apoptosis after treated by geldanamycin.3 The expressions of Akt and Raf-1 protein of SKOV3 cell treated with geldanamycin for 48h were analyzed by flow cytometry and immunocytochemistory method.4 MTT colorimetric method was performed to evaluate the effect of Geldanamycin combined with DDP on SKOV3 cells. Jin’s formula was performed to determine whether the combination of geldanamycin with DDP could produce synergistic cytotoxic effect or not.More than 0.85 of the interaction index was thought to be as a synergic effect,while less than 0.85 was thought to be as antagonism.Results:1 The proliferation of SKOV3 cells could be inhibited by the different concentrations of geldanamycin in a time- and dose- dependent way.The highest inhibition rate reached （74.72±3.16）%and IC50 was 496.57nmol/L after SKOV3 cell were treated GA for 48h.2 After treated with geldanamycin for 48h,cells cycle was arrested in G₂/M phase.3 SKOV3 cells untreated with geldanamycin were fusiform,diamond and satiation by microscopy,while the cells treated with geldanamycin were shrinked and broken,became irregular in shape,spreading in a thin and long pseudopodium in both end points,vacuolus could also be seen in some cells cytoplasm,floating and cast-off cells increased.Giemsa staining showed that the SKOV3 cells untreated with geldanamycin were fusiform,diamond,and they were satiation,furthermore,the cell nucleus staining were uniform and clear,no floating cells.But SKOV3 cells treated with geldanamycin became rounding,and the cytoplasm was concentrated,the staining cell nucleus was uneven,there were also caryocinesis phenomenon,showing the typical apoptotic morphological changes. 4 The expressions of Akt and raf-1 proteins were tested by immunocytochemistory and flow cytometry.The expressions of Akt and raf-1 proteins decreased significantly with the increasing of the concentrations of geldanamycin（P＜0.05）.5 MTT results showed that Geldanamycin combined with DDP could inhibit the proliferation of SKOV3 cells.20,40, 200,400nmol/L geldanamycin combined with DDP produced synergic cytotoxic effects to SKOV3 cells,and these synergic cytotoxic effects were further be testified by interaction index from Jin’s formula.Conclusions:1 Geldanamycin could induce cytotoxic effects on the proliferation of human ovarian cancer SKOV3 cells in a time-and dose- dependent manner.2 Geldanamycin could cause cells cycle arrested in G₂/M phase as well as apoptosis in SKOV3 cells.3 The cytotoxic effects of geldanamycin to inhibit the proliferation and induce apoptosis to SKOV3 cells were partly due to down-regulating the expression of Akt and raf-1.4 Geldanamycin combined with DDP could inhibit the proliferation of SKOV3 cells and produce synergic cytotoxic effects to SKOV3 cells.更多还原