【作者】 李玲；

【导师】 石红；

【作者基本信息】 大连医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的：观察选择性表皮生长因子受体酪氨酸激酶抑制剂吉非替尼(ZD1839)单药及联合顺铂(DDP)或多西紫杉醇(DTX)对人卵巢透明细胞癌ES-2细胞系的抑制效应,并初步探讨其作用机制。比较ZD1839联合DDP或DTX与传统的TP(顺铂联合多西紫杉醇)方案对ES-2细胞系的抑瘤作用,为卵巢透明细胞癌临床化疗寻求一种新的化疗方案。方法：用MTT法检测化疗药物作用24h、48h对人卵巢透明细胞癌ES-2细胞系的抑制率(Fa),分析比较单药及联合用药的抑瘤效果;流式细胞术检测ZD1839作用前后人卵巢透明细胞癌细胞的周期分布和早期、晚期及总体凋亡率。结果：(1) DDP、ZD1839和DTX单用及两两联合应用均能抑制ES-2细胞系的生长,且具有时间和浓度依赖性(P<0.01)。DDP、ZD1839和DTX24h的IC50值分别是35.31ug/mL、2.77ug/mL和3.59ug/mL。单药组中,ZD1839组的IC50最低,抑瘤效果最强(F=14129.74,P<0.01)。联合用药组中,ZD1839联合DDP或DTX比DDP联合DTX的IC50低,抑瘤效果强(P<0.01)。(2)DDP和ZD1839合用24h后,当Fa<0.6时,合用指数CI<1,两药是协同作用；当Fa≥0.6时,合用指数CI>1,两药之间是拮抗作用。合用48h后,当Fa<0.8时,合用指数CI<1,两药是协同作用。DTX和ZD1839合用24h后,当Fa≤0.5时,合用指数CI<1,两药是协同作用：合用48h后,当Fa≤0.6时,合用指数CI<1,两药是协同作用。(3)实验组早期、晚期及总体凋亡率均显著高于对照组(F=115.62,81.48,327.07;P<0.01,0.01,0.01)。单药组中,ZD1839组的早期凋亡率显著高于DDP组和DTX组(17.8% vs 9.65%、9.17%；F=47.34,P<0.01)。ZD1839联合DDP组的总体凋亡率最高(50.43%),DDP组的总体凋亡率最低(16.94%)。(4)应用2.5ug／mL的ZD183924h后,G1期细胞数比对照组增加了1.05倍(98.23% vs 43.07%)。结论：(1)ZD1839能够抑制人卵巢透明细胞癌ES-2细胞系的生长,并且能够增强顺铂、多西紫杉醇的抑瘤作用。该抑制作用具有时间和浓度依赖性。DDP.ZD1839和DTX24h的IC50依次是35.31ug/mL.2.77ug/mL和3.59ug/mL.(2)单药组中,ZD1839组的IC50最低,抑瘤效果最强。提示临床单用ZD1839治疗OCCA可能增强化疗疗效。(3)该抑瘤作用可能与ZD1839使ES-2细胞停滞于G1期并诱导肿瘤细胞凋亡尤其是早期凋亡有关。(4)ZD1839联合DDP或DTX对ES-2细胞系的抑瘤作用显著高于传统的TP(顺铂联合多西紫杉醇)方案；提示对OCCA特别是TP化疗耐药的患者,ZD1839联合方案可能更有效。更多还原

【Abstract】 Objective:To observe the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (ZD1839), whose inhibitory effect of monotherapy and combination with cisplatin and docetaxel to the human ovarian clear cell adenocarcinoma (OCCA) cell line ES-2. Meanwhile, to explore its mechanism. Use ZD1839 combined with DDP or DTX and traditional TP (DDP combined DTX) regime, to compare their inhibition efficacy to ES-2. To find a new effective regime to OCCA’s clinic chemotherapy.Methods:Use MTT method to assay chemotherapeutic drugs’ inhibition rate (Fa) to OCCA cell line ES-2; Analyze and compare the inhibition effect of monotherapy and combination therapy. Use flow cytometry to assay OCCA cell line ES-2’cell cycle distribution and its early, late and total apoptosis rate.Results:(1) DDP, gefitinib and docetaxel, whose monotherapy and combination can inhibit the growth of ES-2 cells, and the inhibition was time and concentration dependent (P<0.01). The 24h ICSO of DDP, gefitinib and docetaxel were 35.31ug/mL,2.77ug/mL and 3.59ug/mL, respectively. Among the monotherapy groups, ZD1839 group had the lowest IC50, and the strongest inhibitory effect (F=14129.74, P<0.01). Among the combined Groups, ZD1839 combined DDP or DTX group had lower IC50 and stronger inhibitory effect than the group DDP combined DTX’s (P< 0.01). (2) 24 hours after combined DDP with ZD1839, when Fa<0.6, the combination index CI< 1, and the two drugs were synergistic; when Fa≥0.6, the combination index CI> 1, and the two drugs were antagonistic. After 48 hours combination, the two drugs were synergistic.24 hours after ZD1839 combined with DTX, when Fa≤0.5, the combination index CI<1, and the two drugs were synergistic. After 48 hours combination, when Fa<0.8, the combination index CI< 1, and the two drugs were synergistic. (3)The early, late and total apoptosis rates of experimental groups were significantly higher than the control group’s (F=115.62,81.48,327.07; P <0.01,0.01, 0.01). Among the monotherapy groups, Group gefitinib’s early apoptosis rate was significantly higher than the other two groups (17.8% vs 9.65%, 9.17%; F=47.34, P<0.01). Among the six experimental groups, gefitinib combined DDP group’s total apoptosis rate was 50.43%, which was the highest of all the groups. Conversely, DDP group had only 16.94% total apoptosis rate, which was the lowest. (4) After application 2.5ug/mL of the gefitinib 24 hours, the G1 phase cells were increased 1.05 times than the control group (98.23% vs 43.07%).Conclusions:(1) Gefitinib can inhibit human ovarian clear cell adenocarcinoma ES-2 cell line’s growth, and it can also enhance the antitumor ability of DDP and docetaxel. The inhibition was time and concentration dependent. The 24h IC50 of DDP, gefitinib and docetaxel were 35.31ug/mL,2.77ug/mL and 3.59ug/mL, respectively.(2) Among the monotherapy groups, the IC50 of ZD1839 group was the lowest and the inhibitory effect was the strongest. It indicated that using ZD1839 in the clinical treatment of OCCA may enhance the effect of chemotherapy.(3) This antitumor mechanism may be related to the detention of G1 phase and induction of tumor cell apoptosis.(4) The inhibition of gefitinib combined DDP or docetaxel to ES-2 cell line were significantly higher than the traditional TP (DDP and DTX) regimen. For OCCA patients especially those resistant to TP regimen, the trial of ZD 1839 may get better treatment efficacy.更多还原