摘要
目的观察有丝分裂原活化蛋白激酶（MAPK）信号传导通路抑制剂PD98059和磷脂酰肌醇3激酶（P13K）信号传导通路抑制剂LY294002对子宫内膜癌细胞株Ishikawa细胞和子宫内膜癌裸鼠移植瘤的抑制作用。方法（1）体外实验：实验分为4组，即PD组（加入浓度分别为0、1、50、100μmol／L的PD98059）、LY组（加入浓度分别为0、1、50、100μmol／L的LY294002）、PD＋LY组（加入浓度均为50μmol／L的PD98059和LY294002）、对照组[仅加入二甲基亚砜（DMSO）]，分别培养24、48和72h。应用四甲基偶氮唑蓝（MTT）比色法检测各组Ishikawa细胞的增殖情况，流式细胞仪检测各组Ishikawa细胞的细胞周期比例和细胞凋亡率。（2）体内实验：建立子宫内膜癌裸鼠移植瘤模型，随机分为4组（每组6只）：PD组（注射PD9805950mg／kg）、LY组（注射LY29400250mg／kg）、PD＋LY组（注射PD9805950mg／kg和LY29400250mg／kg）、对照组（注射等量的生理盐水），每周2次，共3周；观察移植瘤的生长情况，并计算抑瘤率；用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸标记（TUNEL）法检测移植瘤组织的细胞凋亡情况，以细胞凋亡指数表示；免疫组化法检测移植瘤组织内磷酸化细胞外信号调节激酶（p-ERK）和磷酸化蛋白激酶B（p-Akt）蛋白的表达。结果（1）PD98059和（或）LY294002处理后，PD组、LY组Ishikawa细胞增殖的抑制作用均呈明显的时间和浓度依赖性（P〈0．05），且PD＋LY组细胞增殖的这种抑制作用明显高于PD组、LY组（P〈0．05）。LY组细胞S期、G0／G1期比例的变化呈明显的时间和浓度依赖性（P〈0．05）；PD组细胞各期比例的变化均无时间依赖性（P〉0．05），但G0／G1期、S期比例的变化呈明显的浓度依赖性（P〈0．05）；PD＋LY组与PD组、LY组比较，Go／G．期比例明显增加、S期比例明显减少（P〈0．05）。PD＋LY组细胞凋亡率为（63．3±0．5）％，明显高于PD组、LY组[分别为（30．7±20．1）％和（40．8±1．3）％，P〈0．01]。（2）注射PD98059和（或）LY294002后，随着时间的延长，PD组、LY组、PD＋LY组裸鼠移植瘤体积增长相对缓慢，对照组移植瘤体积增长明显，PD组、LY组、PD＋LY组分别与对照组比较，差异均有统计学意义（P〈0．05）；PD＋LY组分别与PD组、LY组比较，差异也均有统计学意义（P〈0．05），而PD组与LY组比较，差异无统计学意义（P〉0．05）。LY组、PD组、PD＋LY组抑瘤率分别为（32±16）％、（38±17）％、（68±9）％，PD＋LY组明显高于LY组、PD组（P〈0．05）。LY组、PD组、PD＋LY组细胞凋亡指数分别为（13．7±1．5）％、（14．1±1．2）％、（29．0±1．8）％，PD＋LY组明显高于LY组、PD组（P〈0．01）。LY组、PD组、LY＋PD组裸鼠移植瘤组织中p-ERK和p-Akt蛋白的表达强度均明显弱于对照组。结论信号传导通路抑制剂PI）98059、LY294002能够抑制体外及体内子宫内膜癌细胞的生长，并促进其凋亡。
Objective To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulated kinase （p-ERK） and phosphorylated protein kinase B （p-Aki） in endometrial carcinoma xenografts. Methods Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium （MTT） assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups （ n = 6） , normal saline group, PD98059 group （ PD group） , LY294002 group （ LY group） or PD98059 ＋ LY294002 group （PD ＋ LY group） by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferasemediated dUTP-digoxigen in nick and labeling method （TUNEL） testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. Results （ 1 ） The proliferation of Ishikawa cells were suppressed after treated by PD98059 and （or） Y294002, in which A570 values of cells decreased showing both time-dependent and concentration-dependent manner （ LY294002 ： Fgroup = 9. 801, P = 0. 002; Ftime = 10. 398, P = 0. 001. PD98059 I Fgroup = 8. 213, P = 0. 015 ; Ftime = 6. 839, P = 0. 036 ）. Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G0/G1 phase（Ftime = 35. 049, P = 0. 004; Fsroup = 32. 024, P 〈 0. 01 ） increased and percentage of S phase cells （ Ftime = 7. 789, P = 0. 049 ; Fgroup = 30. 132, P 〈0. 01 ） decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD ＋ LY group, in which there were significant difference [ （ 63.3 ±0. 5 ） % vs （30. 7±20. 1 ） % vs（40. 8 ±1.3 ） % ; F = 6211. 059, P 〈 0. 01 ]. （2） Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously （ F = 23. 545, P 〈 0. 01 ）, and the inhibited rate of the tumor was higher in PD ＋ LY group than that in PD group or LY group [ （ 68 ±9 ） % vs （32 _ 16） % or （38 ±17 ） % ; F = 10. 283, P 〈 0. 05 ]. （3） HE staining shown that there were different degrees of necrosis for endometrial careinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [ （ 13.7 ± 1.5 ） %, （ 14. 1 ±1.2） %, （ 29. 0±1.8 ） % ; F = 320. 344, P 〈 0. 01 ]. Immunohiscochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY ＋ PD group was the lowest one. Conclusion The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2011年第6期446-452,共7页
Chinese Journal of Obstetrics and Gynecology
基金
河南省高校科技创新人才基金（2010HASTFTDD5）
关键词
子宫内膜肿瘤
细胞系
肿瘤
肿瘤移植
类黄酮物质
色酮类
吗啉类
Endometrial neoplasms
Cell line,tumor
Neoplasm transplantation
Flavonoids
Chromones
Morpholines
