【作者】 姜爱华；

【导师】 刘保安；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的：本文旨在研究c-Met抑制剂SU11274对食管癌Eca109细胞株生物学行为的影响,并通过其作用靶点c-Met蛋白和C-met mRNA与生物钟基因Clock mRNA的可能潜在联系,进一步探讨在其作用过程中是否有时辰效应的存在。方法：(1)体外培养食管癌Eca109细胞株,用免疫细胞化学检测c-Met蛋白的表达。(2)用SU11274(IC50为20nM)处理Eca109细胞,通过MTT生长曲线、划痕实验、transwell侵袭小室以及FCM,观察药物对细胞生长、迁移、侵袭能力以及凋亡率与细胞周期的影响。(3)体外培养食管癌Eca109细胞株,用RT-PCR法分别检测不同时间点Clock与C-met mRNA的表达。(4)用流式细胞仪检测各时间点c-Met蛋白的表达。(5)不同时间点SU11274处理细胞24h后,用流式细胞仪检测细胞周期。结果：(1)Eca109细胞表达c-Met蛋白。(2)SU11274实验组细胞生长,迁移和浸润能力低于对照组,凋亡率高于对照组,增殖指数低于对照组。(3)48h内Clock基因表达呈节律性波动(P<0.05)。(4)24h内Clock与C-met基因表达呈相似节律性波动(P<0.01),。(5)24h内c-Met蛋白表达呈节律性波动(P<0.05),出现表达高峰的时间点与C-met mRNA表达高峰时间点一致。(6)不同时间点SU11274处理细胞后,细胞增殖指数抑制率不同(P<0.01)。结论：(1)c-Met抑制剂SU11274可以抑制Eca109细胞的生长和增殖,减弱迁移和浸润能力,增加细胞凋亡。(2)证实体外培养的食管癌Eca109细胞Clock的表达随时间变化存在节律性。(3)首次发现食管癌Eca109细胞C-met mRNA与c-Met蛋白在24小时内的表达呈节律性波动。(4)在不同时间点经SU11274处理后,细胞增殖指数抑制率不同,提示提示在食管癌靶向治疗中要考虑时辰效应。更多还原

【Abstract】 Objective:To study the impact of SU11274-c-Met protein inhibitor on the biological behavior of Eca109 cells, and via the potential relationships between its target-c-Met protein, c-met mRNA and clock mRNA to explore whether time effect exists during the treatment by SU11274 on Eca109 cells.Method:(1) Eca109 cells were cultured in vitro and c-Met protein was tested by immuncytochemistry. (2) Cell proliferation, movement, invasion, apoptosis rate and cell cycle were detected with MTT, cell scratch healing test, transwell assay and FCM after treated with SU11274 (20nM was used as the IC5o). (3) Clock and C-met mRNAs were assayed by RT-PCR. (4) The expression of c-Met protein at different time was detected by FCM. (5) The cell cycles after treated with SU11274 at different time points were detected by FCM.Result:(1) Eca109 cell expressed c-Met protein. (2) After the cells were treated with SU11274, the abilities of growth, migration and invasion were decreased contrast with those in control groups, and the apoptosis rates were higher than that in control groups, while the percentages of cells at S stage in DNA duplication were less. (3) RT-PCR results showed that expression of Clock mRNA was oscillating during 48h (P＜0.05). (4) The Clock and C-met mRNA expressed with circadian rhythms in 24h (P<0.01), and the expression in both of them showed similarity. (5) The c-Met protein expressed with oscillation in 24h (P<0.05) which was in accord with that of C-met mRNA. (6) The decreasing extent of proliferation index was different when Eca109 cells were treated by SU11274 at different time points.Conclusion:(1) The c-Met inhibitor SU11274 can inhibit Eca109 cells growing, infiltrating and migration, increase cell apoptosis rate and restrain cells transiting to S from Gl stage in cell proliferation cycle. (2) Eca109 cell cultured in vitro express Clock with rhythm. (3) Eca109 cells expressed C-met mRNA and c-Met protein with rhythms. (4) The inhibition rates of proliferation index were different when Eca109 cells were treated at different time points, suggesting that time effect exist in the treatment for esophageal cancer by SU11274 and be taken seriously.更多还原