【作者】 沈阳；

【导师】 许春芳；

【作者基本信息】 苏州大学 ， 消化内科学， 2011， 硕士

【摘要】 第一部分沙利度胺对胰腺癌细胞株SW1990生长及血管生成的影响目的研究沙利度胺(Thalidomide,THD)对胰腺癌细胞株SW1990细胞体外生长及血管生成的影响,并探讨其对人胰腺癌细胞株SW1990可能的作用机制。方法采用浓度为3.125、6.25、12.5、25、50、100、200和400μg/ml的THD处理人胰腺癌SW1990细胞,应用MTT法检测OD值,计算各实验组和对照组SW1990细胞的抑制率,并计算最适宜的沙利度胺IC50;取浓度为50、100、200μg/ml的THD处理人胰腺癌SW1990细胞,流式细胞术(FCM)检测细胞凋亡和细胞周期的变化;RT-PCR检测Bcl-2、Bax和VEGF的mRNA表达水平;免疫印迹法(Westen Blot)检测Bcl-2、Bax和VEGF的蛋白表达水平。结果(1)MTT法显示:THD可明显抑制SW1990细胞的生长,并随着药物浓度的增加和作用时间的延长而明显增强(P<0.05)。(2)流式细胞术显示:SW1990细胞经THD作用48小时后,细胞出现明显凋亡,且以早期凋亡为主。在50μg/ml浓度下早期凋亡率为(8.77±3.30)%,而在200μg/ml浓度下为(29.43±2.25)%,与THD浓度呈正相关;且随着药物浓度的增加,SW1990细胞G0/G1期比例上升,从(46.55±2.44)%上升到(58.83±2.33)%,S期细胞比例下降(P<0.05)。(3)RT-PCR检测Bcl-2、Bax和VEGF的mRNA表明:SW1990细胞经不同浓度THD作用48小时后,Bcl-2、VEGF的mRNA的表达呈浓度依赖性下降,Bax-mRNA的表达呈浓度依赖性增加,各浓度组与对照组比较差异均有统计学意义(P<0.05)。(4)Western Blot结果显示:SW1990细胞经不同浓度THD作用48小时后,VEGF、Bcl-2蛋白表达呈浓度依赖性下降,Bax蛋白表达呈浓度依赖性升高,各浓度组与对照组比较差异均有统计学意义(P<0.05)。结论THD可抑制人胰腺癌SW1990细胞的生长,且呈时间和剂量依赖性,其机制可能是直接抑制肿瘤细胞增殖;阻滞细胞周期于静止期;诱导细胞早期凋亡;抑制血管生成。第二部分沙利度胺联合吉西他滨对人胰腺癌SW1990细胞生长的影响目的:探讨沙利度胺(Thalidomide,THD)联合吉西他滨(Gemcitabine,GEM)对人胰腺癌SW1990细胞的协同抑制作用及机制。方法以浓度为50μg/ml的沙利度胺联合20μmol/L的吉西他滨处理对数生长期的人胰腺癌SW1990细胞,应用MTT比色法检测OD值,计算对照组和各实验组SW1990细胞的抑制率;AnnexinV/PI双染法检测SW1990细胞早期凋亡情况;RT-PCR检测细胞凋亡因子Bcl-2及Bax的mRNA表达水平;免疫印迹法(Westen Blot)检测凋亡蛋白Bcl-2和Bax的表达水平。结果沙利度胺和吉西他滨都能有效地抑制人胰腺癌SW1990细胞增殖,二者联合作用具有协同效应;50μg/mlTHD作用SW1990细胞48h后细胞早期凋亡为(8.7±1.87)%,20μmol/LGEM组为(27.73±2.40)%,两者联合组为(39.4±3.24)%;RT-PCR检测抗凋亡因子Bcl-2-mRNA表达明显下降,促凋亡因子Bax-mRNA表达上升,各组与对照组及单药组与联合组比较均有统计学意义(P<0.05)。Western Blot检测抗凋亡蛋白Bcl-2表达明显下降,促凋亡蛋白Bax表达上升,各组与对照组及单药组与联合组比较均有统计学意义(P<0.05)。结论沙利度胺联合吉西他滨可明显抑制人胰腺癌SW1990细胞增殖,其机制可能是通过促进细胞凋亡而发挥作用。更多还原

【Abstract】 Part One: The studies of thalidomide on cellular growth and angiogenesis of human pancreatic carcinoma cell line SW1990.Objective: To study the effect of thalidomide on cellular growth and angiogenesis of human pancreatic carcinoma cell line SW1990 in vitro,and to explore its possible mechanisms.Methods: After using different concentrations’thalidomide (3.125、6.25、12.5、25、50、100、200 and 400μg/ml) to deal with pancreatic carcinoma SW1990 cells,OD values were evaluated by MTT , and inhibition ratioes were calculated in all groups and suitable IC50 of thalidomide were worked out by calculatation. With using different concentrations’thalidomide (50、100 and 200μg/ml) to deal with SW1990, the apoptosis ratioes and changes of cell cycle were detected by flow cytometry. And the expression of Bcl-2-mRNA、Bax-mRNA and VEGF-mRNA were detected by RT-PCR. And the levels of Bcl-2、Bax and VEGF proteins were detected by Western Blot.Results:(1)Thalidomide could inhibit the growth of the human pancreatic carcinoma cell line SW1990, and the inhibitory effect had dose-and-time dependence(P<0.05). (2)The early stage apoptosis of SW1990 cells was detected by flow cytometry after they were incubated 48 hours with thalidomide.,The early stage apoptosis rate of SW1990 cells was (8.77±3.30)% when they were incubated with 50μg/ml thalidomide, and the rate was (29.43±2.25)% with 200μg/ml thalidomide.Apparently, apoptosis rate was positively correlated to the concentration of thalidomide. With the increase of the concentration of thalidomide, ratio of G0/G1 phase increased homologously, it was (46.55±2.44)% with 50μg/ml thalidomide and (58.83±2.33)% with 200μg/ml thalidomide. And S phase declined homologously(P<0.05).(3)The expression of Bcl-2-mRNA and VEGF-mRNA was downregulated and the expression of Bax-mRNA was upregulated significantly in dose dependence after SW1990 cells were incubated 48 hours with thalidomide, which was detected by RT-PCR(.4)The levels of Bcl-2 and VEGF proteins decreased and the level of Bax protein was upregulated significantly in dose dependence after SW1990 cells were incubated 48 hours with thalidomide, which was detected by Western Blot. Comparisons of all thalidomide groups and control group have significant difference(P<0.05). Conclusion:Thalidomide could inhabit the growth of human pancreatic carcinoma cell line SW1990 in a dose-and-time dependent manner, and its mechanisms may directly inhibit tumor cells proliferation, arrest cell cycle in stillness stage, induce early stage apoptosis, and inhibite angiogenesis.Part two: Effect of THD in combination with GEM on human pancreatic carcinoma cell line SW1990Objective: To investigate the effect and mechanism of action in pancreatic carcinoma cell line SW1990 treated by THD in combination with GEM.Methods: SW1990 cells were cultivated with 50μg/ml of thalidomide and 20μmol/L of gemcitabine. OD values were detected by MTT colormetric assay and their inhibition ratios were calculated and early apoptosis of them were detected by AnnexinV/PI double staining method and the expression of Bcl-2mRNA and BaxmRNA were detected by Semi-quantitative RT-PCR.Result: Both thalidomide and gemcitabine could inhibit effectivly SW1990 cells’proliferation. Thalidomide combined with gemcitabine had synergistic effect. The early stage apoptosis rate of SW1990 cells was (8.7±1.87)% when they were incubated with 50μg/ml thalidomide, it was (27.73±2.40)% when 20μmol/L of gemcitabine, it was(39.4±3.24)% when thalidomide combined with gemcitabine.The level of Bcl-2mRNA was downregulated, and the expression of Bax-mRNA was upregulated,which was detected by RT-PCR. Comparisons of treated groups and control group, Bcl-2mRNA or Bax-mRNA have respectively significant difference(P<0.05), and comparisons of single drug groups and combination group, they have also respectively significant difference(P<0.05). The level of Bcl-2 protein was downregulated, and the level of Bax protein was upregulated,which was detected by Western Blot. Comparisons of treated groups and control group, Bcl-2 or Bax protein have respectively significant difference(P<0.05), and comparisons of single drug groups and combination group, they have also respectively significant difference(P<0.05).Conclusion:The combination of thalidomide and gemcitabine could obviously inhibit the proliferation of human pancreatic carcinoma SW1990 cells, its mechanisms maybe promote early stage apoptosis.更多还原