摘要
第一部分三氧化二砷诱导人胆囊癌GBC细胞凋亡及其机制研究
背景与目的:胆囊癌由于缺乏特异性的临床表现,确诊时多属晚期,手术切除率较低;且常规化疗药物对胆囊癌的疗效较差,因此需寻找治疗胆囊癌新的有效药物。三氧化二砷(As_2O_3)在治疗急性早幼粒细胞白血病中取得了突破性成果,近年来它在实体瘤的研究中颇受关注。本研究旨在探讨As_2O_3,对人胆囊癌GBC细胞系诱导凋亡的生物学效应及BcL-2在其中的作用。方法:用Hoechest33258染色、流式细胞仪和DNA凝胶电泳研究As_2O_3,诱导细胞凋亡情况。用Western blot分析As_2O_3对凋亡相关蛋白Bcl-2、Bcl-xL、Bid、Bak、Bax、cleaved-Caspase-3和cleared-Caspase－9的表达影响。构建Bcl-2的表达质粒,并用Lip02000转染GBC细胞,并检测As_2O_3诱导细胞凋亡情况。结果:As_2O_3可诱导GBC细胞凋亡。蛋白水平的检测表明As_2O_3,可使细胞Bcl-2、Bcl-xL的表达下降,Bid、Bak和Bax表达升高,并促进Caspase-3和Caspase-9的剪切。过表达Bcl-2可抑制As_2O_3诱导的凋亡。结论:As_2O_3可诱导胆囊癌细胞的凋亡,并主要通过下调Bcl-2基因的表达来实现。
第二部分三氧化二砷抑制人胆囊癌细胞生长及机制研究
背景与目的:恶性肿瘤发生和发展固有的特点是肿瘤细胞无节制的生长,这与细胞周期的调控异常密切相关。细胞周期D类蛋白与细胞周期调控密切相关,过表达的细胞周期蛋白D1(Cyclin D1)参与了胆囊癌的发生和发展,转录因子Spl在多种与细胞生长相关基因的转录调控中起激活作用,本研究旨在探讨As_2O_3对人胆囊癌GBC细胞系生长的生物学效应及Cyclin D1、转录因子Spl在其中的作用。方法:不同浓度的As_2O_3作用于胆囊癌细胞GBC-SD,MTT法检测细胞生长情况,流式细胞仪检测细胞周期分布。Western blot和RT-PCR检测CyclinD1、D2、D3、CDK4和CDK6的蛋白和mRNA表达。构建Cyclin D1的表达质粒并转染GBC细胞,观察Cyclin D1过表达时细胞周期的变化。构建Cyclin D1启动子pGL3并转染GBC细胞,测定不同浓度As_2O_3作用下pGL3相关的荧光素酶活性。Western blot测定转录因子Spl蛋白表达并构建其表达质粒转染GBC细胞,测定转染前后Cyclin D1启动子相关的荧光素酶活性。结果As_2O_3能抑制胆囊癌细胞生长,作用呈时间、剂量-效应关系;流式细胞仪检测发现As_2O_3可将增殖过程中的胆囊癌细胞阻滞于G_1期:Western blot及RT-PCR检测显示Cyclin D1蛋白和mRNA的表达下降;As_2O_3能降低Cyclin D1启动子的表达,作用呈剂量-效应关系,4μmol/L的As_2O_3处理后Cyclin D1的启动子活性下调近70%,过表达Cyclin D1逆转了As_2O_3对细胞周期的改变。As_2O_3能降低转录因子Spl的表达及Cyclin D1启动子的活性,外源性Spl能逆转As_2O_3对Cyclin D1启动子活性的抑制作用。结论:As_O_3能明显抑制体外人胆囊癌GBC细胞的生长并主要通过Spl介导的Cyclin D1表达下调来实现。
Part I: Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2
Background and Purpose: Gallbladder carcinoma, lacking of specific clinical manifestation, usually diagnosed at advanced stages of the diseases and fewer can be resected in operation. Chemotherapy has not shown significant activity in gallbladder carcinoma, so new agent should be applied to its treatment. Arsenic trioxide, verified as a breakthrough in the management of acute promyelocytic leukemia, has been applied to the research of a variety of solid tumors. This study is to investigate the biological effect of As_2O_3 on human gallbladder carcinoma cell GBC cells and and identify the contribution of Bcl-2 in the As_2O_3-induced apoptosis. Methods: Hoechest33258 staining, DNA gel electrophoresis and flowcytometric were used to determine apoptosis. Western blot was performed to analysis apoptotic proteins expression. GBC cells were transiently transfected with constructed Bcl-2 plasmid and its apoptosis was detected again. Results: The treatment of As_2O_3 in gallbladder carcinoma cells could induce apoptosis in a dose-dependent manner and downregulate the expression of anti-apoptotic protein Bcl-2. Moreover, Bcl-2 overexpression could protect gallbladder carcinoma cells from As_2O_3-induced apoptosis. Conclusions: As_2O_3 induces gallbladder carcinoma cell apoptosis via down regulation of Bcl-2
Part II: Arsenic Trioxide-mediated Growth Inhibition in Gallbladder carcinoma Cells via down-regulation of Cyclin D1 transcription mediated by Sp1 transcription factor
Background and Purpose: The internal characteristic in the development and progression of malignant tumor is non-limited growth, which is closely linked with the abnormal regulation of cell cycle. The family of cyclin D have played a major role in the regulation of cell cycle and the overexpression of cyclin D1 has participated the development and progression of gallbladder carcinoma. In additional, can stimulate the activity of varieties of the cell-growth-related gene. This study is to investigate the inhibiting effect of arsenic trioxide(As_2O_3) on human gallbladder carcinoma (GBC) cells as well as the whats and the hows of cyclin D1 and Sp1 transcription factor during the effect of As_2O_3. Methods: GBC cells were cultured with different concentrations of As_2O_3, the proliferative activity of cells was detected by MTT methods, the cell cycle status were detected by flow cytometry as well as Western blot and RT-PCR were performed to analyse the protein and mRNA expression of Cyclin D1,D2,D3,CDK4 and CDK6.GBC cells were transient transfected with overexpression of Cyclin D1 derived from the constructed plasmid as well as the As_2O_3-induced alteration of cell cycle was detected again. GBC cells were transient transfected with Cyclin D1 promoter construct pGL3 and then treated by different dose of As_2O_3. The Sp1 protein expression was also measured and its plasmid was constructed and transiently transfected into GBC cells as well as the luciferase activity of Cyclin D1 promoter was measured pre- and post the transfection. Results: The treatment of As_2O_3 in gallbladder carcinoma cells could inhibit the growth of cell in a time- and dose-dependent manner, make cells arrested in G_1 and downregulate the expression of Cyclin D1. Cyclin D1 overexpression played the negative role of As_2O_3-induced alteration of cell cycle. The activity of Cyclin D1 promoter was downregulated by As_2O_3 in a dose-dependent manner and decreased about 70 percent when treated with 4μmol/L As_2O_3. Moreover, the Sp1 was downregulated by As_2O_3 in a dose-dependent manner and the exogenous Sp1 transcription factor played the negative role to the effect of As_2O_3. Conclusion: As_2O_3 can significantly inhibit the growth of human gallbladder carcinoma cell via down-regulating the expression of Cyclin D1 mediated by Sp1 transcription factor.
引文
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