【作者】 林辉；

【导师】 钟德玝；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 目的：1.用光学显微镜、MTT、细胞集落试验及透射电镜等不同方法,观测不同浓度的蜈蚣水提取物对人胆管癌细胞株QBC939体外抑制作用。计算其IC50值。2.建立胆管癌细胞株QBC939裸鼠皮下瘤模型,用10g/kg蜈蚣水提取物灌胃处理胆管癌细胞株QBC939移植瘤裸鼠,比较治疗组及对照组皮下瘤模生长情况。3.取治疗组及对照组人胆管癌细胞株QBC939裸鼠皮下移植瘤瘤体行蛋白质组学分析,寻找差异性蛋白。4.根据蛋白质组学结果,取胆管癌细胞株QBC939裸鼠皮下瘤体行BCL-2及BAX的半定量RT-PCR及Western blot分析,探讨蜈蚣水提取物促进胆管癌细胞凋亡的可能机制。方法：1.光镜观测蜈蚣水提取物对人胆管癌细胞株QBC939作用后的形态学改变及集落变化,计算其IC50值。透射电镜观测IC50值蜈蚣水提取物处理人胆管癌细胞株细胞QBC93948小时,肿瘤细胞结构改变情况。2.建立人胆管癌细胞QBC939裸鼠皮下移植瘤模型,移植瘤术后第12天,采用10g/kg蜈蚣水提取物灌胃处理移植瘤裸鼠,每日一次,共21次。观察记录肿瘤变化情况绘制生长曲线图。移植瘤术后第35天解剖移植瘤裸鼠,观测皮下移植瘤体积、重量及H.E染色情况。3.用蛋白质组学技术检测蜈蚣水提取物处理前后两组人胆管癌细胞QBC939皮下移植瘤蛋白表达的差异。4.上述两组人胆管癌细胞QBC939皮下移植瘤的标本行RT-PCR及Western blot检测Bcl-2及Bax的变化情况。探讨蜈蚣水提取物促进胆管癌细胞凋亡的可能机制。结果：1.蜈蚣水提取物有抑制胆管癌QBC939细胞生长的作用,随着蜈蚣水提取物浓度逐渐增加,其抑瘤作用递增。其IC50值为12.5mg/ml.蜈蚣水提取物能抑制人胆管癌细胞株QBC939细胞集落的形成。电镜观察用半数抑制剂量的蜈蚣水提取物处理人QBC939胆管癌细胞48h后,可见明显的凋亡小体。2.成功建立人胆管癌细胞株QBC939裸鼠移植瘤动物模型；与对照组相比,10 g/kg蜈蚣水提取物灌胃对裸鼠QBC939胆管癌皮下移植瘤具有抑制作用；H.E染色可见实验组肿瘤细胞可见空泡化及大量核固缩,而对照组少见。3.蛋白质组学检测显示,对比QBC939皮下移植瘤细胞38个差异性蛋白。有27个下调,有11个上调, CK8、CK18及Bcl-2相关蛋白表达下调,Galectin-1和Galectin-7表达上调。4.RT-PCR及Western blot检测实验组的Bax上调而Bcl-2下调。结论：1.蜈蚣水提取物有抑制胆管癌QBC939细胞生长的作用,随着蜈蚣水提取物浓度逐渐增加,抑瘤作用递增,其48小时IC50值为12.5mg/ml。2：成功建立人胆管癌细胞株QBC939裸鼠移植瘤动物模型,10g/kg蜈蚣水提取物对裸鼠QBC939胆管癌皮下移植瘤具有抑制作用。3：蜈蚣水提取物的治疗引起胆管癌QBC939皮下移植瘤细胞蛋白质组学的38个差异。CK8、CK18及Bcl-2相关蛋白表达下调,Galectin-1和Galectin-7表达上调。4：蜈蚣水提取物对胆管癌QBC939的抑制作用,可能是通过上调Bax及下调Bcl-2途径引起胆管癌细胞的凋亡来实现的。更多还原

【Abstract】 Objective:1.To explore the inhibitory effects of Centipede Aqueous Extract on cholangiocarcinoma Cell lines QBC939 in vitro.2. To establish the nude mice subcutaneous tumors model of QBC939 cells, The treatment groups were fed with the Centipede Aqueous Extract on the concentration of lOg/kg while the control group were fed with the Saline water. The growth of the two groups were recorded in detail.3. To analysis the proteomics difference between the treatment and the control groups4.To study the possible antitumor mechanisms of Centipede Aqueous Extract on cholangiocarcinoma cell line QBC939 subcutaneous tumors by molecular biology technology.Methods:1. The morphological and colony changes of cholangiocarcinoma cell line QBC939 were observed in different concentration of Centipede Aqueous Extract by light microscopy,and the changes of the cell structure were studied by transmission electron microscopy.2. The model the nude mice subcutaneous tumors of human cholangiocarcinoma cell QBC939 were established.12 days after Subcutaneous transplantation, the treatment group nudes were fed with the Centipede Aqueous Extract on the concentration of lOg/kg while the control group were fed with the Saline water. The changes of tumor size between the treatment and control groups were recorded in details.35 days after subcutaneous transplantation, all the nude mice were anatomized and subcutaneous tumors were observed by H.E staining.3.The protein expression differences of Cholangiocarcinoma QBC939 cell subcutaneous tumors were detected by proteomics.4.To explore the mRNA and protein levels of Bcl-2 and Bax in Cholangiocarcinoma QBC939 cell subcutaneous tumors and its possible mechanism. the samples of the experimental and control groups were detected by RT-PCR and Western blot.Results:1. The growth of cholangiocarcinoma cells QBC939 could be inhibited by Centipede Aqueous Extract. With gradually increasing concentration its anti-tumor effect was also enhanced too. Its IC50 value of 48 hours was 12.5mg/ml. and Centipede Aqueous Extract could also inhibit the colony-forming of cholangiocarcinoma cell line QBC939 cells. Electron microscopy showed that the apoptosis bodies could be seen clearly in Centipede Aqueous Extract treatment cholangiocarcinoma QBC939 cells by the concentration of 12.5mg/ml after 48h. 2. Successfully established were the nude mice subcutaneous tumors model of Cholangiocarcinoma QBC939 cells. After fed with of Centipede Aqueous Extract on the concentration of 10g/kg, Obvious different Growth rates were observed between the treatment group and control groups; Plenty of vacuolization and karyopyknosis cells were seen in experimental group by HE staining,rarely in the control group.3. Compared to 38 proteins differentially expressed significantly in Cholangiocarcinoma QBC939 subcutaneous tumors tissues, among which 27 ribosomal protein genes were down-regulated significantly, while 11 ribosomal protein genes were up-regulated. CK8、CK18及Bcl-2 were down-regulated while Galectin-1 and Galectin-7 genes were down-regulated.4. The result of RT-PCR and Western blot showed that the mRNA and protein levels of Bax were increased and those of Bcl-2 were declined in the experimental groups compared to the control groupsConclusion:1. Cholangiocarcinoma QBC939 cells could be inhibited by Centipede Aqueous Extract in vitro. With gradually increasing concentration its anti-tumor effect was also enhanced too. Its IC50 value was 12.5mg/ml.2. Successfully established were the nude mice subcutaneous tumors model of Cholangiocarcinoma QBC939 cells. Centipede Aqueous Extract with the concentration of lOg/kg might have anti-tumor effect on the nude mice subcutaneous tumors.3. There were 38 proteins differentially expressed significantly in Cholangiocarcinoma QBC939 cells subcutaneous tumors tissues, CK8、CK18 and Bcl-2 were down-regulated while Galectin-1 and Galectin-7 genes were down-regulated.4. The apoptosis mechanism of treatment group Cholangiocarcinoma cells might be that up-regulated Bax and down-regulated Bcl-2 by Centipede Aqueous Extract.更多还原