【作者】 龙江；

【导师】 倪泉兴；

【作者基本信息】 复旦大学 ， 外科学， 2004， 博士

【摘要】 作为一个重要的转录调控因子,胰腺癌中甲基化 CpG 结合域蛋白 1(methyl-CpG binding domain protein 1 ,MBD1)介导的甲基化转录抑制作用可能是造成众多抑癌基因转录表达下降,以至失活的重要原因。探讨 MBD1 在胰腺癌中的表达、调控及其作用有重要的研究价值。 实验研究通过 RT-PCR 检测 MBD1mRNA 在两株胰腺癌细胞株AsPC-1 和 BxPC-3 中的表达,发现均有较高水平的基础表达。BxPC-3为原发癌细胞株,而 AsPC-1 具有很高的淋巴结转移侵袭活性,并同时具有原发癌的生物学特性,因此选择 AsPC-1 细胞株作为研究平台,探讨 MBD1 的表达、意义和调控。我们利用 RNA 干扰技术,设计合成了针对 MBD1 基因的 siRNAs,并在其上下游分别引入 BgLⅡ和HindⅢ限制性酶切位点,通过双酶切连接反应将 MBD1siRNAs 插入质粒载体 Rotro Super 多克隆位点中的 BgLⅡ和 HindⅢ之间,从而成功构建 MBD1siRNAs 真核表达质粒 Rotro Super-MBD1siRNAs,应用RT-PCR 行酶切鉴定,结果显示 MBD1siRNAs 能成功载入质粒。采用脂 质 体 介 导 的 方 法 将 MBD1siRNAs 表 达 质 粒 转 染 胰 腺 癌 细 胞 系AsPC-1,通过 G-418 的筛选获得稳定表达 MBD1siRNAs 的阳性克隆。 应用RT-PCR和Western-blot方法检测AsPC-1细胞中MBD1在基因水平和蛋白水平的表达变化,采用克隆形成实验和MTT法检测肿瘤细胞增殖能力,利用多点微列阵技术观察甲基化相关基因表达的变化。研究结果显示MBD1siRNAs表达质粒能显著抑制胰腺癌细胞AsPC-1中MBD1mRNA 的表达,且 MBD1在蛋白水平亦受到明显抑制;克隆形成实验和MTT测定生长曲线显示细胞生长受到显著抑制,表明MBD1siRNAs能抑制胰腺癌的细胞增殖能力,降低肿瘤细胞的独立生存能力;利用多点微列阵杂交发现在MBD1 mRNA表达下降的同时,其它甲基化相关抑癌基因CDH1、 RB和 E2F5表达上调恢复。 临床研究应用免疫组织化学法和RT-PCR技术分别从蛋白水平和基因水平检测证实MBD1在胰腺癌临床标本中的表达,并观察区域性动脉灌注化疗对MBD1表达的影响,研究结果表明MBD1蛋白在胰腺癌中的表达(阳性率 76.32%)明显高于正常胰腺、癌旁、慢性胰腺炎和胰腺良性肿瘤组织;RT-PCR同样显示MBD1mRNA在胰腺癌组织中的表达明显高于正常胰腺组织和癌旁对照组织(P<0.01)。MBD1的表达水平的高低与性别、年龄、肿瘤部位、肿瘤大小、分化程度和TNM分期之间无显著性差异;而有淋巴结转移的胰 4<WP=5>博士论文 第 5 页 共 100 页腺癌MBD1表达(阳性率92.31%)明显高于无淋巴结转移者(41.67%),其中7例强阳性表达的胰腺癌病理证实均有16组(腹主动脉旁)淋巴结的转移,说明MBD1与胰腺肿瘤转移和侵袭活性密切相关,检测MBD1对预测胰腺癌患者的预后有指导作用。区域动脉灌注介入化疗能明显抑制MBD1基因的表达(P<0.01)。 总之,研究表明MBD1在胰腺癌中的表达明显增高,并与胰腺癌转移侵袭活性相关,是致癌密切相关基因。MBD1siRNAs能抑制胰腺癌AsPC-1细胞株MBD1的表达及其增殖能力,并可上调其它甲基化相关抑癌基因表达,MBD1可能成为一个新的基因治疗靶点。区域性动脉灌注化疗明显抑制MBD1基因的表达,是一有效的临床治疗措施。更多还原

【Abstract】 As an important transcription factor, methyl-CpG binding domain protein1 (MBD1) mediated transcription-suppressing course and might cause descentexpression of numerous tumor suppressor genes, or even result in devitalization ofthose genes. The expression and role of MBD1 were studied in experimental andclinical part respectively. Experimental study: MBD1 mRNA expression level was detected in twopancreatic cancer cell lines, AsPC-1 and BxPC-3, by method of RT-PCR. Theexpression of MBD1 was very high in two cell lines. BxPC-3 was cultured frompancreatic carcinoma in situ, while AsPC-1 was cultured from the ascites of a latestage patient. AsPC-1 not only had strong metastasis vitality but also had biologicalcharacter of the primary cancer. We choose AsPC-1 as our further research patform. By RNA interference (RNAi) technology, the siRNA was designed andsynthesied which aimed at the MBD1 gene. BgLⅡand HindⅢ restriction enzymesites were introduced into the 5’ and 3’of MBD1 gene siRNAs respectively, theninserted into polylinker site of plasmid Rotro Super, MBD1 siRNAs eukaryoticexpression vector Rotro Super-MBD1 was constructed. MBD1 siRNAs had beensuccessfully integrated into the plasmid. MBD1 siRNAs vector was transfected intopancreatic cancer cell AsPC-1 by liposome. Positive clone was obtained by the screenof G-418. MBD1 expression level was detected by RT-PCR, and MBD1 protein was testedby western-blot, growth curve MTT assay and clony forming test were used to assessthe proliferation potency. Multi-dots gene chip was used to detect the expression levelof methylation related cancer suppressor genes. It was demonstrated that MBD1siRNAs could significantly suppress expression of MBD1 mRNA in AsPC-1, whichwas the same for protein expression. Growth curve and clony forming test show thatcell growth is significantly inhibited. It demonstrates that MBD1 siRNAs can inhibitthe proliferation of pancreatic cancer cells, reduce its survival ability and adaptability.The methylated cancer suppressor genes, such as CDH1, Rb and E2F5 wereup-regulated in the multi-dots gene chip experiment. The expression of MBD1 in pancreatic carcinoma was detected at protein levelby immunohistochemistry, at gene level by RT-PCR respectively. MBD1 expressionwas significantly higher (76.32%) in pancreatic carcinomas than that in normalpancreatic tissue, benign pancreatic tumors, corresponding distant pancreas tissues 6<WP=7>博士论文 第 7 页 共 100 页and chronic pancreatitis measured by immunohistochemistry. The expression ofMBD1 mRNA in pancreatic carcinomas was also obviously higher than normalpancreatic tissues, or the distant pancreatic tissues (P<0.01) tested by RT-PCR. Nocorrelation was found between the expression of MBD1 with the sex, age, location,size, differentiation or the staging of pancreatic carcinoma. MBD1 expressed in92.31% pancreatic carcinomas with lymph node metastasis, which is higher than thatin pancreatic carcinomas without lymph node metastasis (41.67%). Among them, 7samples with MBD1 strongly expressed were testified having para-abdominal aortalymph node metastasis (station 16). The influence of intra-arterial chemotherapy onMBD1 gene expression was also studied. MBD1 gene expression declined afterintra-arterial chemotherapy. Conclusion: the expression level of MBD1 was significantly higher in pancreaticcarcinoma and it might have close correlation with metastasis of pancreatic cancer.MBD1 was one of the oncongenes of pancreatic carcinoma. MBD1 siRNAs couldinhibit the expression of MBD1 and the proliferation of pancreatic cancer cellsAsPC-1, up regulating expression level of other methylated cancer suppressor genes,and MBD1 may be a new target of gene therapy for pancreatic carcinoma. Regionalintra-arterial chemotherapy was an effective treatment by inhibiting the expression ofMBD1.更多还原