Change of AFP gene promotion control mechanism with the development of hepatocellular carcinoma : analysis through many clinical cases

AFP基因调控机制随肝细胞癌发生发展的变化：通过大量临床病例分析

• 批准号: 07670566
• 负责人: SHIINA Shuichiro
• 金额: $ 1.47万
• 依托单位: UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670566/
• 关键词: hepatocellular carcinoma; nucleic acid sequence; AFP; AFP gene; transcriptional factor; 肝細胞癌; αFP; 癌化; αFP遺伝子; 生検材料; 遺伝子発現調節機構

We have performed ultrasound-guided percutaneous fine needle biopsy on approximately 150 cases of small hepatocellular carcinoma and have reserved the specimens in liquid nitrogen. A part of each specimen has been reserved with isogen in order to abstract RNA.We have also obtained samples of non-cancerous liver tissue as the control from each cases.We expect that comparison of nucleic acid sequence between the cancerous tissue and the non-cancerous tissue of the same patient will make it clear whether the carcinogenesis has produced any change in the nucleic acid sequence. Thus, the reserved samples were handled with protenase K,DNA was abstracted by the phenol-chroloform method, and the obtained DNA was amplified by PCR using primers set in the promoter and enhancer of AFP gene. Then we tried to determine the nucleic acid sequence of each sample by a sequencer, but we have not had reproducible results yet.We also homogenated the reserved sample by Dounce homogenizer, and centrifuged the homogenate and precipitated the nucleic fragment. Then we homogenated the precipitate in a buffer again, dialyzed it in the buffer, removed unsolvable materials, and obtained the nucleic abstract. Then we tried to examine the presence and quantity difference of transcriptional factors, but we have not found a certain trend yet.We will continue the experiment until we obtain stable results.

我们对大约150例小肝癌进行了超声引导下经皮细针穿刺活检，并将标本保存在液氮中。每例患者的癌组织和非癌组织的核酸序列比较，可以明确癌发生过程中是否发生了核酸序列的改变。因此，用蛋白酶K处理保留的样品，通过酚-氯仿法提取DNA，并使用设置在AFP基因的启动子和增强子中的引物通过PCR扩增所获得的DNA。然后我们尝试用测序仪测定每个样品的核酸序列，但我们还没有重复性的结果，我们还用Dounce匀浆器匀浆保留的样品，离心匀浆并沉淀核酸片段。然后，我们再次将沉淀物在缓冲液中匀浆，在缓冲液中透析，除去不溶性物质，并获得核酸提取物。然后我们尝试检测转录因子的存在和数量差异，但还没有发现一定的趋势，我们将继续实验，直到获得稳定的结果。

项目成果

Kanai F,Shiratori Y,Yoshida Y,Wakimoto H,Hamada H,Kanegae Y,Saito I,Nakabayashi H,Tamaoki T,Tanaka T,Lan KH,Kato N,Shiina S,Omata M.: "Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex

Kanai F、Shiratori Y、Yoshida Y、Wakimoto H、Hamada H、Kanegae Y、Saito I、Nakabayashi H、Tamaoki T、Tanaka T、Lan KH、Kato N、Shiina S、Omata M.：“甲胎蛋白的基因治疗

Matsumura M,Niwa Y,Hikiba Y,Okano K,Kato N,Shiina S,Shiratori Y,Omata M.: "Sensitive assay for detection of hepatocellular carcinoma associated gene transcription (alpha-fetoprotein mRNA) in blood." Biochem Biophys Res Commun. 207. 813-818 (1995)

Matsumura M、Niwa Y、Hikiba Y、Okano K、Kato N、Shiina S、Shiratori Y、Omata M.：“检测血液中肝细胞癌相关基因转录（甲胎蛋白 mRNA）的灵敏测定。”

Matsumura M,et al: "Sensutive assay for detection of hepatocellulan carcinoma・・・・" Biochem Biophys Res Commun. 207. 813-818 (1995)

Matsumura M 等人：“检测肝细胞癌的灵敏测定法……”Biochem Biophys Res Commun。207. 813-818 (1995)

Kanai F.et al: "Gene therapy for α-fetoprotein-producing human hepatoma cells by adenovirus." Hepatology. 23. 1359-1368 (1996)

Kanai F. 等人：“通过腺病毒对产生甲胎蛋白的人肝癌细胞进行基因治疗”，23. 1359-1368 (1996)。