レトロウイルス発現系を用いた遺伝子導入による大腸異常陰窩巣の分子生物学的解析

使用逆转录病毒表达系统通过基因转移对异常结肠隐窝病灶进行分子生物学分析

• 批准号: 12770121
• 负责人: 塚本 徹哉
• 金额: $ 1.41万
• 依托单位: Aichi Cancer Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Young Scientists (A)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12770121/
• 关键词: Aberrant crypt foci; rat; 1,2ーdimethylhydrazine; 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine; hexosaminidase; β-catenin; LightCycler; real time relative quantitative RT-PCR; aberrant crypt foci; 定量的RT-PCR; retrovirus vector; green fluores

大腸異常陰窩巣(aberrat crypt foci, ACF)は、ヒト大腸癌周囲粘膜や、発癌物質を投与した実験動物で多数発生することから、大腸癌の前癌病変と考えられている。F344雄ラットに1,2-dimethylhydrazine(DMH)および2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP)を投与し、ACFの発生と腫瘍への進展における形態および遺伝子変異の解明を試みた。腺管分離法により大腸陰窩を分離し、aberrant crypt(AC)の形態を正常腺管(NC)と比較した。ACは、hexosaminidase (Hex)陰性であり、分離腺管中で、NCとACの同定法を確立した。また、最大径が正常の2倍程度(120μm)の腺管を分離すれば、ほぼ100%の確率でHex(-)のACを単離可能であることを明らかにした。次に、ACからtotal RNAを抽出し、Light Cycler (Roche Diagnostics)を用いてHexαおよびβsubunit (Hexa, Hexb) mRNAの相対的発現量をreal time relative quantitative RT-PCR法(β-actinで補正)にて計測した。ACではNCに比べて、Hexa, Hexbそれぞれ0.266倍(P<0.002)および0.131倍(P<0.001)に発現量が低下しており、ACにおけるHex染色性低下とよく相関し、ACFの転写レベルでの異常が明らかとなった。また、ACFおよび腫瘍のβ-catenin遺伝子をPCR-SSCP解析したところ、ACFでは14/46例(30.4%)、腺腫では7/7例(100%)、癌では6/12例(50.0%)の変異を認めた。β-cateninの変異は、ACFの発生、進展に何らかの役割を果たすことが示唆された。

Aberrat crypt foci, ACF, pericarcinomatous mucosa, cancer, animal, and precancerous lesions of large cancers. F344 male dichotoma 2phildimethylpyrazine (DMH) lead 2-amino-1-methyl-6-phenylimidazo [4recover5Lib] pyridine (PhIP) investment and ACF production systems have improved the performance of the system. Glandular duct separation method, aberrant crypt (AC) shape, normal glandular duct (NC), and so on. AC, hexosaminidase (Hex), glandular canals, NC and AC were confirmed by the same method. Two times the normal diameter (120 μ m), the glandular tube is separated, the Hex (-), the AC, the tube may be separated. The secondary, AC, total RNA, and Light Cycler (Roche Diagnostics) were calculated by the quantitative real time relative quantitative RT-PCR method (β-actin positive) of β-subunit (Hexa, Hexb) mRNA phase. AC was 0.266 times higher than that of Hexa, Hexb, and 0.131 times higher than that of Hex, AC, Hex, and ACF. There were 46 cases (30.4%) of ACF infection, 7 cases (100%) of adenoma, 12 cases (50.0%) of cancer, and 12 cases (50.0%) of cancer. The PCR-SSCP analysis of β-catenin spores was performed in 46 cases (30.4%). The β-catenin, ACF, and the progress of the cutting process have shown that they are instigated to do so.

项目成果

Tsukamoto, T. et al.: "More frequent beta-catenin gene mutations in adenomas than in aberrant crypt foci or adenocarcinomas in the large intestines of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP)-treated rats"Jpn J Cancer Res. 91. 792-796 (2000

Tsukamoto, T. 等人：“2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶的大肠中，与异常隐窝病灶或腺癌相比，腺瘤中的 β-连环蛋白基因突变更为频繁（

Yamamoto, M., Tsukamoto, T., et al.: "p53 knockout mice (-/-) are more susceptible than (+/-) or (+/+) mice to N-methyl-N-nitrosourea stomach carcinogenesis"Carcinogenesis. 21. 1891-1897 (2000)

Yamamoto, M.、Tsukamoto, T.等人：“p53 敲除小鼠 (-/-) 比 (/-) 或 (/) 小鼠更容易发生 N-甲基-N-亚硝基脲胃癌”致癌作用。

Tsukamoto,T. et al.: "Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase alpha and beta subunit mRNAs, in the 1, 2-dimethylhydrazine treated rat colon"Jpn.J.Cancer Res.. 92(in press). (2001)

冢本，T.

Tsukamoto, T. et al.: "Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase alpha and beta subunit mRNAs, in colon of 1,2-dimethylhydrazine-treated rats"Jpn J Cancer Res. 92. 109-118 (2001)

Tsukamoto, T. 等人：“在 1,2-二甲基肼治疗的大鼠结肠中，己糖胺酶改变了异常隐窝，携带减少的己糖胺酶 α 和 β 亚基 mRNA”Jpn J Cancer Res。

Tsukamoto,T. et al.: "More frequent beta-catenin gene mutations in adenomas than in aberrant crypt foci or adenocarcinomas in the large intestines of 2-amino-1-methyl-6-phenylimidazo [4, 5-blpyridine (PhIP) -treated rats"Jpn.J.Cancer Res.. 91. 792-6 (2000

冢本，T.