New method for sensitive in situ telomerase assay and it's application.

灵敏原位端粒酶检测新方法及其应用

• 批准号: 12672255
• 负责人: YAMADA Osamu
• 金额: $ 1.92万
• 依托单位: Tokyo women's Medical university
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672255/
• 关键词: Telomere; Telomerase; in situ; Diagnosis; Cancer

The introduction of the telomeric repeat amplification protocol assay means telomerase can be detected in small samples from almost all types of tumors. However it is not known whether all or only a small subset of tumor cells have telomerase activity. Activated lymphocytes in normal tissue may give false positive results. We used in cell telomeric repeat synthesis and PCR in situ with in situ hybridization to document telomerase activity. The intracellular amplification of telomere sequences was achieved with single primer pairs and the PCR products were detected by subsequent in situ hybridization using digoxigenin-labeled oligonucleotide probes specific for the amplification products.The validity and specificity of our methods were confirmed as follows. (1)Without TS primer, no signal was detected. (2)Without Taq DNA polymerase, no signal was detected. (3)TRAP negative samples gave no signals. (4)Specific indirect in-situ PCR was done. (5)HL60 cells showed down-regulation of telomerase activity after differentiation by VD3 and PHA-stimulated lymphocytes showed up-regulation of telomerase activity. (6)In artificial cell mixtures, there was an approximate correlation between the expected and observed number of positive cells.This indirect method greatly increases the specificity of in situ PCR and enables the simultaneous observation of cell morphology and telomerase activity and could be used to detect minimal residual diseases.

端粒重复扩增试验的引入意味着几乎所有类型的肿瘤的小样本中都可以检测到端粒酶。然而，目前尚不清楚是否所有或只有一小部分肿瘤细胞具有端粒酶活性。正常组织中活化的淋巴细胞可能会出现假阳性结果。我们用细胞端粒重复序列合成和原位聚合酶链式反应结合原位杂交来记录端粒酶活性。用单引物胞内扩增端粒序列，用地高辛标记的寡核苷酸探针对扩增产物进行原位杂交，验证了方法的有效性和特异性。(1)不加TS引物，检测不到任何信号。(2)没有Taq DNA聚合酶，检测不到信号。(3)TRAP阴性样品无信号。(4)特异性间接原位聚合酶链式反应。(5)VD3诱导的HL60细胞端粒酶活性下调，PHA刺激的淋巴细胞端粒酶活性上调。(6)在人工细胞混合物中，预期的阳性细胞数与观察到的阳性细胞数之间存在近似的相关性，这种间接方法大大提高了原位PCR的特异性，使细胞形态和端粒酶活性的同时观察成为可能，并可用于检测微小残留病。

项目成果

Akiyama, M., Yamada, O., Hideshima, T., Yanagisawa, T., Yokoi, K., Fujisawa, K., Eto, Y., Yamada, H., Kenneth C.Anderson.: "TNF α induces rapid activation and nuclear translocation of telomerase in human lymphocytes."B.B.R.C.. 316. 528-532 (2004)

Akiyama, M.、Yamada, O.、Hideshima, T.、Yanagisawa, T.、Yokoi, K.、Fujisawa, K.、Eto, Y.、Yamada, H.、Kenneth C.Anderson.：“TNF α 诱导人淋巴细胞中端粒酶的快速激活和核转位。”B.B.R.C.. 316. 528-532 (2004)

Nakatake, M., Sasaki, N., Murakami Murofushi, K, Yamada, Q.: "Transient post-translational up-regulation of telomerase activity during megakaryocytic differentiation of K562 cells"B.B.R.C.. 314. 1080-1085 (2004)

Nakatake, M.、Sasaki, N.、Murakami Murofushi, K、Yamada, Q.：“K562 细胞巨核细胞分化过程中端粒酶活性的瞬时翻译后上调”B.B.R.C.. 314. 1080-1085 (2004)

Yamada, O., Akiyama, M., Kawauchi, K., Adachi, T., Yamada, H., Kanda, N., Aikawa, E.: "Overexpression of telomerase confers a survival advantage through suppression of TRF1 gene expression while maintaining differentiation characteristics in K562 cells"Ce

Yamada, O.、Akiyama, M.、Kawauchi, K.、Adachi, T.、Yamada, H.、Kanda, N.、Aikawa, E.：“端粒酶的过度表达通过抑制 TRF1 基因表达来赋予生存优势，而

Sawada T., Yamada Q., et al.: "Xenoantigen, an alphaGal epitope-expression construct driven by the hTERT-promoter specifically kills human pancreatic cancer cell line"Cancer Cell International. 2(1). 14-20 (2002)

Sawada T.、Yamada Q.等人：“异种抗原，一种由 hTERT 启动子驱动的 αGal 表位表达构建体，可特异性杀死人胰腺癌细胞系”Cancer Cell International。

Kato, T., Kosaka, K., Kimura, M., Imamura, S., Yamada, O., Iwai, K., Ando, M., Joh, K., Kuroe, K., Ohtake, A., Takao, A., Momma, K., Matsuoka, R.: "Thrombocytopenia in patients with 22q11.2 deletion syndrome and its association with glycoprotein Ib-β."Gen

加藤 T.、小坂 K.、木村 M.、今村 S.、山田 O.、岩井 K.、安藤 M.、约翰 K.、黑江 K.、大竹 A ., Takao, A., Momma, K., Matsuoka, R.：“22q11.2 缺失综合征患者的血小板减少症及其与糖蛋白 Ib-β 的关联。”Gen