Development of full-automatic in situ hybridization system for Diagnosis

全自动原位杂交诊断系统的研制

• 批准号: 08557020
• 负责人: NOMURA Shintaro
• 金额: $ 3.2万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557020/
• 关键词: Digoxigeuin; Virus; DNA; mRNA; Pathological Diagnois; in situ hybrildization; Hybridization; Pathological Diagnosis; ウィルス; in situハイブリダイゼーション; ケニルミネッセンス; TSA; ウイルス検出; 全自動システム; 抗体反応; 銀増感法; 全自動検出システム

We first tested the availability of commercially available "Full automatic histological staining system" and "Full automatic immunostaining system for in situ hybridization system. The combination of both systems did work considarably well in not only to detect viral DNA but mRNAs. However, we recognized that some parts of the instrument need to much more heat resistant for long term use. So we changed some of the parts to the goods resistant to high temperature. Next we investigated the TSA, the amplifier to detect signal. We confirmed the detection limit of the signal raised to approximately 50 times than usual system. This amplifier would be useful for obtaining stable result of in situ hybridization. Finally we tested if we can detect gene amplified product of oncogenes such as ras and sam-1 from sample obtained operation. As a result, we detected amplified sam-1 gene at the efficiency of 30% in the sample. This value was consistent from that obtained by Southern blotting. However, it was difficult to detect amplified gene from pathological samples which were already fixed by formaline. This may due to the regradation of DNA during fixiation. We still need to search optical condition to detect nucleic acid from pathological samples.

我们首先测试了市售的“全自动组织学染色系统”和“原位杂交全自动免疫染色系统”的可用性。这两个系统的组合不仅可以检测病毒 DNA，而且可以检测 mRNA，效果相当好。但是，我们认识到仪器的某些部件需要更加耐热才能长期使用，因此我们将一些部件更换为耐高温的产品，接下来我们研究了检测信号的放大器。发出信号该放大器将有助于获得稳定的原位杂交结果。最后，我们测试了是否可以从获得的样品中检测到致癌基因（如 ras 和 sam-1）的基因扩增产物。我们在样品中以30%的效率检测到扩增的sam-1基因，该值与Southern blotting获得的结果一致，但在已经用福尔马林固定的病理样本中很难检测到扩增的基因。这可能是由于固定过程中 DNA 的再降解造成的。我们仍然需要寻找光学条件来检测病理样本中的核酸。

项目成果

A.Suzuki, S.Nomura, et al.: "Localization of mRNA for trkB isoforms and p75 in the rat retin*l ganglion cells"J. of Neurosci. Res.. 54. 27-37 (1998)

A.Suzuki、S.Nomura 等人：“大鼠视网膜 *1 神经节细胞中 trkB 同工型和 p75 的 mRNA 定位”J.

K.Terai,S.Nomura et al: "The role of osteopositin in the Kinase sensedeling caused by mechanical stress" T.of Bone and Mineral Res. 印刷中.

K. Terai、S. Nomura 等人：“骨沉积素在机械应力引起的激酶感觉缺失中的作用”T. of Bone and Mineral Res。

S.Ohshima,S.Nomura et al: "Ic-b playsas a key role in the development antigen-induced arthikinacing" Proc.Natl.Acad.Sci.U.S.A. 95. 8222-8226 (1998)

S.Ohshima、S.Nomura 等人：“Ic-b 在抗原诱导的关节炎发展中发挥关键作用”Proc.Natl.Acad.Sci.U.S.A.

S. Ohshima, S. Nomura et al.: "IL-6 plays a key role in the development of antigen-induced arthritis (AIA)"Proc. Natl. Acad. Sci.. 95. 8222-8226 (1998)

S. Ohshima、S. Nomura 等人：“IL-6 在抗原诱导性关节炎 (AIA) 的发展中发挥着关键作用”Proc。

N.Yasui: "Distraction ostepgenesis in a rat model three different models of ossification" J.of Bone and Joint Surgery. 79. 824-830 (1997)

N.Yasui：“大鼠模型中三种不同骨化模型的牵引成骨”，《骨与关节外科杂志》。