成果報告書詳細
管理番号20110000001353
タイトル*平成22年度中間年報 「がん超早期診断・治療機器の総合研究開発/超早期高精度診断システムの研究開発:血液中のがん分子・遺伝子診断を実現するための技術・システムの研究開発 血中分子・遺伝子診断自動化システムの研究開発(血中がん遺伝子診断の検体処理自動化システム)」
公開日2011/11/23
報告書年度2010 - 2010
委託先名コニカミノルタテクノロジーセンター株式会社 プレシジョン・システム・サイエンス株式会社 株式会社ファインラバー研究所
プロジェクト番号P10003
部署名バイオテクノロジー・医療技術部
和文要約和文要約等以下本編抜粋:1.共同研究の内容及び成果等 (1) 血液検体評価機能の開発(担当:コニカミノルタテクノロジーセンター株式会社) 1 目的 末梢血中に含有する超微量の循環がん細胞(CTC)を高感度に検出する技術を開発する。従来の免疫学的手法を使った検出法では、検出感度が十分でなかったり、抗体と反応が弱い細胞などを検出できないといった課題がある。本研究では、検体血球中に含まれる超微量のCTCの有無・含有率を、免疫染色とイメージング技術にて高感度検出することを目的とする。 2 目標 白血球/単核細胞内の循環がん細胞(CTC)を、できる限りロスや損傷なく選別し、免疫染色とイメージング技術によって、CTC の有無、含有率、性状を同定することを目指す。最終的には、10 個以下/10mL のがん細胞の検出を実現する。
・H24 年度: 末梢血10mL 中に100 個のCTC モデル細胞を検出できる感度
・H22 年度: 各プロセス技術の選択,プロセス毎に小スケールで確認
英文要約Title: Development of Automatic Testing System for Genetic Diagnosis using Peripheral Blood (FY2010-FY2012) FY2010 Annual Report
1) Development of an evaluation system for blood samples: Technologies for accurate recognition of circulating tumor cells (CTCs) have been developed. We aimed here for detecting less than 100 CTCs in 10 ml peripheral blood samples, and have been developing a system as follows. (a) Pre-treatment of blood: Density-gradient centrifugation was properly adopted for reducing the quantity of RBCs as much as WBCs. (b) Immunostaining of cells: To distinguish CTCs from other cells, we established an immunostaining protocol by choosing biological markers. (cytokeratin for tumor cells and CD45 for WBCs, along with a nuclear staining reagent) (c) Alignment of cells on a plane: A microstructure-fabricated device, a microwell chip, was employed to capture the above cells within a limited area. We confirmed that 500-600 thousand cells (WBCs) were captured and aligned on a single layer of a microwell chip. (d) Optical system for detection of rare cells: We constructed an optical instrument for simultaneous detection and imaging of cells using laser excitation and fluorescence capture. As the next step, we will design a system working with a large quantity of cells.
2) Development of a sample preparation system for genetic diagnostics: It is necessary to standardize the sample preparation procedure and to establish its evaluation method for its practical use in genetic diagnosis. The development of an automated system is indispensable for reliability and accuracy. We aimed here to develop an automated system with a RNA extraction function along with its evaluation function. A rapid RNA extraction protocol with a performance less than 25 minutes per batch was established using the reagents developed including magnetic particles. We confirmed that the purified RNA by using the protocol had a very satisfactory quality and can be used for DNA microarray assay for gene expression analysis, where the value of coefficient of variation (CV) was 7%. Moreover, an enhanced-scalable board system with temperature and motor control, named FiSICS, was developed. The number of components on the board could be reduced by adopting FiSICS, and consequently, we expect a downsizing of the footprint.
3) Development of a sample evaluation system for genetic diagnostics: To manufacture micro-TAS with silicone rubber, we adopted a device for the accumulating sheet-fed laminating method to reduce manufacturing errors in the micro-TAS device. We employed a screen-printing method and developed reagents for flowing samples in the channels. However, there was a difficulty in applying the reagents to silicone rubber sheets because of their very high viscosity. Moreover, the reagents reduced the efficiency of RT-PCR. So, we still continue the improvement of the reagents. Meanwhile, mRNA in breast cancer MCF-7 cells was used for evaluation of the system. We examined gene expression in MCF-7 cells treated with or without estrogen (E2) by RT-PCR on micro-TAS, and observed the electrophoretic peaks at the expected positions and the expected variations in gene expression. From this, we confirmed that the samples for evaluation worked well for micro-TAS.
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