Development of high-throughput analytical system for intracellular molecules with quantum dots

量子点细胞内分子高通量分析系统的开发

• 批准号: 16390107
• 负责人: SASAKI Kohsuke
• 金额: $ 9.22万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390107/
• 关键词: Quantum Dots; Cytomics; Immunocytochemisty; Cytometry; 半導体ナノ粒子; バイオイメージング; stoichiometry

Fluorescent semiconductor quantum dots (QDs) are a novel class of fluorescent probe with optical properties that differ from those of conventional fluorochromes. QDs emit bright fluorescence at visible wavelengths, depending on size of the particle, and in addition this bright fluorescence is resistant to photobleaching. In this project, we aimed to demonstrate that QDs are useful to quantify intracellular materials and applied to cytometry. First, QDs were substituted for organic fluorochromes in immunohistochemistry. Some intracellular/nuclear antigens were detected by secondary antibodies labeled with QDs, but some antigens were not. This may be caused by the difference of physiochemical property between two types of fluoroprobes and we tried various fixations and dying procedure to find optimal condition for immunohistochemistry with QDs. For example, samples needed treatment with Triton X-100 and microwave exposure in sodium citrate buffer (pH6) for the purpose of the detection of … More intranuclear proliferating cell nuclear antigen (PCNA). We plan to determine the optimal methods for various antigens. We verified whether QDs bind to their targets stoichiometrically as well as classical fluorochromes. We labeled PCNA in nuclei of PC-14 cells immunocytochemically with QDs and Alexa 488 concomitantly and measured the level of intranuclear PCNA expression during cell cycle with laser scanning cytometry. At the same time, nuclear DNA stained with propidium iodide (PI) was measured. The bivariate DNA/PCNA content cytograms for QDs and Alexa 488 fluorescence were dome-shaped and very similar to each other. These indicated that immunocytochemistry with QDs under optimal condition provided the data equivalent to those with organic fluorescence dyes and QDs could be useful fluorochromes in cytometry. However, we didn't always succeed to detect fluorescence intensity by LSC or flow cytometer and we need further examination for immunocytochemical methods and optical condition in cytometry. Less

荧光半导体量子点（QDs）是一类新型荧光探针，具有不同于传统荧光染料的光学特性。量子点在可见光波长发射明亮的荧光，这取决于粒子的大小，此外，这种明亮的荧光可以抵抗光漂白。在这个项目中，我们的目的是证明量子点是有用的定量细胞内物质和应用于细胞术。首先，在免疫组织化学中用量子点取代有机荧光染料。部分胞内/核内抗原可被标记有量子点的二抗检测到，部分抗原不能被检测到。这可能是由于两种类型的荧光探针的物理化学性质的差异，我们尝试了不同的固定和死亡程序，以寻找最佳条件的免疫组织化学与量子点。例如，样品需要Triton X-100处理和微波暴露在柠檬酸钠缓冲液（pH6）中，以检测更多的核内增殖细胞核抗原（PCNA）。我们计划确定各种抗原的最佳方法。我们通过化学计量学验证了量子点是否与它们的目标结合，以及经典的荧光染料。用QDs和Alexa 488同时免疫细胞化学标记PC-14细胞细胞核内的PCNA，并用激光扫描细胞术检测核内PCNA在细胞周期内的表达水平。同时用碘化丙啶（PI）染色测定核DNA。QDs和Alexa 488荧光的双变量DNA/PCNA含量细胞图呈圆顶状，彼此非常相似。这表明，在最佳条件下用量子点进行免疫细胞化学可获得与有机荧光染料相当的数据，量子点可作为有用的荧光染料用于细胞检测。然而，LSC或流式细胞仪检测荧光强度并不总是成功的，我们需要进一步研究免疫细胞化学方法和细胞术中的光学条件。少

项目成果

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours : INK4A promoter CpG island methylation is associated with cell proliferation

β-连环蛋白和 pRB 通路成分在恶性卵巢生殖细胞肿瘤中的意义：INK4A 启动子 CpG 岛甲基化与细胞增殖相关

• 发表时间: 2004
• 期刊: J Pathol. 204・3
• 作者: Sumida T;Nagata S;Ohgoshi H;Ishida Y;Kuwabara T;Tsumura T;Tsuji K;Hidaka T;Kaneko M;Yoshino T;Kitoh H;Hashimoto K;Yoshino T;Ikemoto K.;Yoshino T;Yamamoto Y.;Sato H;Sato Y;Kawauchi S;Kitoh H.;Nakamura S;Kawauchi S.
• 通讯作者: Kawauchi S.

Large cell neuroendocrine carcinoma of the uterine cervix with cytogenetic analysis by comparative genomic hybridization: a case study

• DOI: 10.1016/j.humpath.2005.07.022
• 发表时间: 2005-10-01
• 期刊: HUMAN PATHOLOGY
• 影响因子: 3.3
• 作者: Kawauchi, S;Okuda, S;Sasaki, K
• 通讯作者: Sasaki, K

Comparative genomic hybridization analysis for pancreatic cancer specimens obtained by endoscopic ultrasonography-guided fine-needle aspiration

• DOI: 10.1007/s00535-005-1577-0
• 发表时间: 2005-05-01
• 期刊: JOURNAL OF GASTROENTEROLOGY
• 影响因子: 6.3
• 作者: Kitoh, H;Ryozawa, S;Sasaki, K
• 通讯作者: Sasaki, K

Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH

• DOI: 10.1038/modpathol.3800107
• 发表时间: 2004-06-01
• 期刊: MODERN PATHOLOGY
• 影响因子: 7.5
• 作者: Hashimoto, K;Mori, N;Sasaki, K
• 通讯作者: Sasaki, K

Significance of beta-catenin and pPB pathway components in malignant ovarian germ cell tumor : INK4A promoter CpG island methylation is associated with cell proliferation.

β-连环蛋白和 pPB 通路成分在恶性卵巢生殖细胞肿瘤中的意义：INK4A 启动子 CpG 岛甲基化与细胞增殖相关。

• 发表时间: 2004
• 期刊: J Pathol. 204
• 作者: Sumida T;Nagata S;Ohgoshi H;Ishida Y;Kuwabara T;Tsumura T;Tsuji K;Hidaka T;Kaneko M;Yoshino T;Kitoh H;Hashimoto K;Yoshino T;Ikemoto K.;Yoshino T;Yamamoto Y.;Sato H;Sato Y;Kawauchi S
• 通讯作者: Kawauchi S