Development of a Method for Cellular Navigation Using a Carbohydrate Recognition Device in Cell Transplantation

开发细胞移植中使用碳水化合物识别装置进行细胞导航的方法

• 批准号: 07557154
• 负责人: IMAI Yasuyuki
• 金额: $ 4.29万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557154/
• 关键词: Cell transplantation; Cellular trafficking; MMGL; Lectin; Experimental lung metastasis; Adoptive immunotherapy

Cell transplantation is as important as organ transplantation as a subject of medical research. An important problem is how to target transplanted cells into appropriate organs or tissues. Recent progress revealed importance of specific recognition of carbohydrate ligands by endogenous lectins in physiological targeting of cells. In this study, we investigated an artificial targeting of cells into tumor sites using MMGL,which is a transmembrane calcium-type lectin found on tumoricidal macrophages, as a carbohydrate recognition device. We also tried to develop novel methods to analyze cellular targeting.1. Interleukin 2-dependent mouse T cell line, CTLL-2 cells, were transfacted with an expression vector pCEP-4 containing full length cDNA insert encoding MMGL.A stable transfectant cell line, CTL-ML,was obtained. Cell surface expression of MMGL was demonstrated by mAb LOM-14 against MMGL that we developed.2. The CTL-ML cells labeled with a fluorescent dye DiI were intravenously injected … More into mice with established lung metastases produced by mouse ovarian tumor OV2944-HM-1cells. Using sequential frozen sections of lungs, fluorescent cell numbers in the metastatic nodules were compared with those in the normal lung tissues under a fluorescence microscope. CTL-ML cells were found to selectively accumulate in the tumor tissue as compared with mock transfectant CTL-CEP cells.3. For data analysis, image analyzer and confocal microscope were tested. Especially confocal microscope was useful for detection of labeled cells and measurement of sample area.4. MMGL has an cytoplasmic motif which is involved in endocytic pathway. To display MMGL on cell surface more efficiently, we eliminated the motif to reduce down regulation by endocytosis. A stable transfectant CTL-YM,which has altered cytoplasmic motif AENL instead of YENL,was produced. Cell surface expression of MMGL was confirmed. We have not seen significant differences in the cellular accumulation into lung metastatic nodules between CTL-ML and CTL-YM at present. Less

作为医学研究的一个课题，细胞移植和器官移植一样重要。一个重要的问题是如何将移植的细胞靶向到合适的器官或组织中。近年来的研究表明内源性凝集素对糖类配体的特异性识别在细胞生理靶向中具有重要意义。在这项研究中，我们研究了一种人工靶向细胞进入肿瘤部位使用MMGL，这是一种跨膜钙型凝集素上发现的杀肿瘤巨噬细胞，作为碳水化合物识别装置。我们还尝试开发新的方法来分析细胞靶向。将含有MMGL全长cDNA插入片段的表达载体pCEP-4转染小鼠IL-2依赖性T细胞系CTLL-2，获得稳定的转染细胞系CTL-ML。我们开发的抗MMGL的单克隆抗体LOM-14证实了MMGL在细胞表面的表达.静脉注射用荧光染料DiI标记的CTL-ML细胞 ...更多信息 植入小鼠卵巢肿瘤OV 2944-HM-1细胞产生的已确定肺转移的小鼠体内。采用肺连续冰冻切片，在荧光显微镜下比较转移性结节和正常肺组织中荧光细胞的数量。与假转染的CTL-CEP细胞相比，CTL-ML细胞在肿瘤组织中选择性聚集.对于数据分析，测试了图像分析仪和共聚焦显微镜。特别是共聚焦显微镜，对于标记细胞的检测和样品面积的测量是非常有用的. MMGL具有参与内吞途径的胞质基序。为了更有效地在细胞表面展示MMGL，我们消除了基序以减少内吞作用引起的下调。产生了稳定的转染子CTL-YM，其具有改变的细胞质基序AENL而不是YENL。MMGL的细胞表面表达得到证实。目前，我们还没有看到CTL-ML和CTL-YM之间在肺转移结节中的细胞积聚方面的显著差异。少

项目成果

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Kimura, T.：“小鼠巨噬细胞钙型凝集素的钙依赖性构象。碳水化合物结合活性由钙依赖性表位特异性抗体稳定。”

Mizuochi,S.: "Unique tissue distribution of a mouse macrophage C-type lectin." Glycobiology. 7. 137-146 (1997)

Mizuochi,S.：“小鼠巨噬细胞 C 型凝集素的独特组织分布。”

Sakamaki, T., et al.: "Enhancement in accessibility to macrophages by modification of mucin-type carbohydrate chains on a tumor cell line : role of a C-type lectin of macrophages" J.Leukoc.Biol.57. 407-414 (1995)

Sakamaki, T. 等人：“通过修饰肿瘤细胞系上的粘蛋白型碳水化合物链增强巨噬细胞的可及性：巨噬细胞 C 型凝集素的作用”J.Leukoc.Biol.57。

Mizuochi,S.: "Unique tissue distribution of a mouse macrophage C-type lectin." Glycobiology. 7(印刷中). (1997)

Mizuochi, S.：“小鼠巨噬细胞 C 型凝集素的独特组织分布。”7（出版中）。

Sakamaki,T.: "Enhancement in accessibility to macrophages by modification of mucin-type carbohydrate chains on a tumor cell line;role of a C-type lectin of macrophages." J.Leukoc.Biol.57. 407-414 (1995)

Sakamaki,T.：“通过修饰肿瘤细胞系上的粘蛋白型碳水化合物链来增强巨噬细胞的可及性；巨噬细胞 C 型凝集素的作用。”