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

细胞移植是与器官移植同样重要的医学研究课题。一个重要的问题是如何将移植细胞靶向到合适的器官或组织中。近年来的研究进展揭示了内源性凝集素对碳水化合物配体的特异性识别在细胞生理靶向中的重要性。在这项研究中，我们研究了利用MMGL作为碳水化合物识别装置，将细胞人工靶向到肿瘤部位。MMGL是一种发现于杀肿瘤巨噬细胞上的跨膜钙型凝集素。我们也尝试开发新的方法来分析细胞靶向性。转染白细胞介素2 （Interleukin 2-dependent）小鼠T细胞系CTLL-2细胞，表达载体pCEP-4含有编码MMGL的全长cDNA插入物。获得了稳定的转染细胞系CTL-ML。用LOM-14单抗证实了MMGL在细胞表面的表达。将荧光染料DiI标记的CTL-ML细胞静脉注射到小鼠卵巢肿瘤ov2944 - hm -1细胞产生的肺转移小鼠中。使用连续的肺冰冻切片，在荧光显微镜下比较转移性结节与正常肺组织的荧光细胞数量。与模拟转染的CTL-CEP细胞相比，发现CTL-ML细胞在肿瘤组织中选择性积累。为了进行数据分析，测试了图像分析仪和共聚焦显微镜。特别是共聚焦显微镜可用于标记细胞的检测和样品面积的测量。MMGL具有胞质基序，参与胞吞途径。为了更有效地在细胞表面显示MMGL，我们消除了通过内吞作用减少下调的基序。制备了一个稳定的转染CTL-YM，它改变了细胞质基序AENL而不是YENL。证实了MMGL在细胞表面的表达。目前我们尚未发现CTL-ML和CTL-YM在肺转移结节的细胞积聚方面有显著差异。少

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