A STUDY OF CELLULAR NETWORKS WITHIN THE CENTRAL NERVOUS SYSTEM BY USING CONNEXIN45 GENE KNOCKOUT-LACZ GENE KNOCKIN MICE.

使用 CONNEXIN45 基因敲除-LACZ 基因敲除小鼠研究中枢神经系统内的细胞网络。

• 批准号: 14570016
• 负责人: NAKAMURA Kei-ichiro
• 金额: $ 1.92万
• 依托单位: KURUME UNIVERSITY (2003)Kyushu University (2002)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570016/
• 关键词: CONNEXIN45; GAP JUNCTION; KNOCKOUT MOUSE; LACZ; GFP; STEM CELL; IMMUNOCYTOCHEMISTRY; ELECTRON MICROSCOPY; knockout mice; central nervous system; lac Z; confocal laser scanning microscopy

We have investigated connexin45 (Cx45) gene knockout-lacZ gene knockin mice in order, to clarify Cx45 expressing cells in the central nervous system. 1. X-gal staining of those animal tissues enable us to visualize Cx45 expressing cells by the blue coloring. We performed X-gal staining on 1mm thick tissue slices and embedded in Technovit7100 resin. One to five μm thick sections were cut and observed after staining by either toluidine blue or neutral red. In the X-gal staining positive cells, bluish reaction products were observed around cell nucleus. 2. We observed those blueish reaction products by electron microscopy (EM). Slightly electron dense amorphous materials were identified as reaction products, which enable us to identify lacZ positive cells under EM. 3. Quantum dots worked well as marker for immunocytochemistry of light microscopic level for lacZ, however, they were not effective enough for EM level because of their small size. 4. By experiments in which bone marrow cells of GFP expressing transgenic mice were transplanted to wild type donor mice, we found bone marrow derived cells were distributed in tissues as fibroblast-like cells that connected by gap junctions. A part of c-Kit expressing cells were suggested to be bone marrow derived.

我们研究了Cx45基因敲除-lacZ基因敲入小鼠，以阐明中枢神经系统中表达Cx45的细胞。1.这些动物组织的X-gal染色使我们能够通过蓝色显示Cx45表达细胞。我们对1 mm厚的组织切片进行X-gal染色，并包埋在Technovit 7100树脂中。切下1 - 5 μm厚的切片，用甲苯胺蓝或中性红染色后观察。在X-gal染色阳性的细胞中，在细胞核周围观察到蓝色的反应产物。2.用电子显微镜（EM）观察了这些微蓝色的反应产物。轻微电子致密的无定形物质被鉴定为反应产物，这使得我们能够在EM下鉴定lacZ阳性细胞。3.量子点作为lacZ免疫细胞化学的标记物在光镜水平上表现良好，但由于量子点体积小，在电镜水平上表现不佳。4.通过将表达GFP的转基因小鼠的骨髓细胞移植到野生型供体小鼠的实验，我们发现骨髓来源的细胞以成纤维细胞样细胞的形式分布在组织中，这些细胞通过间隙连接连接。部分c-Kit表达细胞被认为是骨髓来源的。

项目成果

HIRATA M: "PIGMENT CELL ORGANIZATION IN THE HYPODERMI OF ZEBRAFISH."DEV DYN. 277. 497-503 (2003)

HIRATA M：“斑马鱼皮下组织中的色素细胞组织。”DEV DYN。

NAKAMURA K: "INTERSTITIAL CELLS OF CAJAL-GAP JUNCTION CONNECTED CELLULAR NETWORKS AS PACEMAKER SYSTEM FOR GASTROINTESINAL MOTILITY."DENSIKENNBIKYOU. 37. 188-193 (2002)

NAKAMURA K：“CAJAL-GAP 连接处的间质细胞连接细胞网络，作为胃肠动力的起搏器系统。”DENSIKENNBIKYOU。

中村 桂一郎: "ギャップ結合による細胞性ネットワーク-消化管運動ペースメーカー細胞を中心にして-"電子顕微鏡. 37. 188-193 (2002)

Keiichiro Nakamura：“基于间隙连接的细胞网络 - 关注胃肠运动起搏细胞 -”电子显微镜。 37. 188-193 (2002)

Matsumoto Y: "Long-term inhibition of Rho-kinase suppresses neointimal formation after stent implantation in porcine coronary arteries : involvement of multiple mechanisms."Arterioscler Thromb Vasc Biol. 24・1. 181-186 (2004)

Matsumoto Y：“长期抑制 Rho 激酶可抑制猪冠状动脉支架植入后的新内膜形成：涉及多种机制。Arterioscler Thromb Vasc Biol。”181-186。

中村桂一郎: "ギャップ結合による細胞性ネットワーク -消化管運動ペースメーカー細胞を中心にして-"電子顕微鏡. 37. 188-193 (2002)

Keiichiro Nakamura：“基于间隙连接的细胞网络 - 关注胃肠运动起搏细胞 -”电子显微镜。 37. 188-193 (2002)