Development of novel therapeutic means to regenerating spinal cord injury using microglia/macrophages

开发利用小胶质细胞/巨噬细胞再生脊髓损伤的新治疗方法

• 批准号: 16390442
• 负责人: TANAKA Junya
• 金额: $ 8.83万
• 依托单位: EHIME 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-16390442/
• 关键词: microglia; astrocyte; oligodendrocyte; transdifferentiation; NG2; regenerative medicine; GFAP; nestin; NG2コンドロイチン硫酸ブロテオグリカン; アストロサイト; 神経細胞; 神経系前駆細胞; オリゴデンドロサイト; 分化転換; 神経組織再生

Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 d, followed by culture in the presence of 70% serum for 2 d. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly … More turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labelled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes.Such multipotent MG called promicroglioblasts (ProMGBs) formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2^+ MG, which appeared to be the source of NG2^+ ProMGB aggregates. The aggregates were MG marker^+/NG2^+/GFAP^+/NCAM^+/S^-100b^- and had alkaline phosphatase activity. The marked accumulation of NG2^+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2^+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2^+ MG in the wounds separated with trypsin-EDTA formed NG2^+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing b-tubulin III^+ cells in vitro easily. These data suggest that NG2^+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain. Less

小胶质细胞被认为是大脑中唯一的中胚层来源的细胞群，尽管它们的作用还没有完全了解。本研究表明，大鼠原代小胶质细胞表达巢蛋白，A2 B5和O 4抗原，这是少突胶质细胞前体细胞的标志物。基于这些发现，我们研究了小胶质细胞是否产生神经元或大胶质细胞。纯化的小胶质细胞在10%胎牛血清存在下培养3d，然后在70%血清存在下培养2d。在两步培养过程中，小胶质细胞变得高度增殖，并强烈表达DNA结合（Id）基因的抑制剂，表明细胞的去分化。去分化的细胞还表达促进分化为神经元或大胶质细胞的转录因子。当去分化细胞转移到聚赖氨酸包被的无血清培养基中时，大量细胞迅速地分化为细胞外基质。 ...更多信息 转化为长突起细胞，表达微管相关蛋白2、突触蛋白I、神经丝蛋白、胶质细胞酸性蛋白或半乳糖苷。当小胶质细胞荧光标记通过乙酰化低密度脂蛋白（LDL）受体或吞噬依赖性机制，荧光轴承神经元，星形胶质细胞，或少突胶质细胞进行了观察。神经球，神经干细胞的聚集体，表达武藏1和表皮生长因子受体，但小胶质细胞衍生的细胞没有。这些结果表明小胶质细胞作为多能干细胞的一种新的功能，可以分化为神经元、星形胶质细胞或少突胶质细胞，这种多能MG称为前小胶质母细胞（promicroglioblasts，ProMGBs），形成细胞聚集体，在无血清培养基中培养后不久，这些细胞就具有神经外胚层表型。免疫组化结果显示，新生大鼠脑内有少量MG表达NG 2硫酸软骨素蛋白聚糖（NG 2）。新生儿脑的原代培养物中含有NG 2 ^+ MG，这似乎是NG 2 ^+ ProMGB聚集体的来源。聚集体为MG标志物^+/NG 2 ^+/GFAP^+/NCAM^+/S^-100 b ^-，并具有碱性磷酸酶活性。在成年大鼠脑中的刺伤附近观察到NG 2 ^+ MG的显著积聚。伤口中积累的NG 2 ^+ MG数量逐渐减少，但这些细胞持续到损伤后150天。此外，GFAP免疫反应性在伤口周围显著增加。用胰蛋白酶-EDTA分离的伤口中的NG 2 ^+ MG在70%血清培养基中形成NG 2 ^+聚集体，然后在无血清培养基中转化为具有神经外胚层表型的细胞。虽然从成熟的大脑中分离出有活力的神经元很困难，但从刺伤中分离出的细胞在体外很容易生成带有突起的b-微管蛋白III^+细胞。这些数据表明，正常发育或病理性脑中的NG 2 ^+ MG参与脑的发生或再生。少

项目成果

Regulation of ADF/cofilin phosphorylation and synaptic function by LIM-kinase

• DOI: 10.1016/j.neuropharm.2004.06.030
• 发表时间: 2004-10-01
• 期刊: NEUROPHARMACOLOGY
• 影响因子: 4.7
• 作者: Meng, YH;Takahashi, H;Jia, ZP
• 通讯作者: Jia, ZP

L-Serine-mediated release of apolipoprotein E and lipids from microglial cells.

L-丝氨酸介导的小胶质细胞释放载脂蛋白 E 和脂质。

• 发表时间: 2004
• 期刊: Experimental Neurology 185
• 作者: Mori K;Yokoyama A;Yang L;Yang L;Maeda N;Mitsuda N;Tanaka J
• 通讯作者: Tanaka J

Human CD34+ cells differntiate into microglia and express recombinant therapeutic protein.

人 CD34+ 细胞分化为小胶质细胞并表达重组治疗蛋白。

• 发表时间: 2004
• 期刊: Proc. Netl. Acad. Sci. U.S.A. 101
• 作者: Asheuer;M.
• 通讯作者: M.

Macrophage/microglia-specific protein lbal binds to fimbrin and enhances

巨噬细胞/小胶质细胞特异性蛋白 lbal 与纤维蛋白结合并增强

• 发表时间: 2004
• 期刊: J.Neurochem. 88
• 作者: Ohsawa;K.;Imai;Y.;Sasaki;Y.;Kanazawa;H.;Kohsaka;S.
• 通讯作者: S.

Human CD34+ cells differentiate into microglia and express recombinant therapeutic protein

• DOI: 10.1073/pnas.0306431101
• 发表时间: 2004-03-09
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Asheuer, M;Pflumio, FO;Cartier, N
• 通讯作者: Cartier, N