Study on the molecular mechanism by which microglia transform into phagocytes

静息状态下的小胶质细胞转变为活跃的吞噬细胞的机制还知之甚少。因此，我的目标是确定与激活的小胶质细胞吞噬活性诱导相关的特异性分子(S)。在第一年，我们试图建立面神经注射神经毒性凝集素RCA60诱导神经细胞死亡的动物模型。然而，由于RCA60在日本不可用，这项实验没有进行。相反，我们使用过氧化氢或过氧亚硝酸盐来诱导神经细胞死亡。第二年，在过氧化氢注射的面神经核中，用减法研究了与诱导吞噬细胞活动相关的分子。差减方法表明，在过氧化氢注射的面神经核中有一些分子被诱导，但大多数分子属于蛋白水解酶、其他酶和胞核…还有更多。尽管反复实验，我们还是没能找到吞噬活性相关蛋白的候选分子。除体内模型外，我们还尝试在体外制备非吞噬的小胶质细胞，并用佛波酯(PMA)处理小胶质细胞，最终成功地制备了非吞噬小胶质细胞。因此，比较吞噬小胶质细胞和非吞噬小胶质细胞之间的蛋白质成为可能。在过去的一年里，我们继续用差减法分析吞噬细胞相关的蛋白质。然而，结果并不显著。另一方面，吞噬小胶质细胞和非吞噬小胶质细胞的蛋白质比较表明，吞噬小胶质细胞中存在一个相对分子质量约为90 kDa、p1 4.8的分子。因此，试图从培养的小胶质细胞中分离出分子(P90)。将小胶质细胞匀浆用于DEAE Sephadex、羟基磷灰石、Sephacryl S200柱层析，然后进行SDS-聚丙烯酰胺凝胶电泳/转印。最后对PVDF膜上的P90进行N-末端氨基酸序列分析。得到的序列为Asp-Asp-Glu-Val-(未打开)。这些结果，包括P90的鉴定和氨基酸序列，都有待证实。较少

Little has been known about the mechanism by which resting microglia change into active phagocytes. Therefore, I aimed to identify the specific molecule(s) associated with the induction of phagocytic activity in activated microglia.In the first year, we tried to establish an animal model of neuronal cell death induced by the injection of neurotoxic lectin RCA6O into facial nerve. However, the experiment was not carried out because RCA60 was not available in Japan. Instead, we used hydrogen peroxide or peroxinitrite to induce neuronal cell death. These radicals were found to be effective for injuring motoneurons in facial nucleus.In the second year, the molecules associated with the induction of phagocytic activity were explored by subtraction method in the hydrogen peroxide-injected facial nucleus. The subtraction method revealed that some molecules are induced in hydrogen peroxide-injected facial nucleus, but the most of the molecules belongs to proteases, other enzymes and cytoskelet … More ons. Despite the repeated experiments, we could not find out candidate molecules for phagocytic activity-related proteins. Apart from the in vivo model, we tried to prepare non-phagocytic microglia in vitro, and finally succeeded to do it by treating microglia with phorbol myristate acetate (PMA). Thus, it became possible to compare the proteins between phagocytic microglia and non-phagocytic microglia.In the last year, we continued to analyze the phagocytes-related proteins by subtraction method. However, the results were not significant. On the other hand, the comparison of proteins between phagocytic microglia and non-phagocytic microglia indicated that a molecule with molecular weight of approximately 90 kDa and pl 4.8 is present in phagocytic microglia. Therefore, the molecule (p90) was tried to be isolated from the cultured microglia. The microglial homogenate was applied to DEAE Sephadex, Hydroxyapatite, Sephacryl S200 column chromatographies, and subsequently SDS polyacrylamide gel electrophoresis/transblotting. Finally, p90 on PVDF membrane was analyzed for N-terminal amino acid sequence. The resultant sequence was Asp-Asp-Glu-Val-----(not opened). These results including the identification and the amino acid sequence of p90 remain to be confirmed. Less