Cloning of cDNA candidates enforcing brain-specific entry in microglia

克隆 cDNA 候选物以实现小胶质细胞中的大脑特异性进入

• 批准号: 13680854
• 负责人: SAWADA Makoto
• 金额: $ 2.11万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680854/
• 关键词: Microglia; gene transformation; brain-specific; drug delivery system; pharge display; blood-brain barrier

The ability to manipulate the expression of genes within the mammalian brain provide unique opportunities to study and potentially treat neurologic disorders. To express certain genes specifically in brain has been done with viral vectors or cells carrying DNA, however, they all needed a surgical operation. Recently we found that microglia could enter specifically in brain when injected intra-arterially, and demonstrated that intra-arterial injection of microglia transfected with the lacZ gene resulted in expression of b-galactosidase up to three weeks post-injection in rat brain. This method will allow us to easily deliver the gene of interest to brain without any effects to other organs. In the present study, we compared migration of systemically injected microglia into normal brain vs. ischaemic brain using a model of ischaemic hippocampal lesion. Microglia were labeled by a fluorescent dye using our standard phagocytosis procedure of microscopic particles and then injected intra- arterially into Mongolian gerbils subjected to ischaemia reperfusion neuronal injury. Delayed death of pyramidal neurons was confirmed by conventional histological analysis and dUTP nick end labeling (TUNEL) method. Clusters of dye-tagged cells migrating into the hippocampal ischaemic lesions were confirmed histochemically to be microglia. Since peripherally injected microglia exhibit specific affinity for ischaemic brain lesions and does not exacerbate ischaemic neuronal injury in the present model, we suggest that microglia may have a potential to be used as a piggy-back ride to deliver therapeutic genes and/or drugs for CNS repair following transitory global ischaemic insult. Now we are trying to isolate gene(s) which product(s) are responsible for brain-specific entry of microglia by pharge-display screening system.

在哺乳动物大脑中操纵基因表达的能力为研究和潜在地治疗神经疾病提供了独特的机会。为了在大脑中特异地表达某些基因，已经用携带DNA的病毒载体或细胞完成了，然而，它们都需要外科手术。最近，我们发现小胶质细胞在动脉注射时可以特异性地进入脑内，并证明动脉注射转LacZ基因的小胶质细胞可导致b-半乳糖苷酶在注射后3周在大鼠脑内表达。这种方法将使我们能够轻松地将感兴趣的基因传递到大脑，而不会对其他器官产生任何影响。在目前的研究中，我们比较了系统注射的小胶质细胞在正常脑和缺血脑中的迁移，使用了缺血性海马区损伤的模型。用荧光染料标记小胶质细胞，用我们标准的微粒吞噬程序将小胶质细胞注入蒙古沙土鼠脑缺血再灌注神经元损伤的动脉内。常规组织学分析和dUTP缺口末端标记法(TUNEL)证实锥体神经元迟发性死亡。迁移到海马区的成簇的染料标记细胞经组织化学证实为小胶质细胞。由于外周注射的小胶质细胞对缺血性脑损伤表现出特异性的亲和力，并且在本模型中不会加重缺血性神经元损伤，我们认为小胶质细胞可能有潜力被用作搭载治疗基因和/或药物来修复短暂性全脑缺血损伤后的中枢神经系统。现在，我们正试图通过Parge-Display筛选系统分离负责小胶质细胞脑特异性进入的基因(S)。

项目成果

Ohara, Y., Himeda, T., Asakura, K., Sawada, M.: "Distinct cell death mechanisms by Theiler's murine encephalomyelitis virus (TMEV) infection in microglia and macrophage"Neurosci. Lett.. 327. 41-44 (2002)

Ohara, Y.、Himeda, T.、Asakura, K.、Sawada, M.：“小胶质细胞和巨噬细胞中泰勒氏鼠脑脊髓炎病毒 (TMEV) 感染的独特细胞死亡机制”Neurosci。

Ishiguro, H., Yamada, K., Sawada.H., Nishii, K., et al.: "Age-dependent and tissue-specific CAG repeat instability occurs in mouse knock-in for a mutant Huntington's disease gene"J.Neurosci.Res.. 65. 289-297 (2001)

Ishiguro, H.、Yamada, K.、Sawada.H.、Nishii, K. 等人：“亨廷顿病突变基因的小鼠敲入中出现年龄依赖性和组织特异性 CAG 重复不稳定性”J.

Kenji F.Tanaka ほか: "Existence of functional b_1-and b_2 -adrenergic receptors on microglia"J Neurosci Res. 70. 232-237 (2002)

Kenji F. Tanaka 等人：“小胶质细胞上功能性 b_1-和 b_2-肾上腺素受体的存在”J Neurosci Res. 70. 232-237 (2002)