Cloning of cDNA candidates enforcing brain-specific entry in microglia

操纵哺乳动物脑内基因表达的能力为研究和潜在治疗神经系统疾病提供了独特的机会。在大脑中特异性表达某些基因已经用病毒载体或携带DNA的细胞完成，然而，它们都需要外科手术。最近，我们发现，小胶质细胞可以进入特定的脑内时，动脉注射，并证明动脉注射的小胶质细胞与lacZ基因转染导致β-半乳糖苷酶的表达在大鼠脑内注射后三周。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。在本研究中，我们使用缺血性海马损伤模型比较了全身注射的小胶质细胞迁移到正常脑与缺血脑。使用我们的标准吞噬程序的微观颗粒的荧光染料标记的小胶质细胞，然后动脉内注射到蒙古沙鼠缺血再灌注神经元损伤。常规组织学分析和dUTP缺口末端标记（TUNEL）法证实锥体神经元的延迟性死亡。迁移到海马缺血性病变的染料标记的细胞簇被证实是小胶质细胞。由于外周注射的小胶质细胞表现出特定的亲和力，缺血性脑损伤，并没有加剧缺血性神经元损伤在本模型中，我们建议，小胶质细胞可能有潜力被用作一个背负式乘坐提供治疗基因和/或药物的中枢神经系统修复短暂的全球缺血性损伤。目前，我们正试图通过药物筛选系统来分离小胶质细胞脑特异性进入的相关基因。

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.