Cloning of cDNA candidates enforcing brain-specific entry in microglia

操纵哺乳动物大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。为了在大脑中特异性地表达某些基因，已经用病毒载体或携带DNA的细胞完成了，但它们都需要外科手术。最近我们发现动脉内注射小胶质细胞可以特异性进入大脑，并证明动脉内注射转染lacZ基因的小胶质细胞可在注射后3周内在大鼠脑中表达b-半乳糖苷酶。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，我们使用缺血海马病变模型，比较了全身注射小胶质细胞在正常脑和缺血脑中的迁移。用荧光染料标记小胶质细胞，并将其注射到缺血再灌注损伤的蒙古国沙鼠动脉内。采用常规组织学分析和dUTP缺口末端标记法（TUNEL）证实锥体神经元延迟死亡。成群的染色细胞迁移到海马缺血病变，组织化学证实为小胶质细胞。由于在目前的模型中，外周注射的小胶质细胞对缺血性脑病变表现出特异性亲和力，并且不会加剧缺血性神经元损伤，因此我们认为，小胶质细胞可能有潜力作为一种搭便车，在短暂的全球缺血性损伤后，为中枢神经系统修复提供治疗性基因和/或药物。目前，我们正试图通过噬菌体展示筛选系统分离出与小胶质细胞脑特异性进入有关的产物基因。

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.