siRNA阻断RGMa基因表达促进脑梗死大鼠下调脑区激活的fMRI研究

Cerebral area of functional down-regulation after cerebral infarction refers to the wider region, and the distant area had expressed the functional activity in a low state. Our earlier studies found that cerebral blood flow and metabolic activity had reduced in the remote area, down-regulation cerebral area is potentially interrelated to neural function recovery, but its pathogenesis is still not clear. To further investigate the mechanism of nerve regeneration inhibitory protein in lower brain regions, this study uses RNAi technology, screening efficient siRNA fragments to effect on RGMa mRNA, construct RGMa shRNA adenovirus expression vector, and transfected rat brain, combined with immunohistochemistry, RT-PCR and in situ hybridization techniques, to observe siRNA blocking RGMa gene expression and promote cerebral area of functional down-regulation. By using magnetic resonance imaging, we had obversed consequent of RNAi interference by continuous dynamic variety imaging parameters in vivo measurements. Thus, confirming the role of RGMa in axonal regeneration and recovery of neurological function in terms of the integrity of neural pathways. Thereby provides a theoretical basis of genetic intervention for clinical evaluation and treatment of cerebral infarction sequela, as well as find effective targets to promote recovery of neurological function.

脑梗死后下调脑区是指梗死灶以外（包括远隔区域在内）较为广泛的脑组织处于一种机能活动低下状态。本课题组前期研究发现，脑梗死发生后远隔区域局部脑血流量减少，代谢活动降低，且下调脑区是潜在的与神经功能恢复有关的脑功能区，其发生机制目前尚不明确。为进一步探讨神经再生抑制蛋白在下调脑区中的作用机制，本研究拟采用RNAi技术，筛选作用于RGMa mRNA的高效片段siRNA，构建RGMa的shRNA腺病毒表达载体，并转染脑梗死大鼠脑组织，结合免疫组化、RT-PCR和原位杂交等技术，观察siRNA阻断RGMa基因表达促进下调脑区激活的效果。并通过fMRI对siRNA阻断RGMa基因表达促进脑梗死大鼠下调脑区激活效应进行连续动态活体测量，从神经传导通路完整性的角度证实RGMa在神经轴突再生以及神经功能恢复中的作用。从而为临床评价和治疗脑梗死后遗症寻找有效靶点进行基因干预、促进神经功能恢复提供理论依据。

脑梗死后下调脑区是指梗死灶以外（包括远隔区域在内）较为广泛的脑组织处于一种机能活动低下状态。为进一步探讨神经再生抑制蛋白在下调脑区中的作用机制，本研究拟采用RNAi技术，筛选作用于RGMa mRNA的高效片段siRNA，构建RGMa的shRNA腺病毒表达载体，并转染脑梗死大鼠脑组织，结合免疫组化、RT-PCR和原位杂交等技术，观察siRNA阻断RGMa基因表达促进下调脑区激活的效果。并通过DTI对siRNA阻断RGMa基因表达促进脑梗死大鼠下调脑区激活效应进行连续动态活体测量，从神经传导通路完整性的角度证实RGMa在神经轴突再生以及神经功能恢复中的作用。研究发现(1) MCAO模型组、生理盐水组大鼠缺血后，不同时间点小脑半球的RGMa蛋白阳性表达均有所增加。在相应时间点的RGMa蛋白的表达较MCAO模型组有所降低，在差异具有统计学意义 (2) MCAO模型组、生理盐水组大鼠小脑半球缺血后1h后，各个时间点FA值较假手术组降低，均具有统计学差异(P＜0.05 ) ；干预组大鼠小脑半球FA值较MCAO模型组升高，在时间点两组间的FA值差异具有统计学意义( P＜0.05) (3) 各手术组大鼠的mNSS神经功能评分与小脑半球RGMa蛋白表达存在正相关，与FA值呈负相关。从而为临床评价和治疗脑梗死后遗症寻找有效靶点进行基因干预、促进神经功能恢复提供理论依据。