基于转铁蛋白受体的磁共振成像示踪大鼠大脑铁代谢的实验研究

An experimental study on magnetic resonance imaging tracking iron metabolism in rat brain based on transferrin receptor

目的探讨基于转铁蛋白受体(TfR)在磁共振成像(MRI)示踪大鼠大脑铁代谢的应用效果,为临床开发监测阿尔茨海默病治疗效果的方法提供参考。方法选择健康成年SD大鼠12只,随机分为实验组和对照组,每组6只。实验组大鼠左侧大脑半球纹状体注入TfR慢病毒(TfR转染组),右侧大脑半球纹状体注入空载体EGFP慢病毒(EGFP转染组);对照组大鼠两侧大脑半球纹状体均注入等量生理盐水(正常组)。2周后经股静脉注入转铁蛋白-超小超顺磁性氧化铁纳米颗粒共轭物(Tf-USPIO)示踪剂15 mg/kg,分别于注射前及注射后1 d行MRI检查。MRI检查完毕后,予戊巴比妥钠麻醉大鼠,并通过断颈法将其处死,取大脑纹状体组织进行免疫荧光染色和普鲁士蓝染色。通过实时定量聚合酶链反应(PCR)检测大鼠纹状体脑组织TfR mRNA表达情况。结果实时定量PCR结果显示,TfR转染组脑组织TfR mRNA表达水平显著高于EGFP转染组和正常组(P<0.05)。免疫荧光染色和普鲁士蓝染色结果显示,TfR慢病毒成功转染大鼠脑组织,并成功模拟阿尔茨海默病大脑铁聚积的情况。在注射Tf-USPIO示踪剂前后,对照组大鼠MRI检查结果无明显变化;实验组大鼠左侧纹状体可见明显的信号改变,而右侧纹状体未见明显的信号改变。结论Tf-USPIO示踪剂可特异性结合脑组织的TfR,并可通过MRI显示,为临床开发监测阿尔茨海默病治疗效果的方法提供参考。

Objective To explore the application effect of magnetic resonance imaging(MRI)tracking iron metabolism in rat brain based on transferrin receptor(TfR),and to provide reference for clinical development of methods to monitor the therapeutic effect of Alzheimer′s disease.Methods Twelve healthy adult Sprague-Dawley(SD)rats were selected and randomly divided into experimental group and control group,with 6 rats in each group.In the rats of the experimental group,TfR lentiviruses were injected into the striatum of the left cerebral hemisphere(TfR transfection group),and empty vector EGFP lentiviruses were injected into the striatum of the right cerebral hemisphere(EGFP transfection group).In the rats of the control group,the same volume of normal saline was injected into both sides of the striatum of the cerebral hemisphere(normal group).Two weeks later,transferrin(Tf)-conjugate of ultra-small super-paramagnetic iron oxide nanoparticles(USPIO)(Tf-USPIO)tracer was injected into the femoral vein of the rats at dose of 15 mg/kg,and MRI examination was performed for the rats before the injection and 1 day after the injection.After the MRI examination was completed,the rats were anesthetized with sodium pentobarbital and were sacrificed by using cervical dislocation method,and the brain striatum tissues of the rats were collected for immunofluorescence staining and Prussian blue staining.Real-time quantitative polymerase chain reaction(PCR)was used to detect the expression of TfR mRNA of the brain striatum tissues in rats.Results The results of real-time quantitative PCR showed that the expression level of TfR mRNA of the brain tissues in the TfR transfection group was significantly higher than that in the EGFP transfection group and the normal group(P<0.05).The results of immunofluorescence staining and Prussian blue staining showed that TfR lentiviruses were successfully transfected into the brain tissues of the rats and the iron accumulation in the brain of Alzheimer′s disease was successfully simulated.Before and after the injection of Tf-USPIO tracer,there were no significant changes in the MRI examination results of the rats in the control group,and obvious signal changes were observed in the left striatum of the rats in the experimental group and no obvious signal changes were observed in the right striatum of the rats in the experimental group.Conclusion Tf-USPIO tracer can specifically bind to TfR in brain tissues and can be displayed through MRI,providing a reference for the clinical development of methods to monitor the therapeutic effect of Alzheimer′s disease.