低频超声协同声诺维促miR-101靶向治疗AD的实验研究

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects elderly people worldwide. AD is a progressive neurodegenerative disorder mainly characterized by amyloid-beta (Aβ) deposition and neurofibrillary tangles (NFTs). Aβ derives from sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The abnormal enrichment of APP leads to a high risk of AD. Two independent miRNA expression profiles have shown that miR-101 is down-regulated in the human AD brain, suggesting that miR-101 down-regulation may play a role in the development of AD. miR-101 is a negative regulator of APP expression and affects the accumulation of Aβ. It was confirmed that cyclooxygenase-2 (Cox-2) was one of a validated miR-101 target. miR-101 could down-regulate APP and Cox-2 expression in hippocampal neurons. One of the plausible age-associated AD animal models, senescence-accelerated mouse prone 8 (SAMP8), have age-related learning and memory deficits. We found APP protein significantly increased in the hippocampus of aged SAMP8 mice, meanwhile miR-101 level decreased significantly. We also found that overexpression of miRNA-101 could significantly inhibit the Aβ formation in N2a cells. However, due to the restrictive blood brain barrier (BBB), drugs and therapy gene are very difficult to be delivered into the brain. Ultrasound-mediated microbubble destruction (UMMD) has evolved as a novel system for non-invasive, organ- and tissue-specific drug and gene delivery.The BBB can be locally disrupted with UMMD with microbubbles. Neuron-specific enolase (NSE) is one of the most abundant proteins in the adult mammalian brain, and is found in mature neurons. NSE promoter fragment has been shown to target the expression of various transgenes to differentiated neurons. Thus, we will construct a miR-101 recombinant plasmid containing NSE promoter and specifically transfect it into the hippocampus of SAMP8 mice by UMMD with SonoVue microbubble. Overexpression of miR-101 in hippocampus neurons play duel effects of down-regulating APP and Cox-2 expression. This technology may offer new opportunities that will allow specific treatment of AD.

人口日趋老龄化使阿尔茨海默病(AD)成为当今社会最为严峻的课题之一。β淀粉样蛋白(Aβ)是造成AD的元凶，然而由于血脑屏障，目前针对Aβ为靶点的治疗手段有限。新近发现miRNA-101在海马组织中表达，是APP（Aβ前体）的负性调节子，在下调APP同时还抑制炎症因子COX-2的表达。我们前期研究显示：快速老化小鼠SAMP8海马区miRNA-101水平显著下降；且miRNA-101过表达可显著抑制N2a细胞Aβ的生成。在此基础上，选择APP为靶点，拟构建神经元特异性NSE启动子控制的miRNA-101表达重组体，应用低频超声协同SonoVue微泡开放靶区血脑屏障，结合NSE启动子，实现静脉给药途径下miRNA-101靶向表达于SAMP8鼠海马区神经元内，进而对Aβ生成和炎症反应起双重抑制作用。有望克服常规药物和基因治疗可控性差、组织细胞特异性不高、毒副作用强等瓶颈，可能成为AD治疗的新手段。

人口日趋老龄化使阿尔茨海默病(AD)成为当今社会最为严峻的课题之一。β淀粉样蛋白(Aβ)是造成AD的元凶，Aβ42聚集到中后期形成老年斑时伴有大量激活的小胶质细胞、炎性因子以及毒性产物，这些物质与Aβ相互激发形成恶性毒性网络加速了老年斑的形成，抗炎治疗是必须措施。然而受制于血脑屏障，目前针对Aβ和颅内炎症为靶点的治疗手段有限。.研究发现miRNA-101在海马组织中表达，是APP（Aβ42前体）的负性调节子，在下调APP同时还抑制炎症因子COX-2的表达。我们的前期研究显示：快速老化小鼠SAMP8海马区miRNA-101水平显著下降；且miRNA-101过表达可显著抑制N2a细胞Aβ的生成。.在此基础上，我们选择APP为靶点，成功构建了神经元特异性NSE启动子控制的miRNA-101重组体（pNSE-miR-101），确定了超声微泡SonoVue载pNSE-miR-101的最佳包载率。进而，应用低频超声协同SonoVue微泡开放靶区血脑屏障（最佳参数为实时造影Flash模式，辐照间隔 5 s、辐照总时长 3 min，Sonovue 0.3ml/25g小鼠体重），Real-Time PCR结果显示，我们实现了静脉给药途径下pNSE-miR-101靶向表达于快速老化鼠SAMP8鼠的海马区神经元内。随后采用半定量RT-PCR和Western Blot检测，结果显示超声辐照侧海马组织中miR-101的上调，对Aβ生成和炎性因子COX-2表达起双重抑制作用，且对COX-1的表达和实验鼠血清及胃组织中PGE2（有利于维持胃黏膜的完整性）的含量无显著性影响。最后，采用Morris水迷宫系统测试实验小鼠空间记忆能力，结果显示：UMMD（Ultrasound-mediated microbubble destruction）介导的pNSE-miR-101转染的SAMP8鼠，与未处理的SAMP8鼠相比，潜伏期显著缩短（p<0.01），搜索平台路径明显改善。综上所述，本项目联合利用无创、可重复输注的UMMD靶向转基因技术、神经元特异性表达启动子NSE和miRNA-101对APP、COX-2负性调控等优势，突破了miRNA-101无创跨越血脑屏障打靶于海马神经元这一瓶颈，并达到对Aβ生成及炎症反应的双重抑制作用，可为临床上AD的安全、特异性基因治疗提供一条新的途径和思路。