【作者】 王健；

【导师】 常津； 武晓丽；

【作者基本信息】 天津大学 ， 生物学， 2023， 硕士

【摘要】 阿尔茨海默症(Alzheimer’s disease,AD)是最常见的神经退行性疾病,早期诊断和干预有助于缓解疾病的进展。目前临床上传统的检测方式如磁共振成像(Magnetic Resonance Imaging,MRI)多针对于疾病中后期阶段的结构性变化,不符合早期诊断的标准,制约了AD成像的发展。基于此,本研究设计了一种识别AD病理标志物乙酰胆碱酯酶(Acetylcholinesterase,ACHE)后开启信号的可激活探针CyBN。CyBN探针由活化部分发光团、自消除结构和ACHE响应部分组成,识别ACHE后探针被特异性激活并释放活化的发光团,其荧光和光声信号均特异性增强,可利用光声和荧光两种模式进行成像检测,兼顾了成像的特异性、灵敏度及组织穿透深度。为阿尔兹海默症的早期诊断提供了新思路。本研究的实验结果如下:(1)成功合成探针CyBN并验证其结构。探针在识别ACHE后“开启”信号,激活探针荧光信号是未激活时的6.8倍,最低检测限为0.2188 U/ml;识别ACHE标志物后光声信号增强4.9倍,最低检测限为0.10635 U/m L。信号的强度与乙酰胆碱酯酶活性正相关,且具有时间依赖性。探针信号不受其它金属离子、酶等非特异性分子的影响,具有较好的生物安全性。(2)CyBN探针在神经细胞PC12中检测ACHE的成像效果优异。MTT细胞实验验证CyBN探针有较好的安全性。CyBN探针荧光信号与PC12细胞中ACHE水平正相关。ACHE抑制剂结果显示,随抑制剂浓度升高,PC12细胞中ACHE活性降低,探针信号显著下降。(3)CyBN探针在APP/PS1阿尔兹海默症动物模型中有较好的荧光/光声成像效果。模型鼠信号与野生型相比强5倍以上,光声和荧光成像结果证实随月龄增加,AD鼠脑内ACHE活性增加,探针信号逐渐增强。离体脑组织光声成像结果进一步证实上述结论。H&E染色发现组织无明显异常,探针的生物安全性良好,离体组织成像显示探针的代谢主要通过肝脏和肾脏两种途径。综上所述,本研究设计的乙酰胆碱酯酶可激活的荧光/光声双模态探针CyBN在体外模拟生理条件,神经细胞PC12及APP/PS1阿兹海默症模型鼠中均有良好成像效果,探针信号仅在识别ACHE后被激活,成像效果与ACHE活性正相关。且探针的生物安全性良好,无明显细胞组织毒性。更多还原

【Abstract】 Alzheimer’s disease(AD)is the most common neurodegenerative disease,early diagnosis and intervention can help mitigate the disease progression.Currently,traditional clinical detection modalities such as Magnetic Resonance Imaging(MRI)mostly target structural changes in the middle and late stages of the disease,which do not meet the criteria for early diagnosis and limit the development of AD imaging.Based on this,we created the activatable probe CyBN,which activates the signal after detecting the acetylcholinesterase(ACHE)pathology marker for AD.The CyBN probe is made up of an ACHE-responsive component that is particularly triggered and releases the activated luminescence cluster after detecting ACHE,a self-eliminating structure,and an activated section of the luminescence cluster that has been activated.Taking into mind the specificity,sensitivity,and tissue penetration depth of imaging,both photoacoustic and fluorescence modes can be used for imaging detection.Fluorescence and photoacoustic signals are also specifically improved.It offers a fresh concept for Alzheimer’s disease early detection.The experimental results are as follows:(1)The activatable probe CyBN was successfully synthesized and its structure was verified.The signal was"turned on"after ACHE recognition and the fluorescence signal is 6.8-fold stronger in vitro tests than inactivated probe,with a minimum detection limit of 0.2188 U/ml;After recognition of markers,the PA intensity increased 4.9-fold than inactivated probe with the minimum detection limit of0.10635 U/m L.The intensity of the signal is positively correlated with ACHE activity and is time-dependent,and the signal was not affected by other non-specific molecules such as metal ions,enzymes,and its biosafety was good.(2)CyBN probe has excellent imaging results in detecting ACHE in neuronal cells PC12.MTT verified that CyBN probe has great safety.CyBN probe fluorescence signal was positively correlated with ACHE level in PC12 cells.ACHE inhibitor results showed that ACHE activity in PC12 cells decreased with increasing inhibitor concentration and probe signal decreased significantly.(3)CyBN probe has great fluorescence and photoacoustic imaging in APP/PS1animal model of Alzheimer’s disease.The signal in the model mouse was more than5-fold stronger than that in the wild type,and the photoacoustic and fluorescence imaging results confirmed that the ACHE activity in the AD mouse brain increased with increasing age,and the probe signal was gradually enhanced.Photoacoustic imaging of isolated brain tissue further confirmed these findings.H&E staining revealed no significant abnormalities in the tissues,showed good biosafety of probes,and isolated tissue imaging showed that the metabolism of the probe was mainly through two pathways:liver and kidney.In summary,we designed a ACHE activatable fluorescent and photoacoustic bimodal probe CyBN,which has great imaging effect in simulated physiological conditions,neuronal cells and APP/PS1 Alzheimer’s disease model mice,with good biological safety in vitro and in vivo.The signal of probe only enhanced after recognition of ACHE and changes with activity of ACHE.The probe is biosafe and has no significant cellular tissue toxicity.更多还原