阿尔茨海默病磁共振诊断磁对比剂及药物、细胞治疗的实验研究
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摘要
研究背景
阿尔茨海默病（Alzheimer’s disease，AD）是一种以慢性进行性痴呆为主要表现的神经退行性疾病，是老年性痴呆最常见的原因之一，其发病率随年龄的增高而不断增高，给社会家庭带来了沉重的经济负担。然而，AD在诊断、治疗等方面仍然比较滞后，本课题通过对目前比较热门的一些技术方法和治疗手段的探讨，来寻找未来可能应用于AD的方法和方向。在AD的诊断方面，磁共振成像（magnetic resonanceimaging，MRI）是一种很有前景的检测AD中β淀粉样蛋白（β-amloid，Aβ）沉积的诊断技术，使用磁共振对比剂既能够提供Aβ沉积的特异性成像以区分其他病理生理结构，又能缩短成像时间。而在AD的治疗方面，早期对血管功能的保护可能成为针对发病机理的治疗新靶点，而晚期神经元的丢失也可能通过细胞移植的方法进行功能重建。
目的
1.开发一种使用超小超顺磁性氧化铁（ultrasmall superparamagnetic iron oxide，USPIO）纳米颗粒连接功能性蛋白的磁共振对比剂，从而给检测Aβ沉积提供方法。
2.用于治疗脑缺血的药物丁苯酞，具有内皮保护作用，通过观察其对Aβ损伤下脑微血管内皮细胞（cerebral microvascular endothelial cells，CMECs）的作用，寻找丁苯酞用于AD治疗的证据并进行初步的机制探讨。
3.神经干细胞（neural stem cells，NSCs）是中枢神经系统疾病细胞移植治疗的最终应用形式，但存在定向分化问题。拟通过观察与CMECs共培养后NSCs在正常条件下和损伤条件下的分化情况，寻找一种能够促使移植治疗应用于神经退行性疾病的解决方案。
方法
1.磁共振对比剂由USPIO纳米颗粒连接Aβ16-20（KLVFF）和跨膜蛋白转导结构域（trans-activating transcriptor-protein transduction domain，Tat-PTD）组成，其中Aβ16-20是能特异性与Aβ沉积结合的探针，Tat-PTD能帮助对比剂穿过血脑屏障（brain bloodbarrier，BBB）。体外实验研究此对比剂的生物膜穿透性、生物毒性、与Aβ沉积的特异性结合能力以及对组织磁共振信号的影响；最终将其注射入AD转基因小鼠来进行活体磁共振成像及组织学验证。
2.建立CMECs原代培养方法并鉴定。使用不同浓度丁苯酞预处理CMECs后予以Aβ损伤，观察其活性和凋亡率的变化，并进一步观察其分泌NO及诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）和内皮型一氧化氮合酶（endothelial nitricoxide synthase，eNOS）水平的变化。
3.共培养原代CMECs和NSCs，观察NSCs的生长状况，移除CMECs后将NSCs分别置于正常培养环境和缺糖缺氧（Oxygen-glucose deprivation，OGD）环境中分化以观察分化比例。鼠胚成纤维细胞（mouse embryo fibroblast，MEF）与NSCs共培养作为对照组。
结果
1.Tat-PTD-USPIO-Aβ16-20的蛋白连接效率达97.75%，体内、体外实验均显示其能穿过生物膜，与Aβ沉积特异性结合，并能降低组织的磁共振信号，与未连接的Aβ16-20及Aβ1-40相比没有发现明显的毒性作用。
2.成功建立了乳小鼠CMECs的原代培养方法，最终阳性率可达95.5%。丁苯酞预处理后的Aβ损伤CMECs，细胞活性升高，凋亡率降低，NO分泌增高，iNOS分泌及胞浆表达均增高，而eNOS分泌及胞浆含量未发现有显著变化。
3.与CMECs共培养的NSCs数量更多，直径更大，不论是在正常培养环境下还是在OGD环境下，神经元分化比例均高于对照。
结论
1.本研究设计的磁共振对比剂能够穿过BBB，并能通过磁共振检测到AD动物模型体内的Aβ沉积，是一种很有潜力的AD诊断技术。此外，还有助于开发和评估新的以减少Aβ沉积为目标的AD药物。
2.丁苯酞可能通过提高iNOS的表达和分泌，从而提高NO水平来对Aβ损伤的CMECs产生保护作用，且大剂量下保护作用更明显和稳定，故而有可能通过保护内皮功能而对AD产生治疗作用。
3. CMECs能够促进NSCs的存活和增殖，不仅能促进其在正常培养条件下，也能促进其在损伤条件下更多的向神经元分化。这为研究NSCs在中枢神经系统疾病的细胞治疗中的定向分化问题提供了新证据。
创新点
1.创造性开发出新型纳米磁共振对比剂，既为AD临床体内诊断应用打下基础，也为新药开发研究提供了评估手段。
2.建立了一种经济、简易、重复性好、纯度高的CMECs原代培养方法。
3.通过AD发病机理的新观点，探讨在临床上应用于脑缺血的药物丁苯酞用于AD治疗的可能，为增加新的适应症提供一定的依据。
4.在细胞移植治疗中的神经干细胞定向分化问题上，着重探讨了在损伤环境中的分化状况，更接近真实的移植状况。
Backgrounds
Alzheimer’s disease (AD) is a neurodegenerative disease with the feature of chronicprogressive dementia and is one of the most common causes for senile dementia. Themorbidity increases with age and great financial load is brought to society and families.However, the diagnosis and therapy of AD remain limited. This study is to find the possiblemethod and direction through discussing the intense research in AD. In terms of diagnosis,magnetic resonance imaging (MRI) is a promising technique in detection of Aβ deposits inAD. It has been proved that using the MRI contrast agent can shorten the scanning time andprovide a specific image to identify various structures. In terms of therapy, the protection ofvascular function may be the new target in the early stage of AD and the celltransplantation may be the development direction to repair the neuron loss in the late stageof AD in the future.
Objectives
1. To develop an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticlescoupled with functional protein as a novel MRI contrast agent to detect the Aβ deposits.
2. To find whether s-(-)-3-n-butylphthalide, which can protect the vascule and is usedfor ischemia clinically, could be applied for AD through the observation of the effects onthe Aβ-damaged cerebral microvascular endothelial cells (CMECs).
3. Neural stem cells (NSCs) are the applied form in cell transplantation for diseases ofcentral nervous system. The increase of neuronal differentiation rate of NSCs remains ablock, particular in a damage environment. The study is to see the effects of CMECs on theneurogenesis of NSCs with or without Oxygen-glucose deprivation (OGD).
Methods
1. We developed a novel MRI contrast agent USPIO nanoparticles coupledAβ(16-20)(KLVFF) as a probe for connecting to Aβ deposits and HIV-1trans-activatingtranscriptor-protein transduction domain (Tat-PTD) for the penetration of blood-brainbarrier (BBB). The penetration, toxicity and binding affinity of the contrast agent in vitrohave been studied. The contrast agents were injected into the transgenic mice for livingbrain MRI imaging and histological studies.
2. Establish the method for primary culture of CMECs. After detecting the specificityof CMECs, they were pre-treated with s-(-)-3-n-butylphthalide on different concentrationsand then were damaged by Aβ. The cell viability and apoptosis rates were observed and theconcentrations of NO, inducible nitric oxide synthase (iNOS) and endothelial nitric oxidesynthase (eNOS) were detected.
3. Survival and proliferation of NSCs were studied after CMECs and NSCs wereco-cultured. Removing the CMECs, NSCs were maintained in differentiation culture withor without treatment of OGD to observe the differentiation rates. Mouse embryo fibroblast(MEF) cells co-cultured with NSCs were used as the control groups.
Results
1. The coupling efficiency of protein can reach97.75%for Tat-PTD-USPIO-Aβ(16-20).The developed MRI contrast agents can pass through biological membranes, bind to the Aβdeposits and decrease the MRI signal of the tissue in vitro and in vivo. Compare toAβ(16-20) or Aβ(1-40) alone, no significant toxicity of Tat-PTD and Aβ(16-20) was foundwhen they coupled with USPIO nanoparticles.
2. Successfully establish the method of primary culture of CMECs from newborn mice and the positive rate was95.5%. In the intervention research of s-(-)-3-n-butylphthalide, thecell viability increased and the apoptosis rates decreased. The secretion of NO and iNOSwere increased. There were no significantly changes of the secretion of eNOS in all groups.
3. NSCs co-cultured with CMECs have an increase in size and number compared tothose co-cultured with MEF cells. They also have an increase in neuronal differentiationrates with or without OGD.
Conclusions
1. The novel developed MRI contrast agent by USPIO nanoparticles coupled withfunctional protein can pass through BBB and detect Aβ deposits in AD animal models byMRI in vivo. This novel MRI contrast agent may help in the evaluation and development ofpotential novel therapeutic interventions to reduce amyloid deposits in AD experimentalanimals.
2. The s-(-)-3-n-butylphthalide could protect CMECs from the damage of Aβ throughincreasing the expression and secretion of iNOS, in turn the NO. The effect was morestabilized under the large concentration. Hence, the s-(-)-3-n-butylphthalide may be acandidate for the therapy of AD through the effect on the protection of vascule.
3. This study provides the evidence that OGD cannot alter the effects of CMECs inpromoting the neuronal differentiation potential of NSCs. These findings may haveimportant implications for the development of new cell therapies for the diseases of centralnervous system.
Innovation points
1. A novel MRI nano-contrast agent was developed and may contribute to the diagnosisof AD and also help in the evaluation and development of potential novel therapeuticinterventions.
2. Established an economical, simple and repetitive primary culture with high positiverate for CMECs.
3. According to the recent opinion of AD, the drug used for ischemia clinically was discussed for the application of AD and that may shorten the development time for new ADdrugs.
3. In the cell transplantation, the study of the neuronal differentiation rate of NSCs in adamage environment is more significant to the transplantation therapy.
阿尔茨海默病（Alzheimer’s disease，AD）是一种以慢性进行性痴呆为主要表现的神经退行性疾病，是老年性痴呆最常见的原因之一，其发病率随年龄的增高而不断增高，给社会家庭带来了沉重的经济负担。然而，AD在诊断、治疗等方面仍然比较滞后，本课题通过对目前比较热门的一些技术方法和治疗手段的探讨，来寻找未来可能应用于AD的方法和方向。在AD的诊断方面，磁共振成像（magnetic resonanceimaging，MRI）是一种很有前景的检测AD中β淀粉样蛋白（β-amloid，Aβ）沉积的诊断技术，使用磁共振对比剂既能够提供Aβ沉积的特异性成像以区分其他病理生理结构，又能缩短成像时间。而在AD的治疗方面，早期对血管功能的保护可能成为针对发病机理的治疗新靶点，而晚期神经元的丢失也可能通过细胞移植的方法进行功能重建。
目的
1.开发一种使用超小超顺磁性氧化铁（ultrasmall superparamagnetic iron oxide，USPIO）纳米颗粒连接功能性蛋白的磁共振对比剂，从而给检测Aβ沉积提供方法。
2.用于治疗脑缺血的药物丁苯酞，具有内皮保护作用，通过观察其对Aβ损伤下脑微血管内皮细胞（cerebral microvascular endothelial cells，CMECs）的作用，寻找丁苯酞用于AD治疗的证据并进行初步的机制探讨。
3.神经干细胞（neural stem cells，NSCs）是中枢神经系统疾病细胞移植治疗的最终应用形式，但存在定向分化问题。拟通过观察与CMECs共培养后NSCs在正常条件下和损伤条件下的分化情况，寻找一种能够促使移植治疗应用于神经退行性疾病的解决方案。
方法
1.磁共振对比剂由USPIO纳米颗粒连接Aβ16-20（KLVFF）和跨膜蛋白转导结构域（trans-activating transcriptor-protein transduction domain，Tat-PTD）组成，其中Aβ16-20是能特异性与Aβ沉积结合的探针，Tat-PTD能帮助对比剂穿过血脑屏障（brain bloodbarrier，BBB）。体外实验研究此对比剂的生物膜穿透性、生物毒性、与Aβ沉积的特异性结合能力以及对组织磁共振信号的影响；最终将其注射入AD转基因小鼠来进行活体磁共振成像及组织学验证。
2.建立CMECs原代培养方法并鉴定。使用不同浓度丁苯酞预处理CMECs后予以Aβ损伤，观察其活性和凋亡率的变化，并进一步观察其分泌NO及诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）和内皮型一氧化氮合酶（endothelial nitricoxide synthase，eNOS）水平的变化。
3.共培养原代CMECs和NSCs，观察NSCs的生长状况，移除CMECs后将NSCs分别置于正常培养环境和缺糖缺氧（Oxygen-glucose deprivation，OGD）环境中分化以观察分化比例。鼠胚成纤维细胞（mouse embryo fibroblast，MEF）与NSCs共培养作为对照组。
结果
1.Tat-PTD-USPIO-Aβ16-20的蛋白连接效率达97.75%，体内、体外实验均显示其能穿过生物膜，与Aβ沉积特异性结合，并能降低组织的磁共振信号，与未连接的Aβ16-20及Aβ1-40相比没有发现明显的毒性作用。
2.成功建立了乳小鼠CMECs的原代培养方法，最终阳性率可达95.5%。丁苯酞预处理后的Aβ损伤CMECs，细胞活性升高，凋亡率降低，NO分泌增高，iNOS分泌及胞浆表达均增高，而eNOS分泌及胞浆含量未发现有显著变化。
3.与CMECs共培养的NSCs数量更多，直径更大，不论是在正常培养环境下还是在OGD环境下，神经元分化比例均高于对照。
结论
1.本研究设计的磁共振对比剂能够穿过BBB，并能通过磁共振检测到AD动物模型体内的Aβ沉积，是一种很有潜力的AD诊断技术。此外，还有助于开发和评估新的以减少Aβ沉积为目标的AD药物。
2.丁苯酞可能通过提高iNOS的表达和分泌，从而提高NO水平来对Aβ损伤的CMECs产生保护作用，且大剂量下保护作用更明显和稳定，故而有可能通过保护内皮功能而对AD产生治疗作用。
3. CMECs能够促进NSCs的存活和增殖，不仅能促进其在正常培养条件下，也能促进其在损伤条件下更多的向神经元分化。这为研究NSCs在中枢神经系统疾病的细胞治疗中的定向分化问题提供了新证据。
创新点
1.创造性开发出新型纳米磁共振对比剂，既为AD临床体内诊断应用打下基础，也为新药开发研究提供了评估手段。
2.建立了一种经济、简易、重复性好、纯度高的CMECs原代培养方法。
3.通过AD发病机理的新观点，探讨在临床上应用于脑缺血的药物丁苯酞用于AD治疗的可能，为增加新的适应症提供一定的依据。
4.在细胞移植治疗中的神经干细胞定向分化问题上，着重探讨了在损伤环境中的分化状况，更接近真实的移植状况。
Backgrounds
Alzheimer’s disease (AD) is a neurodegenerative disease with the feature of chronicprogressive dementia and is one of the most common causes for senile dementia. Themorbidity increases with age and great financial load is brought to society and families.However, the diagnosis and therapy of AD remain limited. This study is to find the possiblemethod and direction through discussing the intense research in AD. In terms of diagnosis,magnetic resonance imaging (MRI) is a promising technique in detection of Aβ deposits inAD. It has been proved that using the MRI contrast agent can shorten the scanning time andprovide a specific image to identify various structures. In terms of therapy, the protection ofvascular function may be the new target in the early stage of AD and the celltransplantation may be the development direction to repair the neuron loss in the late stageof AD in the future.
Objectives
1. To develop an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticlescoupled with functional protein as a novel MRI contrast agent to detect the Aβ deposits.
2. To find whether s-(-)-3-n-butylphthalide, which can protect the vascule and is usedfor ischemia clinically, could be applied for AD through the observation of the effects onthe Aβ-damaged cerebral microvascular endothelial cells (CMECs).
3. Neural stem cells (NSCs) are the applied form in cell transplantation for diseases ofcentral nervous system. The increase of neuronal differentiation rate of NSCs remains ablock, particular in a damage environment. The study is to see the effects of CMECs on theneurogenesis of NSCs with or without Oxygen-glucose deprivation (OGD).
Methods
1. We developed a novel MRI contrast agent USPIO nanoparticles coupledAβ(16-20)(KLVFF) as a probe for connecting to Aβ deposits and HIV-1trans-activatingtranscriptor-protein transduction domain (Tat-PTD) for the penetration of blood-brainbarrier (BBB). The penetration, toxicity and binding affinity of the contrast agent in vitrohave been studied. The contrast agents were injected into the transgenic mice for livingbrain MRI imaging and histological studies.
2. Establish the method for primary culture of CMECs. After detecting the specificityof CMECs, they were pre-treated with s-(-)-3-n-butylphthalide on different concentrationsand then were damaged by Aβ. The cell viability and apoptosis rates were observed and theconcentrations of NO, inducible nitric oxide synthase (iNOS) and endothelial nitric oxidesynthase (eNOS) were detected.
3. Survival and proliferation of NSCs were studied after CMECs and NSCs wereco-cultured. Removing the CMECs, NSCs were maintained in differentiation culture withor without treatment of OGD to observe the differentiation rates. Mouse embryo fibroblast(MEF) cells co-cultured with NSCs were used as the control groups.
Results
1. The coupling efficiency of protein can reach97.75%for Tat-PTD-USPIO-Aβ(16-20).The developed MRI contrast agents can pass through biological membranes, bind to the Aβdeposits and decrease the MRI signal of the tissue in vitro and in vivo. Compare toAβ(16-20) or Aβ(1-40) alone, no significant toxicity of Tat-PTD and Aβ(16-20) was foundwhen they coupled with USPIO nanoparticles.
2. Successfully establish the method of primary culture of CMECs from newborn mice and the positive rate was95.5%. In the intervention research of s-(-)-3-n-butylphthalide, thecell viability increased and the apoptosis rates decreased. The secretion of NO and iNOSwere increased. There were no significantly changes of the secretion of eNOS in all groups.
3. NSCs co-cultured with CMECs have an increase in size and number compared tothose co-cultured with MEF cells. They also have an increase in neuronal differentiationrates with or without OGD.
Conclusions
1. The novel developed MRI contrast agent by USPIO nanoparticles coupled withfunctional protein can pass through BBB and detect Aβ deposits in AD animal models byMRI in vivo. This novel MRI contrast agent may help in the evaluation and development ofpotential novel therapeutic interventions to reduce amyloid deposits in AD experimentalanimals.
2. The s-(-)-3-n-butylphthalide could protect CMECs from the damage of Aβ throughincreasing the expression and secretion of iNOS, in turn the NO. The effect was morestabilized under the large concentration. Hence, the s-(-)-3-n-butylphthalide may be acandidate for the therapy of AD through the effect on the protection of vascule.
3. This study provides the evidence that OGD cannot alter the effects of CMECs inpromoting the neuronal differentiation potential of NSCs. These findings may haveimportant implications for the development of new cell therapies for the diseases of centralnervous system.
Innovation points
1. A novel MRI nano-contrast agent was developed and may contribute to the diagnosisof AD and also help in the evaluation and development of potential novel therapeuticinterventions.
2. Established an economical, simple and repetitive primary culture with high positiverate for CMECs.
3. According to the recent opinion of AD, the drug used for ischemia clinically was discussed for the application of AD and that may shorten the development time for new ADdrugs.
3. In the cell transplantation, the study of the neuronal differentiation rate of NSCs in adamage environment is more significant to the transplantation therapy.
引文
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