优化人α-突触核蛋白核酸疫苗免疫MPTP帕金森病小鼠的神经保护作用及其机制研究
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摘要
研究背景
帕金森病(Parkinson’s disease,PD)是第二个最常见的神经系统变性疾病, 65岁以上人口中的患病率超过1%以上。关注占全国人口10%的60岁以上老年人的健康是个不容忽视的问题。
帕金森病的主要病理性标志物是黑质区出现异常聚集的α-突触核蛋白(alpha-synuclein,α-syn)为主要成分的Lewy小体。α-syn异常聚集参与了PD多巴胺能神经元的变性过程。在家族性和散发性PD的发病机制中,α-syn的异常聚集起到了重要的作用。帕金森病目前尚无根本治疗方法,针对α-syn的疫苗是治疗PD的新方法。近年来,核酸疫苗在退行性病变的防治中显示出诱人的前景。我们课题组前期实验成功地构建了人α-syn(hα-syn)核酸疫苗,并在哺乳动物体内表达,观察到hα-syn核酸疫苗治疗MPTP慢性帕金森病模型小鼠取得了较好的疗效。为了提高核酸疫苗的免疫效果,并对核酸疫苗的神经保护作用机制进行探讨,我们欲对该疫苗作进一步的优化,以期为帕金森病的临床防治提供实验基础和理论依据。
α-syn由大量无序的异构体组成,呈现为非紧密的混合体。自然状态下,α-syn是一种富含疏水区域的天然伸展的蛋白质。其氨基端(1~60)包含四个不完全重复序列,易形成两性螺旋。NAC区(61~95)为中心区,是α-syn序列中疏水性最强的一段。羧基端(96~140)富含酸性氨基酸,如脯氨酸,带有大量负电荷,与正常状态下α-syn保持无规则卷曲状态有关。a-syn蛋白中的这三种结构域是否具有不同的免疫效价?对疫苗的神经免疫防护效果是否有不同的影响?因此对α-syn蛋白结构和功能的了解,会对核酸疫苗的优化构建,以及疫苗的免疫效果造成重要的影响。
我们初期研究中,尽管发现核酸疫苗免疫治疗MPTP慢性帕金森病模型小鼠取得了一定的效果。但是,由于α-syn分子量仅14kD,只有140个氨基酸残基组成的小分子蛋白质。因α-syn分子量小,免疫活性弱,需要较大剂量才能诱导足够的免疫效应。Ig Kappa链信号肽(Ig ksp)可使细胞内hα-syn向细胞外移动,增加hα-syn的分泌能力,必将会有助于抗体效价的提高,促使更多的异常聚集的α-syn蛋白被降解和清除。因此,在hα-syn基础之上增加小鼠Ig ksp基因序列,构建高效分泌型hα-syn真核表达载体,增强免疫治疗效果,提升疫苗的安全性,成为了我们深入研究的重点内容。
异常聚集的α-syn可以通过泛素蛋白酶体系统和溶酶体途径降解和清除。在PD发病中,α-syn的降解通路受损可引起α-syn的异常聚集,形成Lewy小体。若使用蛋白酶体抑制剂抑制酶的活性,可产生剂量依赖性选择性多巴胺(Dopamine,DA)神经元死亡。在清除α-syn的积聚和降解过程中,溶酶体也发挥了重要作用。对于α-syn寡聚体介导的毒性,溶酶体降解通路是一个重要的保护机制。Masliah等发现,使用重组hα-syn接种hα-syn转基因小鼠,产生的抗体可通过溶酶体途径清除α-syn,减少α-syn在DA能神经元胞体和突触的聚集。优化构建的hα-syn核酸疫苗是否也可通过增加异常聚集的α-syn降解清除来发挥神经保护作用?抗原与抗体结合后,是否必定要通过溶酶体或/和蛋白酶体途径代谢分解?它们的分解代谢特点是什么?需要作进一步的研究。
免疫炎症反应与PD的发病机理有着密切的关系,帕金森病可以说是一个动态的炎症变化过程。小胶质细胞(microglia,MI)被激活,大量增殖是中枢神经系统疾病炎症反应的一个最重要表现。黑质内MI被激活,特别是吞噬了异常聚集的α-syn之后可被持续激活,引起DA能神经元变性,导致帕金森病发生。MI被激活、增殖,可产生炎症细胞因子,如α-肿瘤坏死因子、活性氧簇、白细胞介素-1β、白细胞介素-6等。炎症细胞因子反过来又可加剧MI的激活,共同参与脑局部炎症过程。因此在PD的病理生理过程中,炎症发挥了重要作用。优化核酸疫苗对神经细胞起到保护作用,那么核酸疫苗对PD病理过程中的炎症反应会产生哪些影响?在优化核酸疫苗免疫治疗过程中,对MI活性和炎症反应的调节情况如何?对这些问题的深入了解,将有助于我们对核酸疫苗的治疗作用和神经保护机制的理解。
研究目的
根据α-syn结构中折叠的特点,构建三种高分泌表达型hα-syn蛋白的优化核酸疫苗;通过接种MPTP慢性PD模型小鼠,观察核酸疫苗的防治效果和神经保护作用,并对其作用机制进行探讨。本实验的主要内容包括:①将扩增出的人α-syn(1-140)基因、α-syn(84-140)基因、α-syn(34-140)基因与Ig ksp基因克隆到质粒pVAX1上,优化构建成三种重组质粒。在哺乳动物细胞中表达并鉴定其生物学活性;②观察三组疫苗诱导体液免疫的效果、不同特点和免疫特异性;③大量制备三种优化核酸疫苗接种PD小鼠模型,观察三种疫苗的神经保护作用以及对溶酶体功能的影响,炎症反应的影响和调控等;④将微量溶酶体抑制剂与蛋白酶体抑制剂,通过脑立体定向技术注射中脑黑质区以阻断α-syn的降解通路,观察溶酶体抑制剂不同剂量、不同时间段对DA神经元功能的影响和小鼠行为学改变特点;⑤溶酶体和蛋白酶体通路阻滞对核酸疫苗免疫保护作用的影响,运用免疫组化等技术,了解hα-syn核酸疫苗诱导特异性神经保护性免疫所产生的抗体,降解清除过表达α-syn,阻抑PD的病理进程的机制。
方法
1.三种重组质粒的优化构建和表达
从重组质粒中扩增人α-syn1-140、84-140、34-140基因,根据已知序列合成Ig ksp基因;将上述融合基因序列克隆到质粒pVAX1上,构建成三组重组质粒:A组(pVAX1-Igk-hαS1-140)重组质粒;B组pVAX1-Igk-hαS84-140)重组质粒;C组pVAX1-Igk-hαS34-140)重组质粒。进行限制性内切酶酶切分析和DNA测序鉴定。转化大肠杆菌DH5α,抽提质粒后转染COS-7细胞,用Western Blot法检测其生物学活性。
2.重组质粒对体液免疫的诱导
分别用A、B、C三种优化核酸疫苗、空质粒pVAX1免疫正常的C57/BL小鼠。采用股四头肌肉注射,100μg/只/次,每三周加强免疫一次,共免疫5次。在每次免疫后二周各采血一次,分离血清用于抗体测定。并用免疫后的小鼠血清和能表达α-syn的小鼠脑组织行免疫组化反应及中和反应,观察免疫血清抗体的特异性。
3.观察优化核酸疫苗免疫防治的神经保护作用
正常C57/BL小鼠注射MPTP 25mg/kg·d,每周2次,共10次,建立MPTP慢性PD小鼠动物模型。建模成功后分别接种三种优化核酸疫苗。观察免疫治疗小鼠模型行为学改变特点,DA细胞酪氨酸羟化酶(TH)和α-syn蛋白表达水平;并且检测免疫保护作用后的炎性反应,以及了解对溶酶体组织蛋白酶D (Cathepsin D,Cath D)活性的影响。
4.观察溶酶体和蛋白酶体抑制剂对正常小鼠DA神经元作用特点于小鼠右侧中脑黑质(SN)区,分别立体定向微量注射不同剂量溶酶体抑制剂磷酸氯喹溶剂,或者蛋白酶体抑制剂。观察阿朴吗啡(apomorphine,APO)诱导的小鼠旋转行为改变以及行为学变化;于溶酶体抑制剂注射后不同时间段,检测小鼠黑质区TH阳性细胞数、α-syn和Cath D表达水平。
5.探讨溶酶体和蛋白酶体通道阻滞后,核酸疫苗对α-syn降解和清除的影响
同上方法建立慢性模型成功后接种优化核酸疫苗pVAX1-Igk-hαS1-140,共3次。于第三次接种的前三天,小鼠右侧SN区,分别立体定向微量注射溶酶体抑制剂或蛋白酶体抑制剂。检测通道阻滞后,小鼠SN区TH和细胞核形态学的变化,了解对细胞凋亡的影响。
结果
1.重组质粒pVAX1-Igk-hαS1-140、pVAX1-Igk-hαS84-140、pVAX1-Igk-hαS34-140的成功优化构建和表达
成功扩增了hα-syn1-140、84-140、34-140基因,并合成了Ig ksp基因。分别将两者的融合基因克隆到表达载体pVAX1上,经酶切分析和DNA测序证明三组重组质粒的大小、方向和序列完全正确,成功构建了三种重组质粒。经Western blot检测具有较好的生物学活性。
2.重组质粒诱导的体液免疫
大量制备A、B、C三种重组质粒和空质粒pVAX1。重组质粒免疫动物后产生了较高的抗体滴度,pVAX1-Igk-hαS1-140为(3.76±0.33)×103、pVAX1-Igk-hαS84-140为(3.87±0.12)×103、pVAX1-Igk-hαS34-140为(3.79±.022)×103,三组之间差异没有显著性意义(p>0.05),与空质粒pVAX1(0.09±0.01)×103组相比,差异有显著意义(p<0.05)。重组质粒免疫小鼠的血清能与α-syn过表达小鼠的脑组织发生特异性免疫组化反应,被α-syn蛋白中和的血清则不发生反应。
3.核酸疫苗免疫保护作用的观察
与空质粒pVAX1组比较,三种优化核酸疫苗均可以提高小鼠的爬杆能力,提高TH细胞数目达60-65%,减少α-syn表达量44-47%,增加Cath D表达量46-50%,差异均有显著性(P<0.05),但三组疫苗之间上述检测指标差异没有显著性(P>0.O5)。三组核酸疫苗与PBS对照组相比,TNF-α表达量减少27-55%(P<0.05)。B组、C组与A组相比,差异亦有有显著性(p<0.05),显示B组、C组安全性更高。
4.溶酶体和蛋白酶体抑制剂对正常小鼠多巴胺神经元的作用特点
①溶酶体抑制剂只有在400μmol/L浓度时才可出现较明显的由药物诱发的旋转行为,7-8次/分。而蛋白酶体抑制剂组小鼠清醒后即出现明显的旋转行为,甚至不需要药物就可自发出现旋转症状;②毁损侧与黑质区内TH阳性细胞数目正常相比有明显的不同程度减少。脑立体定向注射后的第三周内可以观察到,随着溶酶体剂量25μmol/L~400μmol/L增加,DA神经元的损害程度呈正比例上升。从溶酶体100μmol/L立体定向注射一周到四周可以观察到,多巴胺的数量随时间逐渐恢复。③毁损侧与正常黑质区比较,Cath D阳性细胞数目存在不同程度的明显减少。注射溶酶体25μmol/L~400μmol/L的第三周内,Cath D阳性数量随剂量的增加而明显减少,同时TH损害程度也增加。而且Cath D阳性细胞数与TH阳性细胞数成正比,Cath D阳性表达量与TH损害程度呈正比例关系。
5.α-syn降解通道阻滞后,核酸疫苗的作用特点
当α-syn降解通道阻滞后,与未注射侧相比,溶酶体抑制剂损害较轻,蛋白酶体抑制剂损害略重,溶酶体加蛋白酶体抑制剂相对损害较重,但是在注射局部仍然有TH阳性数目的表达。TH与Hoechst荧光染色显示,抑制剂组小鼠脑黑质部位有大量阳性细胞核固缩呈不规则形,核碎裂,部分裂解为多个凋亡小体。各组小鼠中脑黑质核形态中可以看出,与未注射侧的核形态,PBS组与它没有明显区别。与未注射侧相比,溶酶体抑制剂损害较轻,蛋白酶体抑制剂损害略重,溶酶体加蛋白酶体抑制剂相对损害较重。
结论
1.成功优化构建了三组真核表达质粒并具有较好的生物学活性。
2.三组核酸疫苗免疫小鼠均产生了较高的抗体滴度,并且具有抗α-syn特异性。
3.三组核酸疫苗免疫均可减少慢性PD小鼠模型脑内异常聚积的α-syn表达,改善溶酶体的功能,阻扰MI激活所致前炎症因子的释放,从而减轻神经毒素对神经细胞的损害作用。并且pVAX1-Igk-hαS84-140与pVAX1-Igk-hαS34-140核酸疫苗可能具有更安全的免疫效果。
4.溶酶体和蛋白酶体抑制剂对正常小鼠DA神经元有损害作用,但两种抑制剂的损害具有不同的作用特点。溶酶体抑制剂对DA神经元的损害具有剂量和时间依赖性。DA神经元损害程度与Cath D的活性呈正相关。单侧溶酶体抑制剂损伤小鼠,注射APO可以诱导出旋转行为,但是没有蛋白酶体明显。
5.核酸疫苗可能通过其它的降解清除途径,具有一定的拮抗溶酶抑制剂和蛋白酶体抑制剂对多巴胺神经元损害的作用,可减少细胞凋亡,对神经元起到保护作用。
Backgound
Parkinson's disease (PD) is the second most common neurodegenerative disease, which affects over 65 years old among more than 1% of the population. It’s can’t be ignored which concerned about the health of the elder of more than 60 years making up about 10% of total population.
The major pathological markers of PD is the abnormal accumulation ofα-synuclein (α-syn) in substantia nigra(SN) as the main component of Lewy bodies. Abnormalα-syn aggregation in PD involved in dopaminergic (DA) neuron degeneration process, and abnormal accumulation ofα-syn in familial and sporadic PD pathogenesis has played an important role. So far a fundamental treatment hasn’t been found for the PD. In recent years, the DNA vaccine in the prevention and treatment of degenerative diseases show attractive prospects. Our research’s pre-experiment successfully constructed a humanα-syn DNA vaccine which experimented on mammals, and obverted thatα-syn DNA vaccine can obtain a better effect on chronic MPTP mouse PD model. Therefore, further optimization of vaccine to improve performance, as well as the protection mechanisms of neurons will be a focus on research.
In the natural state, a-syn conformation consists of a large number of disordered non-closed to the isomer mixture, is riches in hydrophobic region of natural extension protein: N-terminal (1~60), it contains four incomplete repeats, which are easy to form spiral amphoteric alone. NAC area (61~95), which is a sequence of a-syn in the strongest of hydrophobic section; Carboxy-terminal (96~140), which is rich in acidic amino acids such as proline, with large number of negative charge, and maintain a-syn to a random coil in the normal state. So do these three structures have different immune potency? Do they have an impact on the different immunization? It remains our exploration and study.
At the early stage of our research, in spite of hα-syn DNA vaccine immunotherapy to chronic MPTP mouse PD model has achieved successfully. However,α-syn is the molecular weight of 14kD, containing 140 amino acid residues which composed of small molecule protein, because ofα-syn covers small molecular weight, as well as immunological activity of the weak, all those made it needs a larger quantity inducing a sufficient immune response. Ig Kappa chain signal peptide (Ig ksp) can increased the movement from the intracellular to the extracellular cell of hα-syn, and increased secretion capacity, it will contribute to the improvement of antibody titer to degrade and remove more abnormalα-syn protein. Therefore, add mouse Ig ksp gene sequence based on hα-syn, building a highly efficient secretion of hα-syn-based eukaryotic expression vector, and enhancing the effect of immunotherapy, increasing the safety of vaccines, those is the key research project.
The study clearly shows that the abnormalα-syn aggregation can be degraded by ubiquitin-proteasome system, as well as lysosomal degradations.The block ofα-syn pathway of degradation may lead to an abnormalα-syn aggregation to result in the formation of Lewy bodies. The application of protease inhibitors which inhibit proteasome activity can produce dose-dependent selective death of DA neurons. Lysosome also plays a key role in the process ofα-syn degradation. The Lysosomal pathway is a major protective mechanism againstα-syn oligomer-mediated toxicity, and lysosomal degradation pathway could remove the accumulation ofα-syn. Masliah found that the recombinant hα-syn immunized the hα-syn transgenic mice produced the antibody againstα-syn that can be removed by means of lysosomal pathway, will decrease the aggregation ofα-syn in DA neuron cell body and synapse. Does the optimization of hα-syn DNA vaccine can play a neuroprotective effect by increasing the degradation of abnormal accumulation ofα-syn? The combination of antigen and antibody bound to catabolism through the lysosomal pathway and UPS? It also needs a further study.
The Immune inflammatory response links closely to the pathogenesis of PD. So PD can be considered to be a dynamic process of imflammation change. The activation and proliferation of MI is the most important embodiments of central nervous system (CNS) response. The activation of MI in SN can be sustained by the abnormalα-syn, which would contribute to the degeneration of DA neurons and finally lead to the occurrence of PD. The activatation and proliferation of MI can induce inflammatory cytokines, such asα-tumor necrosis factor (TNF-α), reactive oxygen species, IL-1β, IL-6, etc. Conversely, the inflammatory cytokines can exacerbate the activation of MI, which involve in the process of brain inflammation. For this reason, the inflammation acts a main role in the pathophysiology of PD process. If the DNA vaccine palys a role in the protection of neurons, what is the impact imposed by the inflammatory characterisctis in PD pathology? We will get a better understanding of the mechanism of vaccine if we have the clarification of the process of the activated MI and the regulation of inflammatory response.
Research purposes
According to the folding feature of a-syn, we construct the three groups of the optimized high secreted human alpha-synuclein DNA vaccine and immunize the chronic MPTP mouse PD model, to observe the neurological effects of immune protection, and explore the role of mechanisms. The main contents including:①Amplified humanα-syn 1-140、84-140、34-140 gene with the Ig ksp gene is cloned into the plasmid pVAX1, get the three groups of the recombinant plasmid,then expression in mammalian cells and identification of its biological activity;②Observe the effectiveness of immunization,the specificity and different characteristics of immunization;③Immunize the mouse model of PD, observe the neuroprotective effect ,lysosomal function, the effects of inflammatory response and regulation after immunization in the process of PD;④Observe the effect of Proteasome inhibitors and lysosome inhibitors on DA neurons in normal mice by stereotactic brain injection into SN in order to blockα-syn degradation pathway, observe the different doses and time of lysosomal inhibitors for the characteristics of behavior,TH,Cath D on the DA neurons ;⑤Observe the lysosome and the proteasome on the role of the hα-syn DNA vaccine-induced protection of neuron-specific antibodies generated by immunization, the characteristic of degrading and removing the overexpression ofα-syn or the characteristic of apoptosis.
Methods
1. The construction and expression of recombinant plasmid. Amplify the gene ofα-syn 1-140、84-140、34-140 from the recombinant plasmid in human and synthesize the sequence Ig ksp,then the two fusion gene will be cloned into plasmid pVAX1 to construct the three groups of recombinant plasmid namely, A group is pVAX1-Igk-hαS1-140; B group is pVAX1-Igk-hαS84-140; C group is pVAX1-Igk-hαS34-140.Restriction endonuclease digestion analysis and DNA sequencing.Transfer the recombinant plasmid into E.coliDH5α, extract the plasmid and transfecte COS-7 cells to assay its biological activity using Western blot.
2. The activity of recombinant plasmid induced humoral immune We use the recombinant plasmid A, B, C of optimized DNA vaccines immunize the healthy C57/BL mice in the musculus quadriceps, every time 100μg/100ul, every three weeks to strengthen the immunity, totally five times. Take those mice blood before the first of immunization and two weeks after immunization; separate the serum for antibody determination. Immune mouse blood serum and the expression of mouseα-syn brain tissue neutralization reaction are used in order to observe immune serum’s specificity.
3. Observe the neuroprotective effect optimizated DNA vaccine. C57BL mouse is injected MPTP twice every week, totally 10 times in order to the establishment of MPTP chronic mouse PD model; immunize three groups of vaccines. Observate the number of DA TH cell and the expression levels ofα-syn, the inflammatory reaction as well as the lysosome cathepsin D.
4. Observation the characteristics of lysosome and proteasome inhibitors on DA neurons in health mice stereotactic injection into the right SN with different doses of lysosome inhibitor chloroquine phosphate(CQP), as well as the proteasome inhibitor MG-132.Observate the apomorphine(APO)-induced rotational behavior change; detecte the number of SN TH positive cells , the levels ofα-syn expression and cath D changes in different time periods after injection of lysosome inhibitors or proteasome inhibitors.
5. Explore the vaccine’s influence to the degradation and elimination of a-syn after the block of the lysosome and the proteasome pathway C57BL mouse is injected with MPTP, 25mg/kg?d,twice every week, totally 10 times. Immunize the the chronic MPTP PD mouse with the vaccine of pVAX1-Igk-hαS1-140.Three days before the third immunization, stereotactic micro-injection into the right SN with 100μmol/L lysosome inhibitors and proteasome inhibitor. Detecte the SN TH, cell morphological changes and the impact on apoptosis
Results
1. Construction and expression of recombinant plasmidsΑ-syn 1-140, 84-140, 34-140 genes are amplified successfully, and Ig ksp gene is synthesized and the fused gene of the two is cloned into the pVAX1. The size, direction and sequence were completely correct with endonuclease digestion analysis and DNA sequencing.The recombinant plasmids are constructed successfully, and expressed effectively in mammalian cells COS-7with better biological activity.
2. The induction of humoral immunity by recombinant plasmids
Three recombinant plasmids and empty plasmid pVAX1 are preparated largescally. A higher antibody titer was produced in animals, pVAX1-Igk-hαS1-140:(3.76±0.33)×103,pVAX1-Igk-hαS84-140:(3.87±0.12)×103,pVAX1-Igk-hαS34-140:(3.79±0.22)×103. There was no obvious differences between them (p>0.05), while compared to empty plasmid (p<0.05). The minimized serum can generate immunohistochemical reaction with mouse brain tissue over-expressedα-syn.
3. Observe the protective effect of immunity by DNA vaccine Compare with empty plasmid, three vaccines elevate the ability of crawling pole, elevate TH-positive cells to 60-65%,diminute a-syn to 44-47%, and augmente Cath D 46-50%(p>0.05).Compared with PBS,the TNF-a decrease 27-55% (P<0.05). B、C vaccine show the higher safety.
4. observe the characteristics of lysosome, proteasome inhibitors effect on DA neurons in healthy mice .(1) there have rotational behaviors 1 or 2 times occasionally below the 200μmol / L of lysosome inhibitor.More pronounced rotary behavior was shown in only 400μmol / L group. There is significant difference between proteasome inhibitor and 400μmol / L lysosome inhibitor group. Rotational behavior is shown in proteasome inhibitor group after the analepsia even needn’t APO, while lysosome inhibition group needed it. (2)An obvious variablely reduction in the number of TH-positive neurons in the damaged lateral SN is indicated with TH immunohistochemistry. In the third week after stereotactic injection, the degree of the damaged TH is aggratated according to the dose from the lysosome 25μmol/L to 400μmol/L.The gradual return of the amount of dopamine is discovered after stereotactic injection 100μmol/L from one week to forth week. (3) Contrast with the normal brain tissue in the SN of the results of cathepsin D, the damaged side of the SN region of cath D-positive cells exists in varying decreased degrees significantly. The number of the positive expression of cathepsin D is significantly reduced with the accumulating dose in the Lysosomes from the third week of 25μmol/L to 400μmol/L dose of stereotactic group, contrast with the number of damaged TH, we found that cathepsin D expression with the TH-positive of damage is directly proportional.
5 Explore the characteristics of a-syn on channel blockers after DNA vaccine
No significant difference is found between PBS group and others in the number of TH-expression neurons in the damaged lateral SN. To compare with normal lateral, the degree of damage is lighter in the lysosome inhibitor group, heavier in the proteasome inhibitor group, heaviest in the lysosomal and proteasome inhibitor group but still local expression of the number of TH-positive is observed in the injection. TH and Hoechst staining showe that part of a large number of positive nuclei in SN were irregular shrinkage, nuclear fragmentation, some cracking to multiple apoptotic bodies. Mice in each group in the SN can be seen in the nuclear morphology, and non-injection side of the nuclear morphology, PBS group were not significantly different. And non-injected side compared with less damage to the lysosome inhibitor, proteasome inhibitor slightly heavy damage, and lysosomal proteasome inhibitor increases the relative heavier harm.
Conclusion
1. The three eukaryotic expression plasmids pVAX1-Igk-hαS1-140, pVAX1-Igk-hαS84-140, pVAX1-Igk-hαS34-140 were successfully constructed and showed better biological activity.
2. Optimized DNA vaccines produced higher antibody titers and the specificity ofα-syn.
3. Optimized DNA vaccine were clear abnormal brain accumulation ofα-syn in the chronic mouse model of PD, and reduced the over-expression ofα-syn. PVAX1-Igk-hαS84-140 fusion plasmid showed better immune effect than the other two groups.
4. Lysosome inhibitor can induce rotational behaviors, but less than proteasome. Lysosome inhibitor and proteasome inhibitor on DA neuronal damage depend on the dose and time. The positively correlated between the damage levels of DA neurons and the expression of cath D.
5. Optimized DNA vaccines have a good neuroprotective effect on DA neurons, and apoptosis damaged by the lysosome inhibitors and proteasome inhibitor.
帕金森病(Parkinson’s disease,PD)是第二个最常见的神经系统变性疾病, 65岁以上人口中的患病率超过1%以上。关注占全国人口10%的60岁以上老年人的健康是个不容忽视的问题。
帕金森病的主要病理性标志物是黑质区出现异常聚集的α-突触核蛋白(alpha-synuclein,α-syn)为主要成分的Lewy小体。α-syn异常聚集参与了PD多巴胺能神经元的变性过程。在家族性和散发性PD的发病机制中,α-syn的异常聚集起到了重要的作用。帕金森病目前尚无根本治疗方法,针对α-syn的疫苗是治疗PD的新方法。近年来,核酸疫苗在退行性病变的防治中显示出诱人的前景。我们课题组前期实验成功地构建了人α-syn(hα-syn)核酸疫苗,并在哺乳动物体内表达,观察到hα-syn核酸疫苗治疗MPTP慢性帕金森病模型小鼠取得了较好的疗效。为了提高核酸疫苗的免疫效果,并对核酸疫苗的神经保护作用机制进行探讨,我们欲对该疫苗作进一步的优化,以期为帕金森病的临床防治提供实验基础和理论依据。
α-syn由大量无序的异构体组成,呈现为非紧密的混合体。自然状态下,α-syn是一种富含疏水区域的天然伸展的蛋白质。其氨基端(1~60)包含四个不完全重复序列,易形成两性螺旋。NAC区(61~95)为中心区,是α-syn序列中疏水性最强的一段。羧基端(96~140)富含酸性氨基酸,如脯氨酸,带有大量负电荷,与正常状态下α-syn保持无规则卷曲状态有关。a-syn蛋白中的这三种结构域是否具有不同的免疫效价?对疫苗的神经免疫防护效果是否有不同的影响?因此对α-syn蛋白结构和功能的了解,会对核酸疫苗的优化构建,以及疫苗的免疫效果造成重要的影响。
我们初期研究中,尽管发现核酸疫苗免疫治疗MPTP慢性帕金森病模型小鼠取得了一定的效果。但是,由于α-syn分子量仅14kD,只有140个氨基酸残基组成的小分子蛋白质。因α-syn分子量小,免疫活性弱,需要较大剂量才能诱导足够的免疫效应。Ig Kappa链信号肽(Ig ksp)可使细胞内hα-syn向细胞外移动,增加hα-syn的分泌能力,必将会有助于抗体效价的提高,促使更多的异常聚集的α-syn蛋白被降解和清除。因此,在hα-syn基础之上增加小鼠Ig ksp基因序列,构建高效分泌型hα-syn真核表达载体,增强免疫治疗效果,提升疫苗的安全性,成为了我们深入研究的重点内容。
异常聚集的α-syn可以通过泛素蛋白酶体系统和溶酶体途径降解和清除。在PD发病中,α-syn的降解通路受损可引起α-syn的异常聚集,形成Lewy小体。若使用蛋白酶体抑制剂抑制酶的活性,可产生剂量依赖性选择性多巴胺(Dopamine,DA)神经元死亡。在清除α-syn的积聚和降解过程中,溶酶体也发挥了重要作用。对于α-syn寡聚体介导的毒性,溶酶体降解通路是一个重要的保护机制。Masliah等发现,使用重组hα-syn接种hα-syn转基因小鼠,产生的抗体可通过溶酶体途径清除α-syn,减少α-syn在DA能神经元胞体和突触的聚集。优化构建的hα-syn核酸疫苗是否也可通过增加异常聚集的α-syn降解清除来发挥神经保护作用?抗原与抗体结合后,是否必定要通过溶酶体或/和蛋白酶体途径代谢分解?它们的分解代谢特点是什么?需要作进一步的研究。
免疫炎症反应与PD的发病机理有着密切的关系,帕金森病可以说是一个动态的炎症变化过程。小胶质细胞(microglia,MI)被激活,大量增殖是中枢神经系统疾病炎症反应的一个最重要表现。黑质内MI被激活,特别是吞噬了异常聚集的α-syn之后可被持续激活,引起DA能神经元变性,导致帕金森病发生。MI被激活、增殖,可产生炎症细胞因子,如α-肿瘤坏死因子、活性氧簇、白细胞介素-1β、白细胞介素-6等。炎症细胞因子反过来又可加剧MI的激活,共同参与脑局部炎症过程。因此在PD的病理生理过程中,炎症发挥了重要作用。优化核酸疫苗对神经细胞起到保护作用,那么核酸疫苗对PD病理过程中的炎症反应会产生哪些影响?在优化核酸疫苗免疫治疗过程中,对MI活性和炎症反应的调节情况如何?对这些问题的深入了解,将有助于我们对核酸疫苗的治疗作用和神经保护机制的理解。
研究目的
根据α-syn结构中折叠的特点,构建三种高分泌表达型hα-syn蛋白的优化核酸疫苗;通过接种MPTP慢性PD模型小鼠,观察核酸疫苗的防治效果和神经保护作用,并对其作用机制进行探讨。本实验的主要内容包括:①将扩增出的人α-syn(1-140)基因、α-syn(84-140)基因、α-syn(34-140)基因与Ig ksp基因克隆到质粒pVAX1上,优化构建成三种重组质粒。在哺乳动物细胞中表达并鉴定其生物学活性;②观察三组疫苗诱导体液免疫的效果、不同特点和免疫特异性;③大量制备三种优化核酸疫苗接种PD小鼠模型,观察三种疫苗的神经保护作用以及对溶酶体功能的影响,炎症反应的影响和调控等;④将微量溶酶体抑制剂与蛋白酶体抑制剂,通过脑立体定向技术注射中脑黑质区以阻断α-syn的降解通路,观察溶酶体抑制剂不同剂量、不同时间段对DA神经元功能的影响和小鼠行为学改变特点;⑤溶酶体和蛋白酶体通路阻滞对核酸疫苗免疫保护作用的影响,运用免疫组化等技术,了解hα-syn核酸疫苗诱导特异性神经保护性免疫所产生的抗体,降解清除过表达α-syn,阻抑PD的病理进程的机制。
方法
1.三种重组质粒的优化构建和表达
从重组质粒中扩增人α-syn1-140、84-140、34-140基因,根据已知序列合成Ig ksp基因;将上述融合基因序列克隆到质粒pVAX1上,构建成三组重组质粒:A组(pVAX1-Igk-hαS1-140)重组质粒;B组pVAX1-Igk-hαS84-140)重组质粒;C组pVAX1-Igk-hαS34-140)重组质粒。进行限制性内切酶酶切分析和DNA测序鉴定。转化大肠杆菌DH5α,抽提质粒后转染COS-7细胞,用Western Blot法检测其生物学活性。
2.重组质粒对体液免疫的诱导
分别用A、B、C三种优化核酸疫苗、空质粒pVAX1免疫正常的C57/BL小鼠。采用股四头肌肉注射,100μg/只/次,每三周加强免疫一次,共免疫5次。在每次免疫后二周各采血一次,分离血清用于抗体测定。并用免疫后的小鼠血清和能表达α-syn的小鼠脑组织行免疫组化反应及中和反应,观察免疫血清抗体的特异性。
3.观察优化核酸疫苗免疫防治的神经保护作用
正常C57/BL小鼠注射MPTP 25mg/kg·d,每周2次,共10次,建立MPTP慢性PD小鼠动物模型。建模成功后分别接种三种优化核酸疫苗。观察免疫治疗小鼠模型行为学改变特点,DA细胞酪氨酸羟化酶(TH)和α-syn蛋白表达水平;并且检测免疫保护作用后的炎性反应,以及了解对溶酶体组织蛋白酶D (Cathepsin D,Cath D)活性的影响。
4.观察溶酶体和蛋白酶体抑制剂对正常小鼠DA神经元作用特点于小鼠右侧中脑黑质(SN)区,分别立体定向微量注射不同剂量溶酶体抑制剂磷酸氯喹溶剂,或者蛋白酶体抑制剂。观察阿朴吗啡(apomorphine,APO)诱导的小鼠旋转行为改变以及行为学变化;于溶酶体抑制剂注射后不同时间段,检测小鼠黑质区TH阳性细胞数、α-syn和Cath D表达水平。
5.探讨溶酶体和蛋白酶体通道阻滞后,核酸疫苗对α-syn降解和清除的影响
同上方法建立慢性模型成功后接种优化核酸疫苗pVAX1-Igk-hαS1-140,共3次。于第三次接种的前三天,小鼠右侧SN区,分别立体定向微量注射溶酶体抑制剂或蛋白酶体抑制剂。检测通道阻滞后,小鼠SN区TH和细胞核形态学的变化,了解对细胞凋亡的影响。
结果
1.重组质粒pVAX1-Igk-hαS1-140、pVAX1-Igk-hαS84-140、pVAX1-Igk-hαS34-140的成功优化构建和表达
成功扩增了hα-syn1-140、84-140、34-140基因,并合成了Ig ksp基因。分别将两者的融合基因克隆到表达载体pVAX1上,经酶切分析和DNA测序证明三组重组质粒的大小、方向和序列完全正确,成功构建了三种重组质粒。经Western blot检测具有较好的生物学活性。
2.重组质粒诱导的体液免疫
大量制备A、B、C三种重组质粒和空质粒pVAX1。重组质粒免疫动物后产生了较高的抗体滴度,pVAX1-Igk-hαS1-140为(3.76±0.33)×103、pVAX1-Igk-hαS84-140为(3.87±0.12)×103、pVAX1-Igk-hαS34-140为(3.79±.022)×103,三组之间差异没有显著性意义(p>0.05),与空质粒pVAX1(0.09±0.01)×103组相比,差异有显著意义(p<0.05)。重组质粒免疫小鼠的血清能与α-syn过表达小鼠的脑组织发生特异性免疫组化反应,被α-syn蛋白中和的血清则不发生反应。
3.核酸疫苗免疫保护作用的观察
与空质粒pVAX1组比较,三种优化核酸疫苗均可以提高小鼠的爬杆能力,提高TH细胞数目达60-65%,减少α-syn表达量44-47%,增加Cath D表达量46-50%,差异均有显著性(P<0.05),但三组疫苗之间上述检测指标差异没有显著性(P>0.O5)。三组核酸疫苗与PBS对照组相比,TNF-α表达量减少27-55%(P<0.05)。B组、C组与A组相比,差异亦有有显著性(p<0.05),显示B组、C组安全性更高。
4.溶酶体和蛋白酶体抑制剂对正常小鼠多巴胺神经元的作用特点
①溶酶体抑制剂只有在400μmol/L浓度时才可出现较明显的由药物诱发的旋转行为,7-8次/分。而蛋白酶体抑制剂组小鼠清醒后即出现明显的旋转行为,甚至不需要药物就可自发出现旋转症状;②毁损侧与黑质区内TH阳性细胞数目正常相比有明显的不同程度减少。脑立体定向注射后的第三周内可以观察到,随着溶酶体剂量25μmol/L~400μmol/L增加,DA神经元的损害程度呈正比例上升。从溶酶体100μmol/L立体定向注射一周到四周可以观察到,多巴胺的数量随时间逐渐恢复。③毁损侧与正常黑质区比较,Cath D阳性细胞数目存在不同程度的明显减少。注射溶酶体25μmol/L~400μmol/L的第三周内,Cath D阳性数量随剂量的增加而明显减少,同时TH损害程度也增加。而且Cath D阳性细胞数与TH阳性细胞数成正比,Cath D阳性表达量与TH损害程度呈正比例关系。
5.α-syn降解通道阻滞后,核酸疫苗的作用特点
当α-syn降解通道阻滞后,与未注射侧相比,溶酶体抑制剂损害较轻,蛋白酶体抑制剂损害略重,溶酶体加蛋白酶体抑制剂相对损害较重,但是在注射局部仍然有TH阳性数目的表达。TH与Hoechst荧光染色显示,抑制剂组小鼠脑黑质部位有大量阳性细胞核固缩呈不规则形,核碎裂,部分裂解为多个凋亡小体。各组小鼠中脑黑质核形态中可以看出,与未注射侧的核形态,PBS组与它没有明显区别。与未注射侧相比,溶酶体抑制剂损害较轻,蛋白酶体抑制剂损害略重,溶酶体加蛋白酶体抑制剂相对损害较重。
结论
1.成功优化构建了三组真核表达质粒并具有较好的生物学活性。
2.三组核酸疫苗免疫小鼠均产生了较高的抗体滴度,并且具有抗α-syn特异性。
3.三组核酸疫苗免疫均可减少慢性PD小鼠模型脑内异常聚积的α-syn表达,改善溶酶体的功能,阻扰MI激活所致前炎症因子的释放,从而减轻神经毒素对神经细胞的损害作用。并且pVAX1-Igk-hαS84-140与pVAX1-Igk-hαS34-140核酸疫苗可能具有更安全的免疫效果。
4.溶酶体和蛋白酶体抑制剂对正常小鼠DA神经元有损害作用,但两种抑制剂的损害具有不同的作用特点。溶酶体抑制剂对DA神经元的损害具有剂量和时间依赖性。DA神经元损害程度与Cath D的活性呈正相关。单侧溶酶体抑制剂损伤小鼠,注射APO可以诱导出旋转行为,但是没有蛋白酶体明显。
5.核酸疫苗可能通过其它的降解清除途径,具有一定的拮抗溶酶抑制剂和蛋白酶体抑制剂对多巴胺神经元损害的作用,可减少细胞凋亡,对神经元起到保护作用。
Backgound
Parkinson's disease (PD) is the second most common neurodegenerative disease, which affects over 65 years old among more than 1% of the population. It’s can’t be ignored which concerned about the health of the elder of more than 60 years making up about 10% of total population.
The major pathological markers of PD is the abnormal accumulation ofα-synuclein (α-syn) in substantia nigra(SN) as the main component of Lewy bodies. Abnormalα-syn aggregation in PD involved in dopaminergic (DA) neuron degeneration process, and abnormal accumulation ofα-syn in familial and sporadic PD pathogenesis has played an important role. So far a fundamental treatment hasn’t been found for the PD. In recent years, the DNA vaccine in the prevention and treatment of degenerative diseases show attractive prospects. Our research’s pre-experiment successfully constructed a humanα-syn DNA vaccine which experimented on mammals, and obverted thatα-syn DNA vaccine can obtain a better effect on chronic MPTP mouse PD model. Therefore, further optimization of vaccine to improve performance, as well as the protection mechanisms of neurons will be a focus on research.
In the natural state, a-syn conformation consists of a large number of disordered non-closed to the isomer mixture, is riches in hydrophobic region of natural extension protein: N-terminal (1~60), it contains four incomplete repeats, which are easy to form spiral amphoteric alone. NAC area (61~95), which is a sequence of a-syn in the strongest of hydrophobic section; Carboxy-terminal (96~140), which is rich in acidic amino acids such as proline, with large number of negative charge, and maintain a-syn to a random coil in the normal state. So do these three structures have different immune potency? Do they have an impact on the different immunization? It remains our exploration and study.
At the early stage of our research, in spite of hα-syn DNA vaccine immunotherapy to chronic MPTP mouse PD model has achieved successfully. However,α-syn is the molecular weight of 14kD, containing 140 amino acid residues which composed of small molecule protein, because ofα-syn covers small molecular weight, as well as immunological activity of the weak, all those made it needs a larger quantity inducing a sufficient immune response. Ig Kappa chain signal peptide (Ig ksp) can increased the movement from the intracellular to the extracellular cell of hα-syn, and increased secretion capacity, it will contribute to the improvement of antibody titer to degrade and remove more abnormalα-syn protein. Therefore, add mouse Ig ksp gene sequence based on hα-syn, building a highly efficient secretion of hα-syn-based eukaryotic expression vector, and enhancing the effect of immunotherapy, increasing the safety of vaccines, those is the key research project.
The study clearly shows that the abnormalα-syn aggregation can be degraded by ubiquitin-proteasome system, as well as lysosomal degradations.The block ofα-syn pathway of degradation may lead to an abnormalα-syn aggregation to result in the formation of Lewy bodies. The application of protease inhibitors which inhibit proteasome activity can produce dose-dependent selective death of DA neurons. Lysosome also plays a key role in the process ofα-syn degradation. The Lysosomal pathway is a major protective mechanism againstα-syn oligomer-mediated toxicity, and lysosomal degradation pathway could remove the accumulation ofα-syn. Masliah found that the recombinant hα-syn immunized the hα-syn transgenic mice produced the antibody againstα-syn that can be removed by means of lysosomal pathway, will decrease the aggregation ofα-syn in DA neuron cell body and synapse. Does the optimization of hα-syn DNA vaccine can play a neuroprotective effect by increasing the degradation of abnormal accumulation ofα-syn? The combination of antigen and antibody bound to catabolism through the lysosomal pathway and UPS? It also needs a further study.
The Immune inflammatory response links closely to the pathogenesis of PD. So PD can be considered to be a dynamic process of imflammation change. The activation and proliferation of MI is the most important embodiments of central nervous system (CNS) response. The activation of MI in SN can be sustained by the abnormalα-syn, which would contribute to the degeneration of DA neurons and finally lead to the occurrence of PD. The activatation and proliferation of MI can induce inflammatory cytokines, such asα-tumor necrosis factor (TNF-α), reactive oxygen species, IL-1β, IL-6, etc. Conversely, the inflammatory cytokines can exacerbate the activation of MI, which involve in the process of brain inflammation. For this reason, the inflammation acts a main role in the pathophysiology of PD process. If the DNA vaccine palys a role in the protection of neurons, what is the impact imposed by the inflammatory characterisctis in PD pathology? We will get a better understanding of the mechanism of vaccine if we have the clarification of the process of the activated MI and the regulation of inflammatory response.
Research purposes
According to the folding feature of a-syn, we construct the three groups of the optimized high secreted human alpha-synuclein DNA vaccine and immunize the chronic MPTP mouse PD model, to observe the neurological effects of immune protection, and explore the role of mechanisms. The main contents including:①Amplified humanα-syn 1-140、84-140、34-140 gene with the Ig ksp gene is cloned into the plasmid pVAX1, get the three groups of the recombinant plasmid,then expression in mammalian cells and identification of its biological activity;②Observe the effectiveness of immunization,the specificity and different characteristics of immunization;③Immunize the mouse model of PD, observe the neuroprotective effect ,lysosomal function, the effects of inflammatory response and regulation after immunization in the process of PD;④Observe the effect of Proteasome inhibitors and lysosome inhibitors on DA neurons in normal mice by stereotactic brain injection into SN in order to blockα-syn degradation pathway, observe the different doses and time of lysosomal inhibitors for the characteristics of behavior,TH,Cath D on the DA neurons ;⑤Observe the lysosome and the proteasome on the role of the hα-syn DNA vaccine-induced protection of neuron-specific antibodies generated by immunization, the characteristic of degrading and removing the overexpression ofα-syn or the characteristic of apoptosis.
Methods
1. The construction and expression of recombinant plasmid. Amplify the gene ofα-syn 1-140、84-140、34-140 from the recombinant plasmid in human and synthesize the sequence Ig ksp,then the two fusion gene will be cloned into plasmid pVAX1 to construct the three groups of recombinant plasmid namely, A group is pVAX1-Igk-hαS1-140; B group is pVAX1-Igk-hαS84-140; C group is pVAX1-Igk-hαS34-140.Restriction endonuclease digestion analysis and DNA sequencing.Transfer the recombinant plasmid into E.coliDH5α, extract the plasmid and transfecte COS-7 cells to assay its biological activity using Western blot.
2. The activity of recombinant plasmid induced humoral immune We use the recombinant plasmid A, B, C of optimized DNA vaccines immunize the healthy C57/BL mice in the musculus quadriceps, every time 100μg/100ul, every three weeks to strengthen the immunity, totally five times. Take those mice blood before the first of immunization and two weeks after immunization; separate the serum for antibody determination. Immune mouse blood serum and the expression of mouseα-syn brain tissue neutralization reaction are used in order to observe immune serum’s specificity.
3. Observe the neuroprotective effect optimizated DNA vaccine. C57BL mouse is injected MPTP twice every week, totally 10 times in order to the establishment of MPTP chronic mouse PD model; immunize three groups of vaccines. Observate the number of DA TH cell and the expression levels ofα-syn, the inflammatory reaction as well as the lysosome cathepsin D.
4. Observation the characteristics of lysosome and proteasome inhibitors on DA neurons in health mice stereotactic injection into the right SN with different doses of lysosome inhibitor chloroquine phosphate(CQP), as well as the proteasome inhibitor MG-132.Observate the apomorphine(APO)-induced rotational behavior change; detecte the number of SN TH positive cells , the levels ofα-syn expression and cath D changes in different time periods after injection of lysosome inhibitors or proteasome inhibitors.
5. Explore the vaccine’s influence to the degradation and elimination of a-syn after the block of the lysosome and the proteasome pathway C57BL mouse is injected with MPTP, 25mg/kg?d,twice every week, totally 10 times. Immunize the the chronic MPTP PD mouse with the vaccine of pVAX1-Igk-hαS1-140.Three days before the third immunization, stereotactic micro-injection into the right SN with 100μmol/L lysosome inhibitors and proteasome inhibitor. Detecte the SN TH, cell morphological changes and the impact on apoptosis
Results
1. Construction and expression of recombinant plasmidsΑ-syn 1-140, 84-140, 34-140 genes are amplified successfully, and Ig ksp gene is synthesized and the fused gene of the two is cloned into the pVAX1. The size, direction and sequence were completely correct with endonuclease digestion analysis and DNA sequencing.The recombinant plasmids are constructed successfully, and expressed effectively in mammalian cells COS-7with better biological activity.
2. The induction of humoral immunity by recombinant plasmids
Three recombinant plasmids and empty plasmid pVAX1 are preparated largescally. A higher antibody titer was produced in animals, pVAX1-Igk-hαS1-140:(3.76±0.33)×103,pVAX1-Igk-hαS84-140:(3.87±0.12)×103,pVAX1-Igk-hαS34-140:(3.79±0.22)×103. There was no obvious differences between them (p>0.05), while compared to empty plasmid (p<0.05). The minimized serum can generate immunohistochemical reaction with mouse brain tissue over-expressedα-syn.
3. Observe the protective effect of immunity by DNA vaccine Compare with empty plasmid, three vaccines elevate the ability of crawling pole, elevate TH-positive cells to 60-65%,diminute a-syn to 44-47%, and augmente Cath D 46-50%(p>0.05).Compared with PBS,the TNF-a decrease 27-55% (P<0.05). B、C vaccine show the higher safety.
4. observe the characteristics of lysosome, proteasome inhibitors effect on DA neurons in healthy mice .(1) there have rotational behaviors 1 or 2 times occasionally below the 200μmol / L of lysosome inhibitor.More pronounced rotary behavior was shown in only 400μmol / L group. There is significant difference between proteasome inhibitor and 400μmol / L lysosome inhibitor group. Rotational behavior is shown in proteasome inhibitor group after the analepsia even needn’t APO, while lysosome inhibition group needed it. (2)An obvious variablely reduction in the number of TH-positive neurons in the damaged lateral SN is indicated with TH immunohistochemistry. In the third week after stereotactic injection, the degree of the damaged TH is aggratated according to the dose from the lysosome 25μmol/L to 400μmol/L.The gradual return of the amount of dopamine is discovered after stereotactic injection 100μmol/L from one week to forth week. (3) Contrast with the normal brain tissue in the SN of the results of cathepsin D, the damaged side of the SN region of cath D-positive cells exists in varying decreased degrees significantly. The number of the positive expression of cathepsin D is significantly reduced with the accumulating dose in the Lysosomes from the third week of 25μmol/L to 400μmol/L dose of stereotactic group, contrast with the number of damaged TH, we found that cathepsin D expression with the TH-positive of damage is directly proportional.
5 Explore the characteristics of a-syn on channel blockers after DNA vaccine
No significant difference is found between PBS group and others in the number of TH-expression neurons in the damaged lateral SN. To compare with normal lateral, the degree of damage is lighter in the lysosome inhibitor group, heavier in the proteasome inhibitor group, heaviest in the lysosomal and proteasome inhibitor group but still local expression of the number of TH-positive is observed in the injection. TH and Hoechst staining showe that part of a large number of positive nuclei in SN were irregular shrinkage, nuclear fragmentation, some cracking to multiple apoptotic bodies. Mice in each group in the SN can be seen in the nuclear morphology, and non-injection side of the nuclear morphology, PBS group were not significantly different. And non-injected side compared with less damage to the lysosome inhibitor, proteasome inhibitor slightly heavy damage, and lysosomal proteasome inhibitor increases the relative heavier harm.
Conclusion
1. The three eukaryotic expression plasmids pVAX1-Igk-hαS1-140, pVAX1-Igk-hαS84-140, pVAX1-Igk-hαS34-140 were successfully constructed and showed better biological activity.
2. Optimized DNA vaccines produced higher antibody titers and the specificity ofα-syn.
3. Optimized DNA vaccine were clear abnormal brain accumulation ofα-syn in the chronic mouse model of PD, and reduced the over-expression ofα-syn. PVAX1-Igk-hαS84-140 fusion plasmid showed better immune effect than the other two groups.
4. Lysosome inhibitor can induce rotational behaviors, but less than proteasome. Lysosome inhibitor and proteasome inhibitor on DA neuronal damage depend on the dose and time. The positively correlated between the damage levels of DA neurons and the expression of cath D.
5. Optimized DNA vaccines have a good neuroprotective effect on DA neurons, and apoptosis damaged by the lysosome inhibitors and proteasome inhibitor.
引文
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