摘要
帕金森病(Parkinson’s Disease,PD)是中枢神经系统常见的一种变性疾病,主要临床特点为:静止性震颤、肌强直、姿势异常和运动减少。该病是影响中老年人生活质量的主要原因之一。中脑黑质多巴胺(DA)能神经元的进行性变性、死亡是PD的主要病理改变。但是到目前为止,PD的发病机制尚不明确。
选择性的多巴胺神经元的损害,是PD研究的重要方向。目前的氧化应激学说、兴奋性氨基酸学说、钙的细胞毒作用学说以及A9区多巴胺神经元特异蛋白的表达,均从不同角度解释了上述现象。但是,针对上述机制采用抗氧化、抗兴奋性氨基酸等措施后,并不能显著缓解PD的发病和A9区的病程进展。因此,目前认为,现有学说不能完全解释PD中选择性的多巴胺神经元损害,仍有其他不明因素参与了PD的发病过程,并在疾病的进展中起重要作用。而与特异性损害密切相关的免疫系统,在近年来受到日益增多的重视。在免疫和PD发病机制关系的研究中,细胞免疫和体液免疫是最受关注的领域。
细胞免疫方面:已有的研究发现,T细胞亚群的比例在PD患者中有改变,PD患者的外周血中CD4+T淋巴细胞/CD8+T淋巴细胞比值降低,其中CD8+T淋巴细胞数量增加。进一步研究发现CD4+CD45RA+幼稚性T细胞的比例下降,而CD4+CD45RO+记忆性T细胞显著增高,且γδ+T细胞显著增高。目前已有多项研究结果提示,T细胞重要表面抗原CD4和CD8可能参与了PD发病,但尚无直接证据。
体液免疫方面:在众多的研究中,PD患者血清中是否存在针对黑质的自身抗体是备受争议的,且长期以来该课题得以高度关注。然而,到目前为止尚无明确结论。虽然如此,多数研究者仍然确信有一种自身抗体可能参与了黑质多巴胺神经元的损伤过程。鉴定该抗体及抗原,对PD发病机制的研究具有重要意义,且可能为PD的治疗提供新思路。
基于以上关于PD的细胞免疫和体液免疫研究现状,我们在以往研究的基础上,观察了在不同剂量γ射线全身照射下,外周血淋巴细胞减少后对PD的发病情况的影响;然后利用分别敲除CD4和CD8的小鼠PD模型,初步分析了敲除CD4和CD8对PD严重程度的影响;同时在实验中利用PD小鼠的血清和临床病例的血清,进行了初步的黑质靶抗原的分析。本研究分为三部分进行:
第一部分:实验中使用不同剂量γ射线(0Gy、0.5Gy、2.0Gy和3.5Gy)全身照射对小鼠进行预处理,之后使用MPTP腹腔注射建立PD小鼠模型。用高效液相检测小鼠纹状体多巴胺和其代谢产物的水平、免疫组化检测黑质神经元数量和胶质细胞活化,同时检测了射线照射后外周血淋巴细胞数量改变。
结果提示:2.0Gy和3.5Gyγ射线进行全身预照射后,外周血淋巴细胞数量迅速而明显的下降;而0.5Gyγ射线进行全身预照射后,在观测的7天内,外周血淋巴细胞数量总体评价较无照射对照组稍增高。高效液相提示,0.5Gy的γ射线进行全身预照射后,纹状体多巴胺含量较对照组降低,而2.0Gy的γ射线进行全身预照射后,纹状体多巴胺含量较对照组增高,3.5Gy的的γ射线进行全身预照射后,纹状体多巴胺含量与对照组相比无明显差异。
我们认为:1、不同的低剂量γ射线全身预照射可以对MPTP致C57BL/6小鼠多巴胺神经元损伤的严重程度有不同影响,并非都可以提供神经保护作用。我们认为在本实验中发现的2.0Gyγ射线进行全身预照射的神经保护作用,可能是因为外周血中淋巴细胞的大量减少导致的;与之相反,0.5Gyγ射线进行全身预照射后,外周血中淋巴细胞轻度增多,导致MPTP注射后的纹状体多巴胺含量进一步降低。2、以往关于“射线的神经保护”的研究中认为,辐射的毒物兴奋效应(radiation hormesis)可能是神经保护作用产生的机制,但是在本研究中提示,该机制未明显参与神经保护。
第二部分:实验中分别利用CD4敲基因小鼠、CD8敲基因小鼠和野生型小鼠,在接受MPTP腹腔注射后,观察各组小鼠在MPTP处理后PD严重程度的改变。衡量严重程度的指标使用:纹状体多巴胺和其代谢产物的水平、免疫组化检测黑质神经元数量、Western-Blot检测的纹状体TH蛋白水平。同时对比观察了不同组小鼠黑质胶质细胞活化情况。
结果提示:C57BL/6小鼠在MPTP腹腔注射后10天,HPLC-EC法检测提示纹状体DA,DOPAC和HVA均明显下降;免疫组化发现黑质DA能神经元数量减少,小胶质细胞和星形胶质细胞活化;Western-blot提示纹状体TH含量下降。分别敲除CD4+/CD8+ T细胞后,两组敲基因动物均出现DA能神经元损伤程度较野生型小鼠减轻的现象。但是在胶质细胞活化方面却有不同的表现:与WT相比,CD4-Ko组小胶质细胞活化稍增强,而CD8-Ko组活化明显增强;CD4-Ko组星形胶质细胞活化程度比WT组轻,CD8-Ko组星形胶质细胞活化程度比WT组明显增强。
因此敲除CD4+或CD8+ T细胞,可减轻帕金森病的多巴胺神经元损伤。我们认为敲除CD4+或CD8+ T细胞后,可能导致小胶质细胞的活化加重,从而减轻了多巴胺神经元的损伤。此外,我们还发现,CD4-Ko组造模后10天星形胶质细胞活化程度比WT组轻,而CD8-Ko组星形胶质细胞在造模组和生理盐水对照组均明显活化,程度比WT组重。因此,星形胶质细胞活化程度可能参与影响多巴胺神经元的损伤程度。但是上述两种胶质细胞影响DA能损伤程度的机制本研究中未进一步涉及。
第三部分:实验中通过免疫组化,ELISA,Western-Blot的方法,初步分析了PD动物模型和PD患者血清中是否存在针对黑质产生自身抗体的情况,并尝试使用二维电泳的方法筛选黑质蛋白中自身抗体特异性靶抗原。
研究中我们发现:免疫组化中患PD病小鼠的自体血清可使脑冰冻切片的神经细胞弥漫性着色,与健康血清相比,似乎在黑质区有一定阳性细胞出现;ELISA提示患PD病小鼠的自体血清与黑质部分蛋白匀浆的结合性较正常小鼠增强。Western-blot中提示有可疑的35kDa的蛋白条带和患病小鼠的自体血清结合。
本研究结果提示在C57BL/6小鼠帕金森病模型中,外周血中可能存在和黑质神经细胞有结合性的蛋白,这种蛋白也可能是一种针对黑质神经元的抗体。从以上的实验结果中进一步严格实验条件,将有助于阐明自身抗体在PD发病中的作用。
从上述实验结果中我们得到结论如下:
1、低剂量射线的全身预照射,可以轻度改变MPTP对C57BL/6小鼠黑质DA能神经元的毒性强度。该神经保护作用的机制可能是外周血淋巴细胞数量、放射损伤以及辐射的毒物兴奋效应的综合结果;其中,外周血淋巴细胞数量与MPTP对C57BL/6小鼠黑质DA能神经元的毒性强度相关,可能是影响毒性过程的重要因素;辐射的毒物兴奋效应在MPTP损害C57BL/6小鼠黑质DA能神经元过程中,作用非常微弱。
2、CD4和CD8可能参与了PD的发病机制。CD4或CD8分子的敲除,可以减轻MPTP导致的亚急性PD的严重程度,但并不能明显阻止多巴胺神经元数量的减少;其机制可能通过改变胶质细胞的功能,达到影响PD发病以及严重程度的作用。
3、黑质神经元自身抗体的存在可能参与了PD的发病,黑质中35kDa蛋白可能是自身抗体的靶抗原。
Parkinson's disease (PD) is a disabling neurodegenerative disorder which was known by uncontrollable resting tremor, rigidity, gait imbalance and slowness of movement. It is one of the common diseases which damaged the life quality of older persons. Parkinson's disease is characterized by a progressive degeneration of dopamine (DA) neurons projecting from the substantia nigra pars compacta (SNpc) to striatal motor loci. But till now, the pathogenesis of Parkinson's disease is still unclear.
Selective injury to dopamine neurons was focused on in the past years. Nowadays, many factors were thought taking part in this selective injury such as oxidative stress, excitatory amino acids, calcium overloading and specific protein expressed on domanine neurons in area nine(A9). However the selective injury to A9 could not be diminished obviously by removing those factors. So there must be some other factors affectting the selective injury. And an immunity cause for PD has been discussed for years.
Immune abnormalities have been described in PD during the past 30 years. Disturbed cellular and humoral immune functions have been reported in patients with PD.
In cellular immunity: Changes of lymphocyte subpopulations in cerebrospinal fluid and peripheral blood has been known for many years in Parkinson's disease. There was an decrease of the CD3+CD4+/CD3+CD8+ ratio. Researchers thought it was the increase of CD3+CD8+ cell that leaded the decrease of the CD3+CD4+/CD3+CD8+ ratio. In further studies, T helper cell analysis revealed a decreased percentage of CD45RA+ "naive" and an increased percentage of CD45RO+ "memory" T cell subset from CD4+ T cells in peripheral blood of patients with Parkinson's disease. An elevated gamma-delta(+) T cell population was also found in PD. Although there were lots of reports on cellular immunity, the role of CD4 and CD8 was still unclear.
In humoral immunity: The studies on the serum autoantibody against substantia nigra neuron were always debatable. Though there were some negative results, researchers still believed the serum from PD patients containing some kinds of autoantibody, which could react with the substantia nigra neuron and damage the dopaminergic neurons. Identification of the autoantibody and antigen could provide a new way to postpone the development of PD.
Based on the former research on cellular and humoral immune functions in PD, we prepared for our study. Firstly we investigated the injury of dopamine neurons induced by MPTP after being pretreated by different Low dose irradiation, and analysed the relationship between peripheral blood lymphocyte and injury of dopamine neurons. Then the role of CD4 and CD8 in the PD development was investigated by using CD4 knock-out mice and CD8 knock-out mice. At last, we tried to identify the target antigen by using the serum from PD mice and PD patients. So this study contains three parts as following.
Part 1: After pretreated with a single low dose(0.5Gy, 2.0Gy or 3.5Gy) total-bodyγ-irradiation (TBI), C57BL/6 mice were administered with MPTP(15 mg/kg, four times, 2 h apart) intraperitoneally (i.p.). The numbers of blood lymphocytes were counted with haemacytometer manually before TBI and on the first day, third day and sixth day after TBI respectively.Then the animals were sacrificed by cervical dislocation on the seventh day. The brains were rapidly removed. Then the striatum and midbrain were dissected and processed for High Performance Liquid Chromatography (HPLC) and immunohistochemistry with antibodies against tyrosine hydroxylase (TH), CD11b or GFAP to show dopamine neuron, microglia and astrocyte respectively.
As a result, in the group pretreated with 2.0Gy TBI, neuroprotection was found by HPLC determination of the striatal dopamine, with lower lymphocyte number. Contrarily, in the group pretreated with 0.5Gy TBI, with higher lymphocyte number, dopaminergic neuron toxicity was enhanced. So it was probably the decrease of lymphocyte, not the radiation hormesis, which rendered the potential neuroprotection. And it was the balance between radiation injury and lymphocytopenia neuroprotection that decided the effect of low doseγ-irradiation on MPTP induced dopaminergic neurotoxicity.
Part 2: The dopaminergic neurotoxicity induced by MPTP was compared among widetype, CD4 knockout (CD4-Ko) and CD8 knockout (CD8-Ko) C57 mice. MPTP were freshly prepared in saline and administered in mice intraperitoneally (i.p. 30mg/Kg.d×5d). The striatum were dissected and processed for HPLC and TH Western-Blotting. The midbrain were processed for immunohistochemistry with antibodies against tyrosine hydroxylase (TH), CD11b or GFAP to show dopamine neuron, microglia and astrocyte respectively.
As a result, there was no evidently difference in DA and its metabolism level detected by HPLC among control saline groups of widetype group, CD4 knockout group and CD8 knockout group (P>0.05). DA and its metabolism decreased obviously on the 10th days after MPTP administration in each group. DA in striatal tissues was higher in CD4 knockout group and CD8 knockout group than that of WT group (P<0.05). Astrocyte and microglia were activated in all groups after MPTP injection. Microglia was more active in CD4 knockout mice slightly and CD8 knockout mice notably than that of WT mice. Astrocyte was more active in CD8 knockout mice, yet less active in CD4 knockout mice than that of WT mice.
We found the knockout of CD4 or CD8 could provide neuroprotection in MPTP induced PD. But the neuroprotection was slightly and could not affect the number of dopaminergic neurons obviously. And the neuroprotection was maybe related to the activatation of the astrocyte and microglia which could change the level of cytokine and neurotrophic factors .
Part 3: The studies on the serum autoantibody against substantia nigra neuron was proceeded in our experiments with immunohistochemistry, enzyme-linked immunosorbentassay (ELISA) and Western-Blotting. Two-dimensional gel electrophoresis (2-DE) was carried out to screen the antigen from substantia nigra.
Autoserum from mice of all groups could react with nerve cells in brain slice widespreadly. But the autoserum from PD mice could react with nerve cell of substantia nigra more actively than autoserum from normal mice. Similar results were found in ELISA and Western-Blotting. And a 35kDa protein from substantia nigra was found reacting with autoserum. It suggested that the serum from PD mice induced by MPTP could react with nerve cell of substantia nigra. And a 35kDa protein from substantia nigra was probably the target.
In conclusion:
1. Low dose TBI could change MPTP induced dopaminergic neuron toxicity slightly. It was probably the balance among radiation injury, neuroprotection of lymphocytopenia and radiation hormesis that decided the effect of low dose TBI on MPTP induced dopaminergic neuron toxicity. The number of lymphocyte in peripheral blood, which was probably the most important role, was related to dopaminergic neuron injury induced by MPTP. And the effect of radiation hormesis was quite weak in the dopaminergic neuron injury induced by MPTP.
2. CD4 and CD8 may take part in the pathogenesis of PD. The knockout of CD4 or CD8 could provide slight neuroprotection in MPTP induced dopaminergic neurons toxicity without affecting the number of dopaminergic neurons obviously. The neuroprotection was maybe related to the activatation of the astrocyte and microglia which could change the level of cytokine and neurotrophic factors .
3. There is probably some kind of autoantibody against nerve cell of substantia nigra, which may take part in the pathogenesis of PD. A 35kDa protein in substantia nigra is probably the target of autoantibody.
引文
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