【作者】 刁欢；

【导师】 王枫； 季爱玲；

【作者基本信息】 第四军医大学 ， 营养与食品卫生学， 2010， 硕士

【摘要】 帕金森病(Parkinson’s disease,PD)是一种常见于神经退行性疾病,研究发现,小胶质细胞介导的脑内炎症反应可能是引起黑质多巴胺能神经元变性的决定性因素。小胶质细胞是中枢神经系统的免疫反应细胞,活化状态的小胶质细胞对神经元具有保护作用,但过度活化的小胶质细胞会产生严重的神经毒性作用。多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)在生物系统中具有广泛的功能,大量研究表明,n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acids,n-3 PUFAs)具有抗心血管疾病,抗炎性作用、抗氧化作用以及抗细胞凋亡等多种生理功能。近年来一些研究认为,n-3 PUFAs在保护中枢神经系统中起着重要作用,这可能和n-3 PUFAs抗炎作用有关。EPA和DHA能抑制脂多糖等诱导的NF-κB以及炎性因子的表达,并与减少其抑制亚基I-κB的磷酸化有关。脂多糖(lipopolysaccharide,LPS)是革兰阴性杆菌细胞内毒素的有效成分,是一种强烈的致炎因子,能够诱导机体的炎症反应。本实验以SD成年雄性大鼠为研究对象,采用脑立体定位注射LPS的方法建立帕金森病动物模型,并在此基础上进一步探讨炎症反应诱导的小胶质细胞激活在帕金森病的发病过程中的作用及其信号转导途径以及PUFAs对帕金森病的保护作用。实验方法1.采用脑立体定位注射LPS方法,注射不同浓度LPS(2.5μg、5μg)14 d后,经腹腔注射阿朴吗啡观察大鼠行为学改变,判断动物模型是否成功,通过免疫荧光染色检测多巴胺神经元损伤和小胶质细胞的活化状况;2.通过预先注射PDTC,采用免疫荧光染色观察小胶质细胞OX-42和NF-κB p65的活化,Western blot检测NF-κB p65蛋白水平的表达;3.设立NS对照组、LPS组、30%鱼油组和60%鱼油组,通过阿朴吗啡诱导行为学变化,免疫荧光染色检测TH阳性细胞、OX-42的活化,Western blot检测TH和NF-κB p65蛋白水平的表达。实验结果1.脑立体定位术向大鼠脑黑质内注射LPS 14 d后,经腹腔注射阿朴吗啡,对照组NS无明显行为异常,两组LPS组大鼠向注射侧旋转圈数明显大于NS组(P<0.05)。免疫荧光染色检测黑质酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞数和小胶质细胞OX-42活化情况的结果显示,同NS对照组相比,LPS 2.5μg组和5μg组TH阳性细胞明显减少,其中以5μg组TH阳性细胞数减少最为显著;LPS 2.5μg组可见部分小胶质细胞表现为细胞胞体变大,突起变粗,而LPS 5μg组OX-42阳性小胶质细胞数明显增多,染色深,胞体增大变圆,突起变短,变粗。2.LPS注射14 d后,经腹腔注射APO后,PDTC处理组大鼠旋转圈数明显小于LPS组,免疫荧光检测结果显示,PDTC组有部分小胶质细胞表现为细胞胞体变大,突起变粗;与PDTC组相比,LPS组OX-42阳性小胶质细胞数增多,染色深,胞体增大变圆,突起变短,变粗。Western blot检测结果提示,同NS组相比,PDTC组NF-κB p65蛋白表达无显著差异,LPS组NF-κB p65蛋白表达显著增加(P<0.05)。3.NS组通过APO诱导没有出现明显的行为学变化,LPS组在诱导后出现向注射侧旋转行为,并且伴有较为明显的前肢抬举性震颤,而30%鱼油组和60%鱼油组通过APO诱导后,两组大鼠旋转圈数明显少于LPS组(P<0.05)。免疫荧光染色检测各组黑质TH结果显示,与NS对照组相比,30%鱼油组和60%鱼油组TH阳性细胞数的差异不显著;而LPS组TH阳性细胞数明显减少(P<0.05)。免疫荧光染色检测小胶质细胞的活化程度,两组鱼油组OX-42阳性小胶质细胞数较少;LPS组OX-42阳性小胶质细胞数增多。Western blot检测TH蛋白的表达水平:两组鱼油组同NS组相比无显著差异,LPS组TH蛋白的表达水平则显著降低(P<0.05);NF-κB p65蛋白的表达水平,两组鱼油组与NS组相比无显著差异,LPS组的NF-κB p65蛋白表达明显增高(P<0.05)。结论1.黑质内注射LPS可以激活小胶质细胞和对多巴胺神经元产生损伤作用。2.LPS可以使NF-κB p65表达明显增高;PDTC则能抑制NF-κB p65的表达。3.膳食添加鱼油能够对LPS诱导的多巴胺能神经元的损伤产生保护作用,其机制可能与抑制小胶质细胞激活并降低NF-κB表达有关。更多还原

【Abstract】 Parkinson’s disease (PD) is a common nerve degenerative disease. It has been reported that microglia mediated inflammatory reaction in brain play a key role in causeing degeneration of substantia nigra dopaminergic neuron. Microglia is the immunoreaction cell of central nervous system, and activated microglia has protective effect on neuron, but the overavtivity microglia may cause serious neurotoxicity.polyunsaturated fatty acids (PUFAs) have extensive functions in biosystem. Many studies have shown that n-3 PUFAs had many kinds of effects, such as the protection of angiocardiopathy, anti-inflammation, antioxidation and the inhibitory effect on apoptosis apoptosis. Recent studies considered that n-3 PUFASs had important effect on the protection of central nervous system, which may be associated with anti-inflammatory effect of n-3 PUFAs. EPA and DHA can decrease I-κB phosphorylation, which contributes to the inhibition of the expression of NF-κB and other inflammatory cytokines induced by LPS. LPS is effective constituent in Gram-negative bacteria cell toxin. It is a strong proinflammatory factor, and can induce inflammatory reaction of organism.SD adult male rats were used to establish the Parkinson’s disease animal model of LPS by stereotaxic surgery in the present study. The effect of microglia cell-activating induced by inflammatory reaction and its role in the development of PD were investigated. We further observed the protective effect of PUFASs on PD.Methods1. Brain stereotactic surgery injection. Fourteen days after injection of different concentrations of LPS(2.5μg、5μg), apomorphine was injected through intraperitoneal, and behavioral changes of the rats were observed to estimate the successful animal model. The density of the dopaminergic neurons and the activation of microglial cells were detected by immunofluorescence fluorescein stain.2. With pre-injection of PDTC, the activation of microglia OX-42 and NF-κB p65 were observed by immunofluorescence fluorescein stain. The protein expression of NF-κB p65 was detected by Western blot.3. The animals were divided into NS control group , LPS control group, 30% fish oils group, and 60% fish oils group. in each group, ethology variation were measured after apomorphine injection. The number of TH positive cells and the activation of microglia OX-42 were detected by immunofluorescence fluorescein stain. The protein expression of TH and NF-κB p65 were detected by Western blot. Results1. Fourteen days after LPS injection by brain stereotactic surgery in rats brain substantia nigra, apomorphine was administrated through intraperitoneal injection. NS control group has no obvious abnormal behavior, the rotation turns to the injection side of two LPS groups was significantly more than NS group(P<0.05). With immunofluorescence detection, the number of TH positive cells of LPS 2.5μg group and 5μg group were significantly decreased compare to the NS group, the most significant reduction was in LPS 5μg group. A few microglia cells body in LPS 2.5μg group become lager, and apophysis become coarsen, but in LPS 5μg group, the number of activated microglial cells obviously increased, cell body become lager and more stainable, and apophysis become coarsen.2. 14 d after LPS injection, through intraperitoneal injection APO, rotation turns of PDTC treatment group were significantly less than LPS group, immunofluorescence detection results show that a few microglia cell body become lager, and apophysis become coarsen in PDTC group. Compare with the PDTC group, number of activated microglial cells in LPS group was significantly increases, cell body become lager and more stainable, and apophysis become coarsen. Western blot detection results shown that, the protein expression of NF-κB p65 was no significant difference between NS group and PDTC group. The protein expression of NF-κB p65 was significantly increased in LPS group(P<0.05).3. NS group has no obvious behavior changes after APO injection, LPS group rotated to the injection side, and accompanied by obvious forelimb trembler. The rotation turns of 30% fish oil group and 60% fish oil group were obvious less than the LPS group (P<0.05) after APO induction. Immunofluorescence detection TH results show that,compared with NS group, the number of TH positive cells in 30% fish oil group and 60% fish oil group were no significantly difference. The number of TH positive cells in LPS group was decreased(P<0.05). With immunofluorescence fluorescein stain detection activation of microglia, we found that the number of activated microglial cells in two fish oil groups, the number of activated microglial cells in LPS group was significantly increased compare to the NS group. Western blot detection of TH proteins level shown that the protein expression of TH was no obvious difference in two fish oils groups. But the protein expression of TH was significantly reduced in LPS group(P<0.05)compare to the NS group; The protein expression of NF-κB p65 in two fish oil groups were no obvious difference. The protein expression of NF-κB p65 in the LPS group was significantly higher than NS group(P<0.05).Conclusions1. Injection LPS in substantia nigra activated microglia and induced dopaminergic neuron injury.2. LPS increased the expression of NF-κB p65 , and PDTC can inhibit the expression of NF-κB p65.3. Dietary Supplement fish oil had protective effect on DA neuron injury induced by LPS, which may be associated with the inhibition of microglia activation and reduction of the expression of NF-κB.更多还原