帕金森病IgG诱导多巴胺能神经元损伤机制的初步研究

<title>eliminary study on mechanism underlying toxicity of IgG from Parkinson's disease on mesencephalic dopaminergic cultures

目的采用帕金森病(PD)患者的血清IgG建立PD的细胞模型,探讨其诱导中脑多巴胺(DA)能神经元损伤的机制。方法采用四唑盐(MTT)检测、免疫细胞化学染色和液闪测定法评价DA能细胞数目、形态和功能的变化,并检测一些毒性物质的作用,肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β):用酶联免疫吸附试验(ELISA法);NO用Griess法;O2-用细胞色素C还原法结果PD IgG在补体参与时,可诱导原代中脑神经元-胶质细胞培养体系的DA能神经元出现选择性损伤(DA摄取力由1012.3 cpm下降至670.5 cpm,P<0.01)。疾病对照组和健康对照组的IgG即使在补体存在时对DA能细胞的数目、形态和功能也无明显影响(P>0.05)。原代培养如抑制胶质细胞,则PD IgG即使在补体存在时对DA能细胞也无明显的毒性作用(P>0.05)。来源于毒性物质的检测、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)、抗TNF-α、抗IL-1β、L-硝基-精氨酸甲酯的数据表明,PD IgG诱导的DA能神经元的损伤受O2-(4.65 nmol／106细胞)的介导。结论PD IgG可诱导原代培养的中脑DA能神经元的选择性损伤,受补体、胶质细胞和活性氧自由基的介导,提示PD的发病可能涉及免疫机制。

<abstract>jective To develop an in vitro model of Parkinson's disease (PD) with PD serum IgG and investigate the mechanism by which PD IgG (60 μg/ml) injures mesencephalic dopaminergic neurons. Methods IgGs was purified from serum samples of patients with idiopathic Parkinson's disease and other neurological diseases ( DC) , and healthy people ( NC) by using ferric ammonium sulfate precipitation, ion exchange chromatography and filtration dialysis. Rat mesencephalic cells were exposed to IgGs in presence or absence of complement. Cell viability was measured by MTT assay. The number and morphology of cells were determined by tyrosine hydroxylase (TH) and p-tubulin immunocytochemistry. Cell bioactivity was detected by [3H] DA and [3H] GABA uptake analyses. Also, the amount of some key cytotoxic factors was examined, including TNF-α and IL-1β ( ELISA), NO ( Griess method) , and superoxide ( cytochrome c method) . Results The number, morphology and bioactivity of rat embryo mesencephalic neuron-glia cells were not altered following exposure to PD IgG or complement respectively (P > 0. 05). After the exposure to PD IgG together with complement, the number of TH ( + ) neurons and activity of DA uptake were significantly decreased from 1012. 3 cpm to 670. 5 cpm (P <0. 01) , while total cell viability was not markedly altered ( P > 0. 05 ) . Morphologically, the neurites of TH ( + ) neurons became fewer and shorter. In contrast, the number of p-tubulin( + ) neurons and activity of GABA uptake did not change obviously ( P > 0. 05 ). For primary mesencephalic neuron-enriched cultures, the exposure to PD IgG did not result in significant changes in the number, morphology and activity of TH( + ) neurons even in the presence of complement (P >0. 05). Also, no alternations in the number, morphology and activity of DA neurons were found following the exposure of primary mesencephalic neuron-glia to the IgGs from DC and NC, even in the presence of complement ( P > 0. 05 ). Finally, in sharp contrast to the lack of co-treatment of PD IgG and complement in stimulating the production of TNF-α, IL-1β and NO, PD IgG significantly stimulated the release of superoxide (4. 65 nmol/106 cell) in the presence of complementas compared with the control (P <0.005). CAT (50 U/ml) /SOD (50 U/ml) significantly reduced PD IgG-induced injury of TH( + ) neurons (P <0. 01). However, pretreatment of neuron-glia cultures with neutralizing antibodies specific for rat TNF-α (50 μg/ml) or for IL-1β ( 10 μg/ml) , or 1 mmol/L NG-nitro-L-arginine methyl ester did not afford any significant neuroprotection (P > 0. 01). Conclusions PD IgG might cause a relatively selective injury of mesencephalic dopaminergic neurons, which was mediated by complement, glia and superoxide. This suggests that immunity might be involved in the pathogenesis of Parkinson's disease.