人神经黑色素选择性导致多巴胺能神经细胞进行性变性

Human Neuromelanin Induced Selective and Progressive Dopaminergic Neurodegeneration

目的探讨从中脑黑质致密带(SNpc)提取的人神经黑色素(HNM)对多巴胺(DA)能神经细胞的作用及机制。方法(1)5μg/mLHNM处理中脑神经-胶质细胞混合培养体系10d后,检测DA能神经细胞摄取能力、计数酪氨酸羟化酶(TH)阳性神经细胞数、测量神经突起长度并观察细胞形态变化。(2)对比检测DA能与γ-氨基丁酸(GABA)能神经细胞的摄取能力,并计算此两种神经细胞摄取能力的差异;分别计数DA能神经细胞及全部神经细胞数并计算其差异。(3)检测神经-胶质细胞、纯神经细胞及神经-星形胶质细胞等3种培养体系中DA能神经细胞摄取能力。结果(1)5μg/mLHNM组的DA能神经细胞摄取能力、TH(+)神经细胞数和神经突起长度分别为HNM空白对照组(对照组)的36%、41%和40%,差异均具统计学意义(分别P<0.001,P<0.05,P<0.001);DA能神经细胞形态学发生明显变化。(2)DA能与GABA能神经细胞摄取能力的平均差异为28%(P<0.005);细胞处理第7、10天,两种细胞摄取能力的差异分别为26%和29%(P<0.05,P<0.01);DA能及全部神经细胞数的差异为33%(P<0.01)。(3)在上述3种培养体系中,5μg/mLHNM组DA能神经细胞摄取能力分别降低为对照组的49%、81%和83%,前两者差异具统计学意义(P<0.01)。结论(1)HNM可从功能与形态上损伤DA能神经细胞,且损伤具选择性;(2)小胶质细胞促进了HNM对DA能神经细胞的损伤作用。(3)HNM可能是PD发病的内源性因素之一,为其制造PD新模型提供了一定的理论依据。

Objective To investigate the role and mechanism of human neuromelanin（HNM） extracted from substantia nigra pars compacta（SNpc） on dopamine（DA） neurons. Methods （1） Mixed neuron-glia cultures were treated with 5 μg/mL HNM for 10 d and the effect of HNM on DA neurons was evaluated by detecting the uptake capacity of DA neurons, counting tyrosine hydroxylase（TH）-positive neuronal number, measuring neuronal dendrite length and observing morphological changes. （2） Differece of uptake capacity between DA and γ-aminobutyric acid （GABA） neurons and number between DA and total neurons were compared. （3） Uptake capacities of DA neurons in mixed neuron-glia, neuron-enriched and neuron-astroglia cultures were detected. Results （1）In mixed neuron-glia cultures,5 μg/mL HNM-induced uptake capacity of DA neurons,TH（＋）neuronal number and neuronal dendrite length were 36%,41% and 40% of the control（P〈0.001,P〈0.05,P〈0.001, respectively）.The cell bodies were shrunk and smaller, the cytoplasmic stainings were reduced and the neuronal dendrites were decreased, shorter, thinner,broken and even gone. （2）The difference of DA and GABA uptake elicited by 5 μg/mL HNM for 10 d was 28% （P〈0.005）,and were 26% and 29% after treating the cells for 7 d and 10 d（P〈0.05, P〈0.01）, respectively. The difference of neuronal number between DA and total neurons was 33%（P〈0.01）,which all were statistically significant. （3）DA uptake produced by 5 μg/mL HNM were 49%, 81% and 83% of the control in mixed neuron-glia,neuron-enriched and neuron-astroglia cultures,respectively, and the difference was significant between the first and second group（P〈0.01） and was not significant between the second and third group. Conclusions （1）HNM induced dopaminergic neurodegeneration was functionally and morphologically selective to DA neurons. （2）HNM produced dopaminergic neurotoxicity was potentiated by microglia to some degree. （3）HNM was a pivotal endogenous mechanism for PD and might be a promising model for PD study.