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目的观察钟样受体4(TLR-4)在胚胎期脂多糖(LPS)暴露引起的出生后个体脑内多巴胺(DA)能神经元减少中的作用。方法 TLR-4突变型C57BL/10ScNCr小鼠和野生型C57BL/10ScSn小鼠各15只,在妊娠期第10.5天给小鼠腹腔注射LPS或肽聚糖(PDG),出生后4月龄时收集大脑组织标本(n=5),通过免疫组织化学染色和体视学技术定量DA能神经元和小胶质细胞数量,高效液相色谱法测定DA及其代谢物水平,免疫荧光法结合流式细胞术测定TNF-α和IL-1β蛋白水平。结果出生前接触过LPS的4月龄C57BL/10ScSn小鼠,与注射生理盐水的对照小鼠比较,黑质DA能神经元减少(25.3±2.1)%,纹状体DA含量降低(33.5±5.0)%,黑质小胶质细胞数量增加(294±24)%,黑质和纹状体TNF-α和IL-1β蛋白水平也明显增高;但是出生前接触过LPS的TLR-4突变型C57BL/10ScNCr小鼠脑内无相应变化。出生前接触过TLR-2配体PDG的4月龄C57BL/10ScNCr和C57BL/10ScSn小鼠均出现脑内DA能神经元减少和免疫炎症改变。结论胚胎期接触LPS可通过TLR-4引起出生后个体脑内DA能神经元减少。
Objective To observe the role of clock-like receptor 4 (TLR-4) in the reduction of dopaminergic neurons (DA) in postnatal brain caused by lipopolysaccharide (LPS) in embryos. Methods Fifteen TLR-4 mutant C57BL / 10ScNCr mice and 15 wild-type C57BL / 10ScSn mice were intraperitoneally injected with LPS or peptidoglycan (PDG) on the 10.5th day of pregnancy and collected at 4 months of age Brain tissue samples (n = 5), DA neurons and microglia were quantified by immunohistochemical staining and stereological techniques. The levels of DA and its metabolites were determined by high performance liquid chromatography. Immunofluorescence combined with flow cytometry Cytometry was used to determine the levels of TNF-α and IL-1β. Results Four-month-old C57BL / 10ScSn mice exposed to LPS at birth had reduced DA neurons in the substantia nigra (25.3 ± 2.1)% and striatum DA (33.5 ± 5.0) compared with saline-injected control mice ), The number of substantia nigra microglial cells increased (294 ± 24)%, and the levels of TNF-α and IL-1β protein in substantia nigra and striatum were also significantly increased. However, TLR-4 mutant C57BL / 10ScNCr mouse brain no corresponding change. Both 4-month-old C57BL / 10ScNCr and C57BL / 10ScSn mice exposed to the TLR-2 ligand PDG before birth develop decreased DA neurons and altered immune inflammation in the brain. Conclusions Exposure of embryonic LPS to DA could decrease DA neurons in postnatal human via TLR-4.