小胶质细胞在新生小鼠高氧暴露脑损伤中的作用

发布时间：2018-05-21 17:27

本文选题：高浓度氧 + 新生小鼠　；参考：《重庆医科大学》2014年硕士论文

【摘要】：目的：探讨小胶质细胞在新生小鼠高氧暴露脑损伤中的功能变化及其作用。方法：清洁级健康野生型新生3d小鼠C57BL/10J（PND3）48只，随机分为空气组（A组）、高氧组(O组)；90%高浓度氧暴露48h，PND5断头取脑：（1）蛋白质印迹技术（western-blot，WB）检测capsase-3蛋白表达。（2）免疫荧光双标检测Caspase-3凋亡蛋白表达情况及分析NG2+标记的少突胶质祖细胞凋亡；（3）免疫组化染色观测脑室周围白质小胶质细胞形态变化及Toll样4受体（Toll-like receptor4,TLR4）的表达；（4） Real-time Quantitative PCR（Q-PCR）检测TLR4和TNF-α(Tumor necrosis factor α)mRNA表达变化；新生3d清洁级健康野生型小鼠C57BL/10J（PND3）24只，采用小胶质细胞活化抑制剂米诺环素（Minocycline），腹腔注射（45mg/kg）30min后，90%高浓度氧暴露48h，，分为米诺环素+空气组（AM组）、米诺环素+高氧组（OM组）,PND5断头取脑：（1）Q-PCR检测TLR4和TNF-αmRNA表达变化；（2）免疫荧光双标检测Caspase-3凋亡蛋白表达情况及分析NG2+标记的少突胶质前体细胞凋亡；结果：免疫荧光显示Caspase-3凋亡蛋白在O组的表达增高，O组NG2+标记的少突胶质前体细胞出现细胞凋亡；O组免疫组化染色观测脑室周围白质活化的小胶质细胞数量及TLR4表达明显增高；同时Q-PCR检测结果中，O组中TLR4和TNF-α的mRNA表达明显高于A组（P0.05）； OM组TLR4和TNF-α的mRNA表达低于相对应的O组（P0.05），但高于A组（P0.05）；OM组脑组织Caspase-3明显低于单纯O组；但高于AM组和A组。各处理组间NG2表达无明显差异，O组与OM组可见Caspase-3与NG2+共表达。结论：新生鼠高浓度氧暴露诱导小胶质细胞活化，TLR4受体表达增加，并最终通过氧化应激与炎症反应（TNF-α表达增加）导致未成熟脑胶质细胞凋亡，且凋亡细胞包括少突胶质前体细胞。小胶质细胞参与了新生鼠高浓度氧暴露导致的脑损伤。
[Abstract]:Aim: to investigate the functional changes and effects of microglia on brain injury in neonatal mice exposed to hyperoxia. Methods: C57BL/10J(PND3)48 mice of clean grade healthy wild-type newborn mice were used. The rats were randomly divided into two groups: air group A and hyperoxia group (90% high concentration oxygen exposure 48h). Western blot technique was used to detect the expression of capsase-3 protein. 2) Immunofluorescence double labeling was used to detect the expression of Caspase-3 apoptotic protein and to analyze the oligospermia of NG2 labeled by PND5. The Western blot technique was used to detect the expression of Caspase-3 protein. The morphological changes of microglia and the expression of Toll-like receptor 4 (TLR4) in the white matter around the ventricle were observed by immunohistochemical staining. The expression of TLR4 and TNF- 伪 Tumor necrosis factor were detected by Real-time Quantitative PCR Q-PCR4). Three days after birth, C57BL/10J(PND3)24 of clean grade healthy wild-type mice was found. Minocycline, an inhibitor of microglial activation, was used to detect the changes of TLR4 and TNF- 伪 mRNA expression in TLR4 and TNF- 伪 mRNA after intraperitoneal injection of 45 mg 路kg ~ (-1) 路min ~ (-1) min for 48 h after exposure to 90% high concentration oxygen, which was divided into two groups: minocycline air group (AM group), minocycline hyperoxia group (OM group) and minocycline group (OM group) were treated with minocycline 1 Q-PCR to detect the changes of TLR4 and TNF- 伪 mRNA expression. The expression of Caspase-3 apoptotic protein and the apoptosis of oligodendrocyte precursor cells labeled with NG2 were detected by immunofluorescence double labeling. Results: immunofluorescence showed that the expression of Caspase-3 apoptosis protein was increased in group O. The number of activated microglia and the expression of TLR4 in the white matter around the ventricle were observed by immunohistochemical staining in oligodendrocyte precursor cells labeled with NG2 in O group. At the same time, the mRNA expression of TLR4 and TNF- 伪 in Q-PCR group was significantly higher than that in group A (P 0.05). The mRNA expression of TLR4 and TNF- 伪 in OM group was lower than that in O group, but higher than that in group A (P 0.05) group, but higher than that in group A group (P 0.05) group, but higher than that in AM group and A group. There was no significant difference in the expression of NG2 between O and OM groups. Caspase-3 and NG2 were coexpressed in O group and OM group. Conclusion: the expression of TLR4 receptor in microglia activated by high oxygen exposure in neonatal rats is increased, and the expression of TNF- 伪 is increased through oxidative stress and inflammatory response), which leads to the apoptosis of immature brain glial cells. The apoptotic cells include oligodendrocytes. Microglia are involved in brain damage induced by high oxygen exposure in newborn rats.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R722.1

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