肝脏X受体β对皮质和海马发育中放射状胶质细胞向星形胶质细胞转化的作用及机制研究

发布时间：2018-03-29 21:38

本文选题：肝脏X受体β　切入点：放射状胶质细胞　出处：《第三军医大学》2012年硕士论文

【摘要】：肝脏X受体（Liver X receptors, LXRs）为配体依赖的核受体超家族成员之一，包括LXRα和LXRβ两种亚型。LXRα主要表达于脂类代谢旺盛的器官如肝脏，而LXRβ主要表达于中枢神经系统。在哺乳类动物脑皮质发育中，早期新生神经元位于皮质深层，晚期以“由内而外”的方式迁移至大脑皮质的浅层。研究显示LXRβ在大脑皮质发育中呈发育学表达模式(developmentally expression pattern），可影响胚胎发育中晚期新生神经元的迁移以及大脑皮质板层的形成。在小脑发育中，LXR激动剂可促进小脑颗粒细胞由外颗粒层向内颗粒层迁移，提示LXRβ在新生神经元的放射状迁移中发挥重要作用。放射状胶质细胞（radial glial cells, RGCs）来源于胚胎发育早期的神经上皮，在胚胎发育早期作为干细胞来源之一产生新生神经元，在胚胎发育中期RGCs作为细胞迁移的支架引导新生神经元的迁移，在胚胎发育晚期RGCs开始向星形胶质细胞以及室管膜上皮细胞转化，至生后两周RGCs几乎全部转化为星形胶质细胞和室管膜上皮细胞。在皮质板层发育中如果RGCs分化加速，提前转化为星形胶质细胞，往往导致新生神经元迁移障碍和皮质板层发育异常。我们前期在LXRβ基因敲除小鼠中观察到大脑皮层中RGCs长纤维出现断裂，突起形态出现异常。此外，在小脑发育过程中也观察到LXR激动剂T0901317可以抑制RGCs向星形胶质细胞的提前转化，，提示LXRβ参与了RGCs的转化而影响板层状结构的发育。最近离体和在体实验均证实转化生长因子β1（transforming growth factorβ1，TGF-β1）/Smad4信号通路参与了皮质RGCs向星形胶质细胞的转化。LXR激动剂通过抑制TGFβ1/Smad4信号通路抑制小脑RGCs提前转化。LXRβ敲除是否加速大脑皮层RGCs向星形胶质细胞转化，其机制如何？目前尚不清楚。海马同大脑皮层和小脑一样，也属于经典的板层结构，由分子层、颗粒细胞层及多形层三层结构组成。在海马发育过程中，新生神经元由颗粒下层迁往颗粒层。在迁移过程中RGCs作为导向性支架引导新生神经元迁移。LXRβ是否通过影响海马RGCs而影响海马发育，其机制如何？目前仍不清楚。本实验拟采用RT-PCR和免疫组化分别检测LXRβ在大脑皮质和海马发育过程中的表达模式。采用免疫组化观察不同发育阶段WT小鼠和LXRβ敲除小鼠中Nestin、BLBP以及GFAP在大脑皮质和海马的表达差异；采用Western Blot进一步验证BLBP和GFAP表达水平的改变。采用免疫荧光双标检测WT小鼠和LXRβ敲除小鼠中BLBP-GFAP双标细胞在大脑皮质的表达差异。采用RT-PCR检测WT小鼠和LXRβ敲除小鼠大脑皮层TGFβ1和Smad4在不同发育时间点的表达差异。主要结果如下： 1. LXRβ在E18.5，P2，P7，P10及P14大脑皮质及E18.5，P2海马齿回中有丰富表达，其中在皮层中的表达水平从E18.5至P10逐渐升高。 2.随着大脑发育，WT小鼠皮层内Nestin标记的阳性细胞数在生后逐渐减少，与同龄WT小鼠相比，LXRβ敲除小鼠Nestin阳性细胞数减少以及纤维密度降低。 3.WT小鼠皮层内BLBP标记的RGCs在E18.5时长纤维跨越整个大脑皮层；在P2时部分细胞呈现成熟星形胶质细胞形态；在P7时，具有成熟星形胶质细胞形态的RGCs分布于整个大脑皮层；在P10和P14，具有成熟星形胶质细胞形态的RGCs进一步增多，LXRβ缺失导致这一变化过程进一步加速。 4.Western Blot结果显示，与同龄WT小鼠相比，在P2和P7LXRβ敲除小鼠大脑皮质中BLBP表达含量显著降低(p0.01)，GFAP表达含量显著增高（p0.01）。 5.在P7和P10，与同龄WT小鼠相比，LXRβ敲除小鼠大脑皮质中BLBP-GFAP双标细胞显著增多（p0.01）。 6.在P2和P7时，与同龄WT小鼠相比，LXRβ敲除小鼠大脑皮质中TGFβ1/Smad4表达水平显著增高（p0.05）；在P10和P14时，TGFβ1/Smad4表达水平在两者之间无显著差异（p0.05）。 7.在E18.5和P2时，与同龄WT小鼠相比，LXRβ敲除小鼠海马齿回PCNA阳性细胞显著减少（p0.05），RGCs纤维密度显著降低（p0.05），部分纤维出现断裂。结论：以上研究结果提示LXRβ通过调节TGFβ1/Smad4信号通路而参与调节发育过程中大脑皮质和海马RGCs向星形胶质细胞转化。
[Abstract]:LXR 伪 is mainly expressed in organs such as liver , while LXR 尾 is mainly expressed in the central nervous system . In the development of mammalian brain cortex , early neonatal neurons are located in the deep cortex of the cortex and the late stage is migrated to the superficial layer of the cerebral cortex . In the development of cerebellum , the LXR agonists promote the migration of the cerebellar granule cells from the outer granular layer to the inner granular layer , suggesting that LXR.beta . plays an important role in the radial migration of neonatal neurons .

Neuroepithelial cells ( RGCs ) from early embryonic development were derived from neural epithelial cells in early embryonic development . RGCs were transformed into astrocytes and dymal epithelial cells in early embryonic development . RGCs were transformed into astrocytes in the early stage of embryonic development .

In vitro and in vivo experiments , transforming growth factor - 尾1 ( TGF - 尾1 ) / Smad4 signaling pathway was involved in the transformation of cortical RGCs into astrocytes . LXR agonists inhibited the early transformation of RGCs by inhibiting TGF - 尾1 / Smad4 signaling pathways .

The hippocampus , like the cerebral cortex and cerebellum , also belongs to the classical lamellar structure . It is composed of molecular layer , granular cell layer and multi - layer three - layer structure . During the development of hippocampus , the new neurons migrate from the lower layer to the granular layer . In the course of migration , RGCs is used as the guide bracket to guide the neonatal neuron migration .

The expression patterns of Nestin , BLBP and GFAP in cerebral cortex and hippocampus were observed by RT - PCR and immunohistochemistry respectively .
The expression of BLBP - GFAP double - labeled cells in the cerebral cortex of WT mice and LXR - 尾 knockout mice was detected by Western Blot . The expression of TGF - 尾1 and Smad4 in the cerebral cortex of WT mice and LXR - 尾 knockout mice was detected by RT - PCR .

The main results are as follows :

1 . LXR 尾 was expressed in the cerebral cortex of E18.5 , P2 , P7 , P10 and 14 and in the hippocampus of E18.5 and P2 , in which the level of expression of LXR.beta . was gradually increased from E18.5 to P10 .

2 . With the development of the brain , the number of Nestin - positive cells in the cortex of WT mice decreased gradually , and the number of Nestin - positive cells decreased and the fiber density decreased compared with WT mice of the same age .

3 . The RGCs of the BLBP - labeled RGCs in the cortex of WT mice span the entire cerebral cortex at E18.5 .
At P2 , some cells presented mature astrocytes .
At P7 , RGCs with mature astrocytes were distributed throughout the cerebral cortex ;
In P10 and P14 , RGCs with mature astrocytes morphology increased further , and the deletion of LXR.beta . resulted in further acceleration of this change .

4 . Western Blot showed that the expression of BLBP increased significantly in the cerebral cortex of P2 and P7LXR 尾 knockout mice compared with WT mice of the same age ( p0.01 ) , and the expression of GFAP increased significantly ( P0.01 ) .

5 . At P7 and P10 , the BLBP - GFAP double labeled cells increased significantly in the cerebral cortex of mice compared with WT mice of the same age ( p0.01 ) .

6 . At P2 and P7 , the expression level of TGF - 尾1 / Smad4 in rat cerebral cortex increased significantly compared with WT mice of the same age ( p < 0.05 ) .
There was no significant difference between the expression levels of TGF 尾 1 / Smad4 ( p < 0.05 ) .

7 . At E18.5 and P2 , compared with WT mice of the same age , the positive cells of PCNA - positive cells in the hippocampus of LXR - 尾 knockout mice were significantly decreased ( p < 0.05 ) , and the density of RGCs decreased significantly ( p < 0.05 ) .

Conclusion :

The above results suggest that LXR 尾 is involved in the regulation of TGF - 尾1 / Smad4 signaling pathway in the regulation and development of cerebral cortex and hippocampus RGCs into astrocytes .

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R338

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