本文選題：肝臟X受體β　切入點：放射狀膠質細胞　出處：《第三軍醫(yī)大學》2012年碩士論文

【摘要】：肝臟X受體（Liver X receptors, LXRs）為配體依賴的核受體超家族成員之一，包括LXRα和LXRβ兩種亞型。LXRα主要表達于脂類代謝旺盛的器官如肝臟，而LXRβ主要表達于中樞神經系統。在哺乳類動物腦皮質發(fā)育中，早期新生神經元位于皮質深層，晚期以“由內而外”的方式遷移至大腦皮質的淺層。研究顯示LXRβ在大腦皮質發(fā)育中呈發(fā)育學表達模式(developmentally expression pattern），可影響胚胎發(fā)育中晚期新生神經元的遷移以及大腦皮質板層的形成。在小腦發(fā)育中，LXR激動劑可促進小腦顆粒細胞由外顆粒層向內顆粒層遷移，提示LXRβ在新生神經元的放射狀遷移中發(fā)揮重要作用。 放射狀膠質細胞（radial glial cells, RGCs）來源于胚胎發(fā)育早期的神經上皮，在胚胎發(fā)育早期作為干細胞來源之一產生新生神經元，在胚胎發(fā)育中期RGCs作為細胞遷移的支架引導新生神經元的遷移，在胚胎發(fā)育晚期RGCs開始向星形膠質細胞以及室管膜上皮細胞轉化，至生后兩周RGCs幾乎全部轉化為星形膠質細胞和室管膜上皮細胞。在皮質板層發(fā)育中如果RGCs分化加速，提前轉化為星形膠質細胞，往往導致新生神經元遷移障礙和皮質板層發(fā)育異常。我們前期在LXRβ基因敲除小鼠中觀察到大腦皮層中RGCs長纖維出現斷裂，突起形態(tài)出現異常。此外，在小腦發(fā)育過程中也觀察到LXR激動劑T0901317可以抑制RGCs向星形膠質細胞的提前轉化，，提示LXRβ參與了RGCs的轉化而影響板層狀結構的發(fā)育。 最近離體和在體實驗均證實轉化生長因子β1（transforming growth factorβ1，TGF-β1）/Smad4信號通路參與了皮質RGCs向星形膠質細胞的轉化。LXR激動劑通過抑制TGFβ1/Smad4信號通路抑制小腦RGCs提前轉化。LXRβ敲除是否加速大腦皮層RGCs向星形膠質細胞轉化，其機制如何？目前尚不清楚。 海馬同大腦皮層和小腦一樣，也屬于經典的板層結構，由分子層、顆粒細胞層及多形層三層結構組成。在海馬發(fā)育過程中，新生神經元由顆粒下層遷往顆粒層。在遷移過程中RGCs作為導向性支架引導新生神經元遷移。LXRβ是否通過影響海馬RGCs而影響海馬發(fā)育，其機制如何？目前仍不清楚。 本實驗擬采用RT-PCR和免疫組化分別檢測LXRβ在大腦皮質和海馬發(fā)育過程中的表達模式。采用免疫組化觀察不同發(fā)育階段WT小鼠和LXRβ敲除小鼠中Nestin、BLBP以及GFAP在大腦皮質和海馬的表達差異；采用Western Blot進一步驗證BLBP和GFAP表達水平的改變。采用免疫熒光雙標檢測WT小鼠和LXRβ敲除小鼠中BLBP-GFAP雙標細胞在大腦皮質的表達差異。采用RT-PCR檢測WT小鼠和LXRβ敲除小鼠大腦皮層TGFβ1和Smad4在不同發(fā)育時間點的表達差異。 主要結果如下： 1. LXRβ在E18.5，P2，P7，P10及P14大腦皮質及E18.5，P2海馬齒回中有豐富表達，其中在皮層中的表達水平從E18.5至P10逐漸升高。 2.隨著大腦發(fā)育，WT小鼠皮層內Nestin標記的陽性細胞數在生后逐漸減少，與同齡WT小鼠相比，LXRβ敲除小鼠Nestin陽性細胞數減少以及纖維密度降低。 3.WT小鼠皮層內BLBP標記的RGCs在E18.5時長纖維跨越整個大腦皮層；在P2時部分細胞呈現成熟星形膠質細胞形態(tài)；在P7時，具有成熟星形膠質細胞形態(tài)的RGCs分布于整個大腦皮層；在P10和P14，具有成熟星形膠質細胞形態(tài)的RGCs進一步增多，LXRβ缺失導致這一變化過程進一步加速。 4.Western Blot結果顯示，與同齡WT小鼠相比，在P2和P7LXRβ敲除小鼠大腦皮質中BLBP表達含量顯著降低(p0.01)，GFAP表達含量顯著增高（p0.01）。 5.在P7和P10，與同齡WT小鼠相比，LXRβ敲除小鼠大腦皮質中BLBP-GFAP雙標細胞顯著增多（p0.01）。 6.在P2和P7時，與同齡WT小鼠相比，LXRβ敲除小鼠大腦皮質中TGFβ1/Smad4表達水平顯著增高（p0.05）；在P10和P14時，TGFβ1/Smad4表達水平在兩者之間無顯著差異（p0.05）。 7.在E18.5和P2時，與同齡WT小鼠相比，LXRβ敲除小鼠海馬齒回PCNA陽性細胞顯著減少（p0.05），RGCs纖維密度顯著降低（p0.05），部分纖維出現斷裂。 結論： 以上研究結果提示LXRβ通過調節(jié)TGFβ1/Smad4信號通路而參與調節(jié)發(fā)育過程中大腦皮質和海馬RGCs向星形膠質細胞轉化。
[Abstract]:LXR 偽 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 .

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R338

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