宫内感染/炎症后未成熟脑星形细胞胶质化干扰神经发生的机制研究

Perinatal brain injury and immature brain inflammation caused by intrauterine infection is an important cause of cerebral palsy, and neurogenesis may be one of repair mechanisms and potential therapeutic direction. Our previous study suggested that astrogliosis and neurogenesis in immature brain affected by inflammatory reaction after intrauterine infection/inflammation and the expression of PI3K-Akt signalling in immature brain was increased after intrauterine infection/inflammation. The mechanisim of signal transduction pathway coulpling intrauterine infection/inflammation and perinatal brain damage is unknown, and it’s unclear that how PTEN/PI3K-Akt signalling pathway regulate astrogliosis and neurogenesis in immature brain and the proliferation and differentiation of neural stem/progenitors (NSPs) after intrauterine infection/inflammation. To explore the important role of PTEN/PI3K-Akt signalling pathway in the procedure of astrogliosis interfered the neurogenesis in immature brain caused by intrauterine infection/inflammation, we developed a model of intrauterine infection/inflammation in rats to investigate neurogenesis in neonatal rat brain; and to study the spatio-temporal characteristics of PTEN/PI3K-Akt signalling pathway, we detected the expression of PTEN/PI3K-Akt in different brain region (periventricular white matter, hippocampus, corpus callosum and cerebral cortex) at different time after intrauterine infection/inflammation. Moreover, astrocyte and NSPs were isolated from cultures of fetal rat brain, and the astrocyte proliferation/astrogliosis and the disdifferentiation of NSPs after infection/inflammation were analyzed. We used primary astrocyte and neurosphere cocultures to demonstrate that whether astrocytes inhibit NSPs differentiation through a cell–cell contact or not. We also improved the expression of PTEN gene by transfection and knockout the expression of PTEN gene by TELAN, and investigated the control affection of PTEN/PI3K-Akt signalling pathway to the differentiation and development of NSPs and the expression characteristics of PTEN/PI3K-Akt signalling pathway of in NSPs after infection/inflammation. Furthermore, to determine the effect of PTEN inhibitor bpV(HOpic) or PI3K inhibitor (LY294002) to differentiation and development of NSPs, the NSPs were treated with bpV(HOpic) or LY294002 to detect the characteristics of differentiation, and we also explored whether the activation of PI3K-Akt signalling pathway by bpV(HOpic) or the inhibition of PI3K-Akt signalling pathway by LY294002 could influence neurogenesis in immature brain after intrauterine infection/inflammation or not. Taken together, the present research will provide additional hope that restoring normal development after injuries have occurred and will provide a new neuroprotective therapy to perinatal brain damage.

宫内感染后脑内炎症反应致围产期脑损伤是脑瘫的重要原因，神经发生可能是围产期脑损伤后的一种自身修复机制和潜在治疗方向。我们的前期研究发现宫内感染后未成熟脑出现星形细胞胶质化和神经发生，但星形细胞胶质化是否干扰神经发生及其调控机制尚不清楚。PTEN/PI3K-Akt信号在神经发生时起调控作用，但炎症条件下PTEN/PI3K-Akt信号是否调控星形细胞胶质化与神经发生需进一步研究。本课题拟通过建立宫内感染大鼠模型，探讨PI3K-Akt信号活化是否影响宫内感染后未成熟脑神经发生；并予细胞培养，观察炎症条件下星形细胞是否影响神经前体细胞(NSPs)的增殖分化，且予转染PTEN基因上调PI3K-Akt信号和TELAN技术敲除PTEN基因，研究PTEN/PI3K-Akt信号对炎症条件下星形细胞干扰NSPs增殖分化的调控作用；从细胞分化角度初步阐明宫内感染/炎症导致围产期脑损伤及脑内自身修复作用的机制。

宫内感染后脑内炎症反应致围产期脑损伤是脑瘫的重要原因，神经发生可能是围产期脑损伤后的一种自身修复机制和潜在治疗方向。我们的前期研究发现宫内感染后未成熟脑出现星形细胞胶质化和神经发生，但星形细胞胶质化是否干扰神经发生及其调控机制尚不清楚。本课题通过建立宫内感染大鼠模型，发现宫内感染/炎症后新生大鼠脑组织中Nestin和GFAP的表达高于正常组，而β-Ⅲ-tubulin，NG2和MBP的表达则低于正常组，NeuN的表达在两组间没有明显可见差异；原位核酸分子杂交技术检测和脑组织切片结果显示相比于对照 PETN抑制剂-bpV处理组PTEN和p-PTEN的表达下调，而PI3K和Akt的表达上调；PI3K 抑制剂-LY294002处理组相比于对照PTEN的表达没有明显变化，而PI3K，Akt和p-Akt的表达下调。Morris水迷宫实验结果显示bpV处理的感染组仔代大鼠的潜伏期比对照缩短，穿越原平台的次数增多。悬吊实验结果显示bpV处理的感染组仔代大鼠的悬吊时间比对照变长。斜坡实验结果显示bpV处理的感染组仔代大鼠的转向所需时间缩短。此外，LY294002处理得到的结果与上述完全相反。表明PI3K-Akt信号活化可影响宫内感染后未成熟脑神经发生及仔代大鼠的神经行为。并予细胞培养，过表达PTEN后神经干细胞中Nestin，MBP，NG2和β-Ⅲ-tubulin的表达均下调，干扰PTEN的表达则效果相反；而且过表达PTEN后神经干细胞中PCNA、Ki67、PI3K、Akt和p-Akt蛋白的表达均下调，干扰PTEN的表达则效果相反，提示PTEN抑制神经干细胞的增殖和分化。对LPS诱导的感染/炎症星形细胞培养液中分别加入PI3K-Akt信号的抑制剂和活化剂之后发现，PI3K-Akt信号活化可以促进神经干细胞中β-Ⅲ-tubulin，NG2，MBP和Nestin的表达以及上调PCNA，Ki67，PI3K，Akt和p-Akt蛋白的磷酸化水平；PI3K-Akt信号的抑制则有相反的结果，提示PI3K-Akt信号促进神经干细胞的增殖和分化。这些研究结果表明PTEN/PI3K-Akt信号对炎症条件下星形细胞干扰NSPs增殖分化的调控作用；本课题从细胞分化角度初步阐明宫内感染/炎症导致围产期脑损伤及脑内自身修复作用的机制。