PAX6在七氟醚抑制神经前体细胞增殖中的功能及机制研究

The second trimester is considered to be the safest phase during pregnancy for mom and fetus to undergo non-obstetric surgery. But in fact the most of the neurons are produced by mid-gestation. This brings out a new issue how the inhaled anesthetics especially sevoflurane which is widely used nowadays in large cities in China effect the fetal brain in this period. In our previous study, we found that the expression of Pax6 was suppressed and the cell cycle related protein was changed in the fetus with the inhabitation of proliferation of neural progenitors when the sevoflurane exposure occured in the corticogenesis of brains. Recent researches have showed that the Wnt signaling took part in the neurotoxicity of sevoflurane and Pax6 was a novel downstream target of this pathway. We postulate that sevoflurane exposure of pregnant mice inhibits the expression of Pax6 and its downstream gene cdk6 and ccnd1 through Wnt signaling. The corticogenesis of fetus is finally affected and this leads to the neurocognitive deficits of offspring in the adulthood. So we plan to establish the model of sevoflurane exposure of pregnant mice and progenitors to clarify the mechanism of neurotoxicity of anesthesia. The Tet-on system in vitro will be used to adjust the expression of Pax6 to explore its effect on the cell cycle and proliferation of progenitors. Li will be used to testify if the Wnt pathway is involved. We hope to provide some proof to optimize the anesthesia of pregnant women under non-obstetric surgeries.

临床研究显示胎儿以及儿童期接受麻醉的患儿发生学习障碍的可能性增加。孕中期一直被认为是孕妇接受手术最安全的时期。但实际上从孕3个月起胎儿大脑就进入发育的第一个高峰期，生成大部分的神经元。对于七氟醚抑制神经前体细胞增殖的机制知之甚少。预试验发现孕15.5天的小鼠接受七氟醚麻醉后胎鼠大脑中转录因子Pax6的表达受到抑制，伴随着神经前体细胞增殖的减少以及细胞周期的改变。Wnt通路参与七氟醚对神经前体细胞增殖的抑制作用，而Pax6被证实是Wnt通路一个重要的下游基因。因此，我们提出“七氟醚通过Wnt通路影响转录因子Pax6及其下游基因cdk6和ccnd1的表达，造成神经前体细胞池的减少，最终引起胎鼠成年后行为学异常”的假说。为此，我们通过体内体外研究，尤其是体外建立Tet-on系统调节Pax6的表达，验证七氟醚通过Wnt通路抑制Pax6及其下游细胞周期相关基因的表达从而影响神经前体细胞增殖。

有研究指出孕期吸入麻醉药物的暴露可以影响胎儿大脑发育，进而影响其认知功能。胚胎期大脑神经发育异常可能和七氟醚的神经毒性有关，但具体机制目前不明。本实验的目的在于探索七氟醚是否通过影响胚胎期大脑前额叶皮层神经前体细胞的细胞周期来影响其神经发生进而影响成年期的认知功能。将处于孕中期（孕15.5天）的孕鼠随机分为2组，实验组暴露于2.5%七氟醚+97.5%氧气6小时，对照组暴露于100%氧气6小时。一部分孕鼠继续饲养待其产子，子代生长至产后28天时进行Morris水迷宫测试，检测其认知功能变化。另一部分孕鼠脱颈处死取其胎鼠进行免疫荧光染色，检测其神经元、神经前体细胞、增殖和细胞周期的变化。将得到的研究结果通过体外培养的神经干细胞进一步验证。Morris 水迷宫实验结果提示，孕中期暴露于七氟醚可以导致胎儿在成年期出现学习记忆功能障碍。免疫荧光染色结果显示，七氟醚可以导致胚胎大脑皮质神经前体细胞细胞周期S期延长、细胞周期推出减少，进而引起神经前体细胞增殖抑制、神经前体细胞数量减少、深层新生神经元数量减少。孕中期暴露于七氟醚可以导致子代认知功能障碍。七氟醚对胚胎期大脑的神经毒性主要表现在干扰胚胎期大脑皮质中神经前体细胞的细胞周期进展，引发神经前体细胞增殖和分化障碍，最终导致胚胎大脑皮质神经细胞的种类和数量发生异常。