【作者】 王媛；

【导师】 赵平；

【作者基本信息】 中国医科大学 ， 麻醉学， 2018， 博士

【摘要】 目的:随着胎儿宫内治疗和胎儿手术的快速发展,孕中期接受全身麻醉的孕妇数量日益增多。孕中期应用麻醉药物会对胎儿的神经发育产生怎样的影响?这种影响是否会在生后显现出来?产生这种影响的可能机制是什么?因此,本研究选择了临床上使用较为广泛的全身麻醉药物—七氟醚,拟定研究大鼠孕中期吸入七氟醚对子代学习记忆功能和神经干细胞增殖的影响并初步研究相关机制。研究方法:孕期为14天Sprague-Dawle大鼠按随机数字表随机分成5组(n=56,共280只):对照组(C GROUP),低浓度七氟醚组(L GROUP),高浓度七氟醚组(H GROUP),高浓度七氟醚加氯化锂组(H+Li GROUP),以及单纯氯化锂组(Li GROUP)。在予以七氟醚麻醉前,高浓度七氟醚加氯化锂组及单纯氯化锂组孕鼠接受腹腔注射120mg/kg的氯化锂。如前进行七氟醚吸入麻醉。低浓度七氟醚组孕鼠吸入2%的七氟醚2 h,高浓度七氟醚组以及高浓度七氟醚加氯化锂组孕鼠吸入3.5%的七氟醚2 h,对照组以及单纯氯化锂组孕鼠吸入30%氧气2 h。各组在特定的时间点(6 h后、24 h后、48 h后)取胎鼠脑组织以Western Blot方法检测Wnt信号通路中的关键蛋白GSK-3β,p-Ser9-GSK-3β和β-catenin的表达,以real time PCR法检测Wnt信号通路下游直接调控的靶基因CD44以及cyclinD1 mRNA的表达。在6个特定的时间点(6 h后、24 h后、48 h后、P0、P14和P28)取胎鼠或子代大鼠脑组织制备冰冻切片,以免疫荧光染色法检测NSCs增值情况。脑冰冻切片Nestin和Ki67双染标记胎鼠脑组织或子代大鼠海马区增殖的NSCs,计数Ki67+/Nestin+细胞计算正在增值的NSCs数量;此外,Western blot法检测胎鼠脑组织或子代大鼠海马组织的Nestin,Sox-2和Ki67蛋白表达水平。仔鼠出生4 w后,从每只孕鼠的子代中随机取出4只子代鼠,进行Morris水迷宫测试,检测子代大鼠的空间认知能力。结果:孕中期七氟醚麻醉后,孕鼠血流动力学指标及血气分析指标无明显改变,孕中期七氟醚麻醉对子代运动能力也无明显影响。与对照组相比,低浓度七氟醚组子代大鼠各时间点逃避潜伏期相当,差异无统计学意义(p>0.05)。高浓度七氟醚组子代大鼠在第四天、第五天逃避潜伏期明显延长,差异有统计学意义(p<0.01)。与对照组相比,低浓度七氟醚组子代大鼠平台穿越次数略有增加,差异无统计学意义(p>0.05)。高浓度七氟醚组子代大鼠平台穿越次数明显减少,差异有统计学意义(p<0.05)。对照组及低浓度七氟醚组子代大鼠在第四象限有较明确活动轨迹;而高浓度七氟醚组子代大鼠活动缺乏明确的目的性,活动轨迹平均分布于整个区域。低浓度七氟醚组(L GROUP)在六个观察时间点的胎鼠脑组织或子代大鼠海马组织中凋亡细胞数、凋亡神经干细胞数、增殖干细胞数、Nestin蛋白、Sox-2蛋白和Ki67蛋白表达水平与对照组(C GROUP)相比差异均无统计学意义(p>0.05)。高浓度七氟醚组(H GROUP)子代大鼠在六个观察时间点的凋亡细胞数明显高于对照组(C GROUP),增殖干细胞数、Nestin蛋白水平、Sox-2蛋白水平以及Ki67蛋白水平明显低于对照组(C GROUP),差异有统计学意义(p<0.05)。高浓度七氟醚组(H GROUP)子代大鼠在麻醉后6 h、24 h、48h、出生时、出生后14 d这5个观察时间点的凋亡神经干细胞数明显高于对照组(C GROUP),差异有统计学意义(p<0.05);而在出生后28 d这个时间点高浓度七氟醚组凋亡神经干细胞数较对照组相比无显著性差异(p>0.05)。低浓度七氟醚组(L GROUP)子代大鼠脑组织GSK-3β水平、p-Ser9-GSK-3β水平、β-catenin蛋白水平以及CD44和cyclinD1 mRNA表达水平与对照组(C GROUP)相比差异均无统计学意义(p>0.05)。高浓度七氟醚组(H GROUP)子代大鼠脑组织或海马GSK-3β蛋白水平明显高于对照组(C GROUP),p-Ser9-GSK-3β、β-catenin蛋白水平以及CD44和cyclinD1 mRNA表达水平明显低于对照组(C GROUP),差异有统计学意义(p<0.05)。氯化锂通过增加了p-GSK-3β的量,使GSK-3β活性受到抑制,进而增加了β-catenin的含量来减轻高浓度七氟醚对神经干细胞增殖的抑制作用,但这种保护作用持续时间短暂,并且水迷宫结果提示氯化锂并没有改善高浓度七氟醚所引起的行为学异常。结论:1、孕中期高浓度七氟醚麻醉对子代大鼠空间学习记忆能力造成了损害,表现为水迷宫实验中逃避潜伏期延长,平台区域穿越次数减少,而接受低浓度七氟醚麻醉的孕鼠的子代并没有表现出空间学习记忆能力损伤,说明孕中期应用麻醉药物对子代远期神经功能的损害作用是浓度依赖的。2、孕中期单次应用高浓度七氟醚则明显抑制子代神经干细胞的数量和增殖,促进神经干细胞以及整体神经细胞的凋亡。而单次应用低浓度七氟醚麻醉对子代神经干细胞的增殖及凋亡无明显影响。3、孕中期应用高浓度七氟醚可以升高子代脑内GSK-3β蛋白水平,降低p-Ser9-GSK-3β水平、β-catenin蛋白水平以及CD44和cyclinD1 mRNA表达水平,同时氯化锂通过增加了p-GSK-3β的量,使GSK-3β活性受到抑制,进而增加了β-catenin的含量来减轻高浓度七氟醚对神经干细胞增殖的抑制作用,这些表明高浓度七氟醚可能通过抑制Wnt/β-catenin信号通路损伤海马神经干细胞数量和增值、促进其凋亡,从而影响子代大鼠学习记忆能力。更多还原

【Abstract】 Objective: With the rapid development of the fetal intervention procedures,more and more pregnant women in the second trimester had received general anesthesia.Anesthesiology focus mainly on: Is there any effect on the development of the fetal nervous system by using anesthetic drugs during the second trimester? Will this influence have any manifestation after birth? What is the possible mechanism? The authors set out to assess the effects of the most widely used anesthetics---sevoflurane,on proliferation of neural stem cells and on learning and memory in fetal and offspring rats,and discuss on the related mechanism.Methods: 1.Pregnant SD rats(gestational day 14)were randomly divided into 3groups: the control group(C GROUP),low concentration group(L GROUP)and high concentration of sevoflurane group(H GROUP).Pregnant rats were treatd with 30%oxygen,2% sevoflurane,3.5 sevoflurane for 2 hours,respectively.Learning and memory function in the offspring rats was determined in the Morris water maze.2.Pregnant SD rats(gestational day 14)were randomly divided into 3 groups: the control group(C GROUP),low concentration group(L GROUP)and high concentration of sevoflurane group(H GROUP).Pregnant rats were treated with 30% oxygen,2%sevoflurane,3.5 sevoflurane for 2 hours,respectively.In six specific time point(6 h,24 h,48 h after treatment,P0,P14 and P28 after birth)fetal brain tissues or offspring rats hippocampal tissue were havested and subjected to western blot and immunofluorescence to assess Nestin,Sox-2,Ki67 level and the number of proliferative and apoptotic NSC.3.Pregnant SD rats(gestational day 14)were randomly divided into 5 groups: control group(C GROUP),low sevoflurane group(L GROUP),high sevoflurane group(H GROUP),sevoflurane+lithium group(H+Li GROUP)and lithium group(Li GROUP).Rats received different concentration for 2h.The rat offspring were weaned at 28 days for Mirror Water Maze behavioral studies and fetal brains or postnatal hippocampal tissues were harvested for immunofluorescence staining,real time PCR,and western blot to detect NSC proliferation and the Wnt/β-catenin signaling levels.Results: 1.There were no significant differences in ABG values and hemodynomic levels of the pregnant rats among the three groups.There was also no statistically significant difference in swimming speed of the offsprings among the three groups.The pups of the H GROUP spent more time to find the platform(escape latency)than those of C GROUP(P<0.05),however,no differences were observed between the C GROUP and L GROUP in comparison of the escape latency.In the probe test,the number of crossing over the former platform location in H GROUP pups was fewer and the time spent in the fouth quadrant where the platform located was shorter than the control animals,while no differences were observed between the C GROUP and L GROUP.2.In the H GROUP pups,the expression of Nestin,Ki67 and Nestin and Ki67 positive cells in the fetal brain or offsprings’ hippocampal area decreased compared with the C GROUP.The TUNEL positive cells displayed a tendency of increase in the fetal brain or offsprings’ hippocampal area in the H GROUP pups.Consistent with the findings of immunofluorescence studies,western blot analysis showed that the levels of Nestin,Sox-2 and Ki67 protein markedly decreased in the fetal brain or offsprings’ hippocampal region of the H GROUP pups when compared with the C GROUP.However,there was no difference between L GROUP and C GROUP.3.The results showed that exposure to 3.5% sevoflurane(H GROUP)in mid-gestational rats increased fetal GSK-3β protein levels as well as reducedβ-catenin protein levels,CD44 and cyclin D1 m RNA levels when compared with control group.However,these effects in fetus were attenuated by lithium chloride.Conversely,2% sevoflurane(L GROUP)did not show the same effects.Conclusions: 1.High concentration sevoflurane anesthesia in pregnant rats of second trimester induced learning and memory impairment in offspring rats,manifesting the prolonged escape latency and reduced the number of passing across platforms the region.While low concentration sevoflurane anesthesia showed no learning and memory impairment,indicating that the toxic effect of anesthetics on the developing brain is concentration-dependent.2.High concentration sevoflurane anesthesia in pregnant rats of second trimester reduced neural stem cell proliferation and induced apoptosis in the fetal brain or hippocampus of the offspring rats.3.Sevoflurane exposure in the second trimester inhibits fetal neural stem cell proliferation possibly via Wnt/β-catenin pathway and impairs postnatal learning and memory function in a dose-dependent manner.更多还原