多次七氟烷麻醉对幼鼠海马区树突棘发育的影响及TTLL6介导的微管多聚谷氨酰化机制  

Impact of repeated sevoflurane anesthesia on hippocampal dendritic spine development in neonatal mice and the mechanism of microtubule polyglutamylation mediated by TTLL6

作　　者：于洋[1] 赵悦 冯婧昱 杨月 李雅楠 于佳丰 于泳浩[1] Yu Yang;Zhao Yue;Feng Jingyu;Yang Yue;Li Yanan;Yu Jiafeng;Yu Yonghao(Department of Anesthesiology,Tianjin Medical University General Hospital(Tianjin Institute of Anesthesiology),Tianjin 300052,China)

机构地区：[1]天津医科大学总医院麻醉科(天津市麻醉学研究所),天津300052

出　　处：《中华行为医学与脑科学杂志》2025年第3期193-200,共8页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：国家自然科学基金(82001149)。

摘　　要：目的采用脑组织基因特异性敲除小鼠评价微管蛋白酪氨酸接酶6(TTLL6)介导的微管多聚谷氨酰化和Spastin(一种微管切割蛋白)介导的微管过度剪切在多次七氟烷麻醉致幼鼠海马神经元树突棘发育障碍中的作用。方法选取出生6 d的SPF级雌性C57BL/6J背景的TTLL6脑组织特异性敲除(TTLL6_(CKO):Camk2-Cre+,TTLL6 ^(f/f))小鼠及其对照(TTLL6_(CON):TTLL6 ^(f/f))小鼠各50只,按照随机区组法将TTLL6_(CON)小鼠分为TTLL6_(CON)对照组和TTLL6_(CON)七氟烷组,TTLL6_(CON)小鼠分为TTLL6_(CKO)对照组和TTLL6_(CKO)七氟烷组,每组25只。七氟烷组小鼠在出生后第6、8、10天接受3%七氟烷+60%O_(2)处理,每天2 h;对照组则在相同条件下仅接受60%O_(2)处理。采用Western blot实验检测多聚谷氨酰化微管蛋白(polyGlu-Tubulin)和突触后致密蛋白95(PSD95)表达,免疫荧光法检测TTLL6、Spastin、α微管蛋白(α-Tubulin)表达,高尔基染色及电镜观察海马树突棘密度及突触情况;用Morris水迷宫实验评估空间记忆能力。采用GraphPad Prism 8.0软件进行统计分析。结果(1)行为学结果显示,4组小鼠寻找平台潜伏期的时间和组别交互效应显著(F=8.22,P<0.001)。TTLL6_(CON)七氟烷组小鼠第3~7天逃避潜伏期明显长于TTLL6_(CON)对照组小鼠(均P<0.05),TTLL6_(CKO)七氟烷组小鼠逃避潜伏期与TTLL6_(CKO)对照组比较差异无统计学意义(均P>0.05)。4组小鼠穿越平台次数差异有统计学意义(H=11.95,P=0.007),TTLL6_(CON)七氟烷组小鼠穿越平台次数低于TTLL6_(CON)对照组(P<0.05),TTLL6_(CKO)七氟烷组小鼠与TTLL6_(CKO)对照组比较差异无统计学意义(P>0.05)。(2)高尔基染色及电镜结果显示,4组小鼠树突棘密度及突触数量比较均差异有统计学意义(F=29.00,41.94,均P<0.001)。TTLL6_(CON)七氟烷组树突棘密度[(5.83±0.40)个/10μm]及突触数量[(3.67±0.58)个/10μm]均低于TTLL6_(CON)对照组[(12.87±1.70)个/10μm,(9.33±0.57)个/10μm](均P<0.05),而TTLL6_(CKO)�ObjectiveTo evaluate the role of tubulin tyrosin ligase like-6(TTLL6)-mediated microtubule polyglutamylation and Spastin(a microtubule cleaving protein)-induced excessive microtubule cleavage in the developmental impairment of dendritic spines in neonatal mice following repeated sevoflurane anesthesia,by utilizing TTLL6 conditional knockout mice.MethodsFifty SPF female TTLL6 brain tissue-specific knockout(TTLL6_(CKO):Camk2-Cre+;TTLL6 ^(f/f))and fifty control(TTLL6_(CON):TTLL6 ^(f/f))mice with C57BL/6J background,aged 6 days old were selected.TTLL6_(CKO) mice were divided into TTLL6_(CON) control group and TTLL6_(CON) sevoflurane group,and TTLL6_(CKO) mice were divided into TTLL6_(CKO) control group and TTLL6_(CKO) sevoflurane group,with 25 mice in each group by random block method.Mice in the sevoflurane groups were exposed to 3%sevoflurane with 60%O_(2) for 2 hours daily on postnatal days 6,8,and 10.The mice in control groups received only 60%O_(2) under the same condition.The polyGlu-Tubulin and postsynaptic density 95(PSD95)protein expression were detected using Western blot.The expressions of TTLL6,Spastin,andα-Tubulin were assessed via immunofluorescence.Golgi staining and electron microscopy were employed to observe the density of hippocampal dendritic spines and synaptic conditions.The Morris water maze test was used to evaluate spatial memory capabilities.Statistical analysis was performed using GraphPad Prism 8.0 software.Results(1)Behavioral results showed significant time and group interactions among the four groups in terms of latency to find the platform(F=8.22,P<0.001).Mice in the TTLL6_(CON) sevoflurane group had a significantly longer escape latency on days 3-7 compared with the TTLL6_(CON) control group(all P<0.05),while there was no significant difference between the TTLL6_(CKO) sevoflurane group and the TTLL6_(CKO) control group(all P>0.05).The number of platform crossings differed significantly among the four groups(H=11.95,P=0.007).The TTLL6_(CON) sevoflurane group had significantly fewer cr

关 键 词：七氟烷 认知功能障碍 树突棘发育 微管蛋白 多聚谷氨酰化微管蛋白 微管切割蛋白 小鼠 

分 类 号：R614[医药卫生—麻醉学]