抑制雷帕霉素靶蛋白复合物1信号通路可改善孤独症谱系障碍模型BTBR小鼠的症状

发布时间：2018-04-09 05:02

  本文选题：孤独症谱系障碍　切入点：雷帕霉素靶蛋白复合物　出处：《上海医学》2017年02期

【摘要】：目的探讨抑制孤独症谱系障碍模型BTBR小鼠的中枢雷帕霉素靶蛋白复合物1(mTORC1)信号通路能否改善其交流和社交障碍。方法选择6只6周龄C57BL/6J(B6)小鼠和18只6周龄BTBR小鼠,每笼3只小鼠,自由进食和饮水。将6只B6小鼠(B6组)和6只BTBR(BTBR组)小鼠适应性饲养1周后,进行交流障碍实验和社交行为实验,实验结束后处死两组小鼠,提取其大脑额叶皮层组织抽提蛋白进行后续Western免疫印迹实验。将余12只6周龄BTBR小鼠分成干预组和对照组,每组6只,分别于第三脑室注射2\mL带有短发夹RNA(shRna)-Raptor的慢病毒和带有shRna阴性对照的慢病毒[滴度为1×10~(10)空斑形成单位(PFU)/mL\],注射2周后进行前述的行为实验和Western免疫印迹实验。观察并记录每只小鼠10min内吸嗅蘸有鼠尿、乙醇和0.9%氯化钠溶液的棉签所用的时间,以及社交行为(身体接触和跟随同伴)和非社交行为(自我理毛和区域探索)的发生次数;检测小鼠前额叶皮层Raptor蛋白或RNA,以及磷酸化蛋白S6(pS6)的蛋白表达情况。结果交流障碍实验结果显示,BTBR组小鼠吸嗅鼠尿的时间显著短于B6组小鼠(P0.05),两组小鼠吸嗅0.9%氯化钠溶液和乙醇的时间的差异均无统计学意义(P值均0.05)。社交行为实验结果显示,BTBR组小鼠的身体接触和跟随同伴的次数均显著少于B6组小鼠(P值均0.05),自我理毛的次数显著多于B6组小鼠(P0.05);两组小鼠区域探索次数的差异无统计学意义(P0.05)。Western免疫印迹实验结果显示,BTBR组小鼠前额叶皮层mTORC1信号通路下游pS6的蛋白相对表达量显著高于B6组(P0.05)。经慢病毒干预后,实时定量PCR结果显示,干预组小鼠前额叶皮层Raptor mRNA的相对表达量显著低于对照组(P0.05);Western免疫印迹实验结果显示,干预组小鼠前额叶皮层Raptor蛋白和mTORC1信号下游pS6的蛋白相对表达量均显著低于对照组(P值均0.05);交流障碍实验结果显示,干预组小鼠吸嗅鼠尿的时间显著长于对照组(P0.05),两组小鼠吸嗅0.9%氯化钠溶液和乙醇的时间的差异均无统计学意义(P值均0.05);社交行为实验结果显示,干预组小鼠的身体接触次数显著多于对照组(P0.05),自我理毛次数显著少于对照组(P0.05),两组小鼠跟随同伴和区域探索次数的差异均无统计学意义(P值均0.05)。结论抑制孤独症谱系障碍模型BTBR小鼠的中枢mTORC1信号通路能明显改善其社交障碍和刻板动作,该小鼠的孤独症谱系障碍样表现与其中枢异常激活的mTORC1信号通路明显相关。
[Abstract]:Objective to investigate whether the central rapamycin target protein complex 1 mTORC1 signal pathway can improve communication and social disturbance in BTBR mice model of autism spectrum disorders.Methods six 6-week-old C57BL / 6JJ B6) mice and 18 6-week-old BTBR mice were selected.Six B6 mice and six BTBR(BTBR groups were fed adaptively for one week, then the communication disorder test and the social behavior experiment were carried out. After the experiment, the mice of the two groups were killed.The protein was extracted from the frontal cortex of the brain for Western imprinting.The remaining 12 6-week-old BTBR mice were divided into intervention group (n = 6) and control group (n = 6).We injected 2\ mL lentivirus with short hairpin RNA(shRna)-Raptor into the third ventricle and lentivirus with shRna negative control (titer 1 脳 10 ~ (10)) of plaque formation unit (PFU / P / mL\). After 2 weeks of injection, the behavioral tests and Western Western blot tests were performed.To observe and record the time of sniffing and sniffing with mouse urine, ethanol and 0.9% sodium chloride solution cotton swabs, as well as the occurrence times of social behavior (physical contact and companion) and non-social behavior (self-hairdressing and regional exploration) in each mouse.The expression of Raptor protein and phosphorylated protein S6 pS6 in prefrontal cortex of mice were detected.Results the time of sniffing rat urine in BTBR group was significantly shorter than that in B6 group (P 0.05). There was no significant difference in the time of sniffing 0.9% sodium chloride solution and ethanol between the two groups.The results of social behavior test showed that the number of physical contact and companion following in BTBR group was significantly lower than that in B6 group (P = 0.05), and the number of self-hairdressing was significantly higher than that in B6 group (P 0.05), and there was no difference in the number of regional exploration between the two groups.The results of Western blot analysis showed that the relative expression of pS6 protein downstream of mTORC1 signaling pathway in prefrontal cortex of B6 group was significantly higher than that of B6 group.The results of real-time quantitative PCR showed that the relative expression of Raptor mRNA in the prefrontal cortex of the intervention group was significantly lower than that in the control group.The relative expression of Raptor protein in prefrontal cortex and pS6 downstream of mTORC1 signal in intervention group was significantly lower than that in control group (P < 0.05).The time of sniffing rat urine in the intervention group was significantly longer than that in the control group (P0.05A), and there was no significant difference in the time of sniffing 0.9% sodium chloride solution and ethanol between the two groups (P = 0.05).The number of physical contact in the intervention group was significantly higher than that in the control group (P 0.05), and the number of self-hairdressing was significantly lower than that in the control group (P 0.05). There was no significant difference between the two groups in the number of follow companions and the number of regional explorations (P < 0.05).Conclusion inhibition of the central mTORC1 signaling pathway in BTBR mice with autism spectrum disorders can significantly improve its social disorders and stereotypical movements. The autism spectrum disorders in this mouse are significantly correlated with the abnormal activation of the mTORC1 signaling pathway in the central nervous system.
【作者单位】： 上海交通大学医学院附属精神卫生中心儿少科;
【分类号】：R-332;R749.94
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本文编号：1724942