本文選題：孤獨(dú)癥譜系障礙　切入點(diǎn)：雷帕霉素靶蛋白復(fù)合物　出處：《上海醫(yī)學(xué)》2017年02期

【摘要】：目的探討抑制孤獨(dú)癥譜系障礙模型BTBR小鼠的中樞雷帕霉素靶蛋白復(fù)合物1(mTORC1)信號(hào)通路能否改善其交流和社交障礙。方法選擇6只6周齡C57BL/6J(B6)小鼠和18只6周齡BTBR小鼠,每籠3只小鼠,自由進(jìn)食和飲水。將6只B6小鼠(B6組)和6只BTBR(BTBR組)小鼠適應(yīng)性飼養(yǎng)1周后,進(jìn)行交流障礙實(shí)驗(yàn)和社交行為實(shí)驗(yàn),實(shí)驗(yàn)結(jié)束后處死兩組小鼠,提取其大腦額葉皮層組織抽提蛋白進(jìn)行后續(xù)Western免疫印跡實(shí)驗(yàn)。將余12只6周齡BTBR小鼠分成干預(yù)組和對(duì)照組,每組6只,分別于第三腦室注射2\mL帶有短發(fā)夾RNA(shRna)-Raptor的慢病毒和帶有shRna陰性對(duì)照的慢病毒[滴度為1×10~(10)空斑形成單位(PFU)/mL\],注射2周后進(jìn)行前述的行為實(shí)驗(yàn)和Western免疫印跡實(shí)驗(yàn)。觀察并記錄每只小鼠10min內(nèi)吸嗅蘸有鼠尿、乙醇和0.9%氯化鈉溶液的棉簽所用的時(shí)間,以及社交行為(身體接觸和跟隨同伴)和非社交行為(自我理毛和區(qū)域探索)的發(fā)生次數(shù);檢測(cè)小鼠前額葉皮層Raptor蛋白或RNA,以及磷酸化蛋白S6(pS6)的蛋白表達(dá)情況。結(jié)果交流障礙實(shí)驗(yàn)結(jié)果顯示,BTBR組小鼠吸嗅鼠尿的時(shí)間顯著短于B6組小鼠(P0.05),兩組小鼠吸嗅0.9%氯化鈉溶液和乙醇的時(shí)間的差異均無(wú)統(tǒng)計(jì)學(xué)意義(P值均0.05)。社交行為實(shí)驗(yàn)結(jié)果顯示,BTBR組小鼠的身體接觸和跟隨同伴的次數(shù)均顯著少于B6組小鼠(P值均0.05),自我理毛的次數(shù)顯著多于B6組小鼠(P0.05);兩組小鼠區(qū)域探索次數(shù)的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。Western免疫印跡實(shí)驗(yàn)結(jié)果顯示,BTBR組小鼠前額葉皮層mTORC1信號(hào)通路下游pS6的蛋白相對(duì)表達(dá)量顯著高于B6組(P0.05)。經(jīng)慢病毒干預(yù)后,實(shí)時(shí)定量PCR結(jié)果顯示,干預(yù)組小鼠前額葉皮層Raptor mRNA的相對(duì)表達(dá)量顯著低于對(duì)照組(P0.05);Western免疫印跡實(shí)驗(yàn)結(jié)果顯示,干預(yù)組小鼠前額葉皮層Raptor蛋白和mTORC1信號(hào)下游pS6的蛋白相對(duì)表達(dá)量均顯著低于對(duì)照組(P值均0.05);交流障礙實(shí)驗(yàn)結(jié)果顯示,干預(yù)組小鼠吸嗅鼠尿的時(shí)間顯著長(zhǎng)于對(duì)照組(P0.05),兩組小鼠吸嗅0.9%氯化鈉溶液和乙醇的時(shí)間的差異均無(wú)統(tǒng)計(jì)學(xué)意義(P值均0.05);社交行為實(shí)驗(yàn)結(jié)果顯示,干預(yù)組小鼠的身體接觸次數(shù)顯著多于對(duì)照組(P0.05),自我理毛次數(shù)顯著少于對(duì)照組(P0.05),兩組小鼠跟隨同伴和區(qū)域探索次數(shù)的差異均無(wú)統(tǒng)計(jì)學(xué)意義(P值均0.05)。結(jié)論抑制孤獨(dú)癥譜系障礙模型BTBR小鼠的中樞mTORC1信號(hào)通路能明顯改善其社交障礙和刻板動(dòng)作,該小鼠的孤獨(dú)癥譜系障礙樣表現(xiàn)與其中樞異常激活的mTORC1信號(hào)通路明顯相關(guān)。
[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.
【作者單位】： 上海交通大學(xué)醫(yī)學(xué)院附屬精神衛(wèi)生中心兒少科;
【分類號(hào)】：R-332;R749.94
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本文編號(hào)：1724942