專題3 睡眠、應激和干細胞：細胞與基因調控

HUANG Zhi-li

(Department of Pharmacology,School of Basic Medical Sciences;State Key Laboratory of Medical Neurobiology;Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Shanghai Medical College,Fudan University,Shanghai 200032,China)

S3-2 In the dorsal raphe neucleus,the role of Ca2+,PKC and CaMKⅡin sleep-wake regulation

CUI Su-ying,CUI Xiang-yu,ZHANG Yong-he

(Department of Pharmacology,School of Basic Medical Science,Peking University,Beijing,China)

S3-3面向航天特因環境認知損傷及防護的動物行為實驗技術

劉新民

(中國醫學科學院藥用植物研究所）

專題3 睡眠、應激和干細胞：細胞與基因調控

S3-1 Genetically engineered systems revealed the roles of basal ganglia in sleep-wake regulation

HUANG Zhi-li

(Department of Pharmacology,School of Basic Medical Sciences;State Key Laboratory of Medical Neurobiology;Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Shanghai Medical College,Fudan University,Shanghai 200032,China)

黃志力，復旦大學特聘教授、博士生導師，上海醫學院藥理學系主任。擔任Sleep Biol Rhythms和《中國臨床藥理學與治療學》等雜志副主編、《中國藥理學報》等10個雜志編委或常務編委。研究方向:睡眠與失眠機制、鎮靜催眠藥物開發。發表SCI論文80篇，包括Nature Neuroscience,Light: Science&Applications，PNAS，Neuropsychopharmacology,Journal of Neuroscience等。2014，2015，2016年被列入愛思唯爾中國論文被高引學者榜單。主編《藥理學》，副主編《中藥藥理實驗方法學》等教材及大型參考書10余部。應邀為Trends Neurosci,Prog Neurobiol雜志撰寫綜述，為PNAS,J Neurosci等20個SCI雜志審稿。多次應邀赴美國、德國、法國、意大利、日本、澳大利亞等國家作學術報告。2005年獲日本睡眠學會優秀研究獎，2006年獲國家杰出青年科學基金，2016年獲教育部科學研究優秀成果獎（自然科學二等獎）。

S3-2 In the dorsal raphe neucleus,the role of Ca2+,PKC and CaMKⅡin sleep-wake regulation

CUI Su-ying,CUI Xiang-yu,ZHANG Yong-he

(Department of Pharmacology,School of Basic Medical Science,Peking University,Beijing,China)

張永鶴，博士，教授，博士生導師，北京大學基礎醫學院藥理學系副主任。長期從事睡眠及其共病機制和催眠新藥研發等方面的工作。已在國內外雜志上發表論文80余篇，其中SCI收錄論文60余篇。先后主持了科技部“十一五”國家重大新藥創制項目、國家自然科學基金、教育部博士學科點專項基金-博導類、留學回國人員啟動基金、美國中華醫學基金-專項人才基金、北京大學“十五”211工程項目等科研項目。參編出版著作8本，包括八年制《藥理學》教材、《神經精神藥理學進展》、《神經藥理學研究方法》、《神經精神藥理學》和《新藥藥理學研究方法》等。已授權國際專利7項，國內專利1項。授權專利1項。擔任中國藥理學會副秘書長等學術兼職。

Abstract:OBJECTIVEDorsal raphe nucleus(DRN)is the largest single collection of neurons containing 5-HT in the entire brain and particularly attractive in a wide variety of complex physiological and behav?ioral processes,such as sleep-wake regulation.Calmodulin dependent kinaseⅡ(CaMKⅡ)and protein kinase C(PKC)are important signal-transducing molecules activated by Ca2+.Since the Ca2+modulation in DRN plays an important role in sleep-wake regulation,it should be presumed that the intracellular CaMKⅡ/PKC signaling in DRN may be involved in the regulation of sleep-wake.METHODSThe poly?somnogram consisting of EEG and EMG was recorded for analyzing sleep architecture.Immunohisto?chemisrty and Western-blotting methods were used in this study to investigate the roles of Ca2+,CaMKⅡand PKC in sleep-wake regulation in rat DRN.RESULTSCa2+in the DRN exert arousal effects by reducing the NREMs,SWS and REMs via up-regulating serotonergic functions and activating CaMKⅡ-PKC. However,inhibition of PKC leads to significant promotion of total sleep time especially the NREM sleep, but there were no changes in sleep parameters after the inhibition of CaMKⅡby its inhibitor KN-93 in DRN.CONCLUSIONThe molecular,pharmacological,and behavioral findings of this study demonstrate a novel wake promoting and sleep-suppressing role for the Ca2+/CaMKⅡ/PKC signaling pathway in DRN.Abnormalities in CaMKⅡ are found in patients with several neurological disorders that are associated with disturbed sleep,such as schizophrenia,depression,and Alzheimer′s disease.Several psychotropic drugs modulate CaMKⅡ activity.In addition,PKC is a cellular target of most current mood stabilizing and anti-manic agents and involved in bipolar disorder.The data of the present study raise the question whether PKC or CaMKⅡmodulations may also be effective on the sleep disorders or the mood disorders associated with sleep disorders.

Key words:calmodulin dependent kinaseⅡ;protein kinase C;sleep

Corresponding author:ZHANG Yong-he,E-mail:zhyh@hsc.pku.edu.cn

S3-3面向航天特因環境認知損傷及防護的動物行為實驗技術

劉新民

(中國醫學科學院藥用植物研究所）

劉新民，中國醫學科學院（北京協和醫學院）教授、博導；世界衛生組織傳統醫學顧問；中國航天員中心客座研究員、國家載人航天工程醫學領域專家。從事基于動物行為實驗的中藥神經精神藥理和實驗方法研究三十余年。在國內外學術刊物上發表學術論文150多篇，第一或通訊作者80余篇。培養畢業研究生40余名。

聚焦航天特因環境應激所致認知功能下降和損傷的預測、預警和防護，利用本團隊前期自主研發的包括獎賞、被動回避和自發探索原理在內的認知行為檢測分析系統，開展了狹小空間、節律紊亂和重力效應等多種模擬航天特因環境對動物認知功能損傷評價實驗方法研究，建立了復雜操作任務下多重認知行為作業的定量檢測分析。發現了航天特因模擬環境對認知作為作業損傷的效應累積強度和時間節點，以及不同認知作業的損傷特征。為我國航天特因環境所致認知損傷監測、預警與防護，保障航天飛行的順利進行提供了功能完善、體系完整、高自動化和智能化的動物行為實驗技術。

航天；認知損傷；醫學防護

S3-4 EphB4信號通路對人胚胎神經干細胞自我更新、增殖、分化和凋亡的影響

王 文，劉婷婷，孫芳玲，魏仁平，艾厚喜，郭德玉，田 欣，祝自新，鄭文榮，王宇峰

（首都醫科大學宣武醫院實驗動物室，北京 100053）

王 文，教授，研究員，博士生導師，實驗動物室主任，北京市衛生系統高層次衛生技術人才。從事中藥藥理、神經藥理、天然藥物化學專業研究工作，主要研究方向為中藥、天然藥物對重大疾病干預研究。兼任中國照明學會光生物光化學專業委員副主任委員；中國藥理學會抗衰老及老年癡呆專業委員會秘書長；中華預防醫學會老年病專業委員會秘書長；中國老年學會抗衰老科學委員會秘書長；中國藥理學會補益藥藥理專業委員會副秘書長；中國藥理學會抗炎免疫專業委員會理事；中國醫師協會中西醫結合神經病專業委員會理事；中國藥理學會神經藥理專業委員會委員等。已發表論文100多篇，其中SCI收錄16篇。作為課題負責人，先后主持了衛計委和科技部牽頭的2個國家重大專項“重大新藥創制”和5個國家自然科學基金等十多項科學研究。參編中國科協《2049年的中國：科技展望》白皮書，參譯第12版古德曼和吉爾曼的《治療學的藥理學基礎》等著作。

摘要：目的本實驗旨在研究EphB4是否參與調節了人胚胎神經干細胞的增殖、分化、凋亡活動，并探索其下游信號通路。方法培養原代人胚胎神經干細胞，使用沉默慢病毒與過表達慢病毒轉染細胞，分別下調和上調EphB4蛋白表達水平。檢測EphB4對細胞增殖、分化、凋亡的影響并探索其下游信號通路。結果EphB4基因沉默后，抑制細胞增殖及向神經元分化，促進細胞向膠質細胞分化，對細胞凋亡無影響。EphB4基因過表達后，促進細胞增殖及向神經元分化，抑制細胞向膠質細胞分化，對細胞凋亡無影響。EphB4是通過下游信號通路Abl-Cyclin D1調節細胞增殖，此信號通路不參與EphB4在細胞分化方面的調節。結論EphB4參與調節神經干細胞增殖，而且是決定神經干細胞向神經元分化還是向膠質細胞分化的開關，是調節干細胞增殖、遷移和分化的新信號通路，其很可能成為腦卒中后神經元修復的有效治療靶點。

關鍵詞：EphB4信號；人胚胎神經干細胞；細胞凋亡

S3-5 Preparation and evaluation of silymarin nanosuspensions for protective effects on stress-induced liver injury

WANG Xiao-dan1＊，HAO Ji-fu2＊，CHEN Wei1，WANG Hao1，WANG De-cai1，ZHANG Han-ting1，3

(1.Institute of Pharmacology，2.School of Pharmaccutical Sciences，Taishan Medical University,Tai′an 271016，China;3.Departments of Behavioral Medicine&Psychiatry and Physiology&Pharmacology，West Virginia University Health Sciences Center,Morgantown,WV26506,USA)

Abstract:OBJECTIVETo fabricate Silymarin(SM)nanosuspensions(NSs)and evaluate their protective effect on stress-induced liver injury.METHODSSM nanosuspensions were tailored by combination of the anti-solvent precipitation and high pressure homogenization(HPH);the formulations were optimized by central composite design.The pharmacokinetics and pharmacodynamics of SM-NSs were also performed.RESULTSIn light of the quadratic mathematical equations derived from the Design of Expert Software,the optimal formulation of SM-NSs consisted of PVP 0.34%and F188 0.36%.The morphology of NSs was found to be spherical with a diameter of about 150 nm using transmission electron microscope（TEM）observation.The pharmacokinetics experiment demonstrated that oral administration of SM-NSs signifi?cantly increased its bioavailability compared to the coarse powder(Cmax:9.03±2.39mg·L-1；AUMC0→∞：3757.35±227.19 mg·L-1·h;AUC0→∞：171.84±26.61 mg·L-1·h).In pharmacodynamics,it was found that restraint stress produced oxidative effects and increased serum AST and ALT levels in mice,both of which were significantly inhibited by SM and SM-NSs;in addition,administration of SM-NSs showed more effective prevention against acute liver injury than SM coarse suspensions(r2=0.986,0.984,P＜0.05).CONCLUSIONThe results suggest that fabricated SM-NSs exert potent hepatoprotective effects and attenuate restraint stress-induced liver injury.The study provides an effective approach to improving the property of SM,which can be used for treatment of liver diseases.

Key words:silymarin;nanosuspensions;pharmcokinetics;stress-induced liver injury

Foundation item:The project supported by Natural Science Foundation of Shandong Province (ZR2014HL103,ZR2016HM21,J13LM51);Taishan Medical University Foundation(2014GCC15);and the Foundation of Overseas Distinguished Taishan Scholars of Shandong Province,China

Corresponding author:ZHANG Han-ting,E-mail:hzhang@hsc.wvu.edu,Tel:(0538)6231386

＊Co-first author.

The basal ganglia(BG)act as a cohesive functional unit that regulates motor function,habit formation,and reward/addictive behaviors.However,it is still not well understood how the BG maintains wakefulness and suppresses sleep to achieve all these fundamental functions until genetically engineered systems developed these years.Significant research efforts have recently been directed at developing genetic-molecular tools to achieve reversible and cell-type specificin vivosilencing or activation of neurons in behaving animals.Optogenetic tools can be used both to specifically activate or inhibit neurons of interest and identify functional synaptic connectivity between specific neuronal populations,bothin vivoand in brain slices.Another recently developed system by Roth and colleagues permits the selective and″remote″manipulation(activation and silencing)of neuronal activity via all 3 major GPCR signaling pathways(Gi,Gsand Gq).These so-called″designer receptors exclusively activated by designer drugs″(DREADD)involve mutant GPCRs that do not respond to their endogenous ligands but are responsive to otherwise inert biological compounds.Recently,we demonstrated the essential roles and the neural pathways of the neurons expressing adenosine A2Areceptors or dopamine D1receptors in the BG for sleep-wake regulation using the genetically engineered systems including optogenetics and DREADD.We proposed a plausible model in which the caudate-putamen and the nucleus accumbens integrates behavioral processes with sleep/wakefulness through adenosine and dopamine receptors.

adeno-associated virus;optogenetics;DREADD;basal ganglia;sleep-wake regulation

HUANG Zhi-li,E-mail:huangzl@fudan.edu.cn

The project supported by National Natural Science Foundation of China(81573407, 81302746,81202511,81173031);National Mega-project of Scicence Research of China for New Drug Development(2009ZX09103-124);and Research Fund for the Doctoral Program of Higher Eductaion of China（20100001110048）