專(zhuān)題3 睡眠、應(yīng)激和干細(xì)胞：細(xì)胞與基因調(diào)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(huán)境認(rèn)知損傷及防護(hù)的動(dòng)物行為實(shí)驗(yàn)技術(shù)

劉新民

(中國(guó)醫(yī)學(xué)科學(xué)院藥用植物研究所）

專(zhuān)題3 睡眠、應(yīng)激和干細(xì)胞：細(xì)胞與基因調(diào)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(fù)旦大學(xué)特聘教授、博士生導(dǎo)師，上海醫(yī)學(xué)院藥理學(xué)系主任。擔(dān)任Sleep Biol Rhythms和《中國(guó)臨床藥理學(xué)與治療學(xué)》等雜志副主編、《中國(guó)藥理學(xué)報(bào)》等10個(gè)雜志編委或常務(wù)編委。研究方向:睡眠與失眠機(jī)制、鎮(zhèn)靜催眠藥物開(kāi)發(fā)。發(fā)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(ài)思唯爾中國(guó)論文被高引學(xué)者榜單。主編《藥理學(xué)》，副主編《中藥藥理實(shí)驗(yàn)方法學(xué)》等教材及大型參考書(shū)10余部。應(yīng)邀為T(mén)rends Neurosci,Prog Neurobiol雜志撰寫(xiě)綜述，為PNAS,J Neurosci等20個(gè)SCI雜志審稿。多次應(yīng)邀赴美國(guó)、德國(guó)、法國(guó)、意大利、日本、澳大利亞等國(guó)家作學(xué)術(shù)報(bào)告。2005年獲日本睡眠學(xué)會(huì)優(yōu)秀研究獎(jiǎng)，2006年獲國(guó)家杰出青年科學(xué)基金，2016年獲教育部科學(xué)研究?jī)?yōu)秀成果獎(jiǎng)（自然科學(xué)二等獎(jiǎng)）。

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(dǎo)師，北京大學(xué)基礎(chǔ)醫(yī)學(xué)院藥理學(xué)系副主任。長(zhǎng)期從事睡眠及其共病機(jī)制和催眠新藥研發(fā)等方面的工作。已在國(guó)內(nèi)外雜志上發(fā)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(guó)家重大新藥創(chuàng)制項(xiàng)目、國(guó)家自然科學(xué)基金、教育部博士學(xué)科點(diǎn)專(zhuān)項(xiàng)基金-博導(dǎo)類(lèi)、留學(xué)回國(guó)人員啟動(dòng)基金、美國(guó)中華醫(yī)學(xué)基金-專(zhuān)項(xiàng)人才基金、北京大學(xué)“十五”211工程項(xiàng)目等科研項(xiàng)目。參編出版著作8本，包括八年制《藥理學(xué)》教材、《神經(jīng)精神藥理學(xué)進(jìn)展》、《神經(jīng)藥理學(xué)研究方法》、《神經(jīng)精神藥理學(xué)》和《新藥藥理學(xué)研究方法》等。已授權(quán)國(guó)際專(zhuān)利7項(xiàng)，國(guó)內(nèi)專(zhuān)利1項(xiàng)。授權(quán)專(zhuān)利1項(xiàng)。擔(dān)任中國(guó)藥理學(xué)會(huì)副秘書(shū)長(zhǎng)等學(xué)術(shù)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(huán)境認(rèn)知損傷及防護(hù)的動(dòng)物行為實(shí)驗(yàn)技術(shù)

劉新民

(中國(guó)醫(yī)學(xué)科學(xué)院藥用植物研究所）

劉新民，中國(guó)醫(yī)學(xué)科學(xué)院（北京協(xié)和醫(yī)學(xué)院）教授、博導(dǎo)；世界衛(wèi)生組織傳統(tǒng)醫(yī)學(xué)顧問(wèn)；中國(guó)航天員中心客座研究員、國(guó)家載人航天工程醫(yī)學(xué)領(lǐng)域?qū)＜摇氖禄趧?dòng)物行為實(shí)驗(yàn)的中藥神經(jīng)精神藥理和實(shí)驗(yàn)方法研究三十余年。在國(guó)內(nèi)外學(xué)術(shù)刊物上發(fā)表學(xué)術(shù)論文150多篇，第一或通訊作者80余篇。培養(yǎng)畢業(yè)研究生40余名。

聚焦航天特因環(huán)境應(yīng)激所致認(rèn)知功能下降和損傷的預(yù)測(cè)、預(yù)警和防護(hù)，利用本團(tuán)隊(duì)前期自主研發(fā)的包括獎(jiǎng)賞、被動(dòng)回避和自發(fā)探索原理在內(nèi)的認(rèn)知行為檢測(cè)分析系統(tǒng)，開(kāi)展了狹小空間、節(jié)律紊亂和重力效應(yīng)等多種模擬航天特因環(huán)境對(duì)動(dòng)物認(rèn)知功能損傷評(píng)價(jià)實(shí)驗(yàn)方法研究，建立了復(fù)雜操作任務(wù)下多重認(rèn)知行為作業(yè)的定量檢測(cè)分析。發(fā)現(xiàn)了航天特因模擬環(huán)境對(duì)認(rèn)知作為作業(yè)損傷的效應(yīng)累積強(qiáng)度和時(shí)間節(jié)點(diǎn)，以及不同認(rèn)知作業(yè)的損傷特征。為我國(guó)航天特因環(huán)境所致認(rèn)知損傷監(jiān)測(cè)、預(yù)警與防護(hù)，保障航天飛行的順利進(jìn)行提供了功能完善、體系完整、高自動(dòng)化和智能化的動(dòng)物行為實(shí)驗(yàn)技術(shù)。

航天；認(rèn)知損傷；醫(yī)學(xué)防護(hù)

S3-4 EphB4信號(hào)通路對(duì)人胚胎神經(jīng)干細(xì)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(yī)科大學(xué)宣武醫(yī)院實(shí)驗(yàn)動(dòng)物室，北京 100053）

王 文，教授，研究員，博士生導(dǎo)師，實(shí)驗(yàn)動(dòng)物室主任，北京市衛(wèi)生系統(tǒng)高層次衛(wèi)生技術(shù)人才。從事中藥藥理、神經(jīng)藥理、天然藥物化學(xué)專(zhuān)業(yè)研究工作，主要研究方向?yàn)橹兴?、天然藥物?duì)重大疾病干預(yù)研究。兼任中國(guó)照明學(xué)會(huì)光生物光化學(xué)專(zhuān)業(yè)委員副主任委員；中國(guó)藥理學(xué)會(huì)抗衰老及老年癡呆專(zhuān)業(yè)委員會(huì)秘書(shū)長(zhǎng)；中華預(yù)防醫(yī)學(xué)會(huì)老年病專(zhuān)業(yè)委員會(huì)秘書(shū)長(zhǎng)；中國(guó)老年學(xué)會(huì)抗衰老科學(xué)委員會(huì)秘書(shū)長(zhǎng)；中國(guó)藥理學(xué)會(huì)補(bǔ)益藥藥理專(zhuān)業(yè)委員會(huì)副秘書(shū)長(zhǎng)；中國(guó)藥理學(xué)會(huì)抗炎免疫專(zhuān)業(yè)委員會(huì)理事；中國(guó)醫(yī)師協(xié)會(huì)中西醫(yī)結(jié)合神經(jīng)病專(zhuān)業(yè)委員會(huì)理事；中國(guó)藥理學(xué)會(huì)神經(jīng)藥理專(zhuān)業(yè)委員會(huì)委員等。已發(fā)表論文100多篇，其中SCI收錄16篇。作為課題負(fù)責(zé)人，先后主持了衛(wèi)計(jì)委和科技部牽頭的2個(gè)國(guó)家重大專(zhuān)項(xiàng)“重大新藥創(chuàng)制”和5個(gè)國(guó)家自然科學(xué)基金等十多項(xiàng)科學(xué)研究。參編中國(guó)科協(xié)《2049年的中國(guó)：科技展望》白皮書(shū)，參譯第12版古德曼和吉爾曼的《治療學(xué)的藥理學(xué)基礎(chǔ)》等著作。

摘要：目的本實(shí)驗(yàn)旨在研究EphB4是否參與調(diào)節(jié)了人胚胎神經(jīng)干細(xì)胞的增殖、分化、凋亡活動(dòng)，并探索其下游信號(hào)通路。方法培養(yǎng)原代人胚胎神經(jīng)干細(xì)胞，使用沉默慢病毒與過(guò)表達(dá)慢病毒轉(zhuǎn)染細(xì)胞，分別下調(diào)和上調(diào)EphB4蛋白表達(dá)水平。檢測(cè)EphB4對(duì)細(xì)胞增殖、分化、凋亡的影響并探索其下游信號(hào)通路。結(jié)果EphB4基因沉默后，抑制細(xì)胞增殖及向神經(jīng)元分化，促進(jìn)細(xì)胞向膠質(zhì)細(xì)胞分化，對(duì)細(xì)胞凋亡無(wú)影響。EphB4基因過(guò)表達(dá)后，促進(jìn)細(xì)胞增殖及向神經(jīng)元分化，抑制細(xì)胞向膠質(zhì)細(xì)胞分化，對(duì)細(xì)胞凋亡無(wú)影響。EphB4是通過(guò)下游信號(hào)通路Abl-Cyclin D1調(diào)節(jié)細(xì)胞增殖，此信號(hào)通路不參與EphB4在細(xì)胞分化方面的調(diào)節(jié)。結(jié)論EphB4參與調(diào)節(jié)神經(jīng)干細(xì)胞增殖，而且是決定神經(jīng)干細(xì)胞向神經(jīng)元分化還是向膠質(zhì)細(xì)胞分化的開(kāi)關(guān)，是調(diào)節(jié)干細(xì)胞增殖、遷移和分化的新信號(hào)通路，其很可能成為腦卒中后神經(jīng)元修復(fù)的有效治療靶點(diǎn)。

關(guān)鍵詞：EphB4信號(hào)；人胚胎神經(jīng)干細(xì)胞；細(xì)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）