CAJ | 학술논문

背景紫红质通道蛋白2(ChR2)是从单细胞绿藻莱茵衣藻上分离的一种光敏感通道蛋白,可作为光刺激神经细胞的手段,与电、磁和超声波相比,光在时间和空间上对神经细胞的刺激将更精确. 目的构建负载ChR2基因的重组腺病毒,并鉴定其功能.方法将人胚肾293( HEK293)细胞在含质量分数10％胎牛血清的DF12培养基中进行培养及传代.腺病毒穿梭质粒pSB291-hCHR2-GFP与腺病毒包装质粒pBHGlox △E1,3 Cre共转染HEK293,包装得到少量负载ChR2基因的重组腺病毒(Ad-ChR2),经HEK293细胞扩增、CsCl梯度离心,Tris-HCl透析后得到纯化Ad-ChR2.获取4只出生1d的清洁级Long Evans大鼠视皮层组织,用组织块培养法利用无血清培养基原代培养视皮层细胞,用纯化的Ad-ChR2转染培养的视皮层细胞,当转染成功的视皮层细胞表达绿色荧光时给予460 nm蓝光刺激,应用膜片钳技术记录视皮层细胞的动作电位.结果纯化浓缩后的Ad-ChR2滴度可达7.9×1010PFU/ml,HEK293细胞转染Ad-ChR2 24 h后倒置荧光显微镜下即可见细胞膜上有绿色荧光表达,转染13d可见细胞由扁平变为圆形.无血清培养的视皮层细胞转染纯化的Ad-ChR2后细胞膜上可见绿色荧光蛋白(GFP)的表达,460 nm蓝光刺激后用膜片钳技术可记录到蓝光诱发的视皮质细胞的动作电位.结论本研究成功构建了负载ChR2基因的重组腺病毒表达载体,并证实ChR2能够感染有功能的视皮质细胞,这对视觉可塑性方面的研究非常重要.
배경 자홍질통도단백2(ChR2)시종단세포록조래인의조상분리적일충광민감통도단백,가작위광자격신경세포적수단,여전、자화초성파상비,광재시간화공간상대신경세포적자격장경정학. 목적 구건부재ChR2기인적중조선병독,병감정기공능.방법 장인배신293( HEK293)세포재함질량분수10％태우혈청적DF12배양기중진행배양급전대.선병독천사질립pSB291-hCHR2-GFP여선병독포장질립pBHGlox △E1,3 Cre공전염HEK293,포장득도소량부재ChR2기인적중조선병독(Ad-ChR2),경HEK293세포확증、CsCl제도리심,Tris-HCl투석후득도순화Ad-ChR2.획취4지출생1d적청길급Long Evans대서시피층조직,용조직괴배양법이용무혈청배양기원대배양시피층세포,용순화적Ad-ChR2전염배양적시피층세포,당전염성공적시피층세포표체록색형광시급여460 nm람광자격,응용막편겸기술기록시피층세포적동작전위.결과 순화농축후적Ad-ChR2적도가체7.9×1010PFU/ml,HEK293세포전염Ad-ChR2 24 h후도치형광현미경하즉가견세포막상유록색형광표체,전염13d가견세포유편평변위원형.무혈청배양적시피층세포전염순화적Ad-ChR2후세포막상가견록색형광단백(GFP)적표체,460 nm람광자격후용막편겸기술가기록도람광유발적시피질세포적동작전위.결론 본연구성공구건료부재ChR2기인적중조선병독표체재체,병증실ChR2능구감염유공능적시피질세포,저대시각가소성방면적연구비상중요.
Background Channelrhodopsin-2 (ChR2)is a cation channel isolated from the eyespot of Chlamydomonas algae and has been used to control neuron activity.The light stimulation is a more precise fashion whether space or time than that of electrical,magnetic and ultrasound stimulation. Objective This study was to construct a replication deficient recombinant adenovirus cxpression vector of ChR2 and to determine its function.Methods Human embryo kidney 293 (HEK293) cell line was cultured and passaged in DF12 medium containing 10％ fetal bovine serum(FBS).The ChR2 gene was cloned at the downstream of cytomegalovirus(CMV)promoter of the adenoviral shuttle plasmid pSB291 in sense direction,and the resultant recombinant plasmid pSB291-hChR2- GFP was transfected into HEK293 cell together with plasmid pBHG lox ( deltaE1,3 ) containing adenoviral genome,then small amounts replication deficient recombinant adenovirus expression vector of ChR2 (Ad-ChR2) was obtained.Through amplification gradient centrifugation and dialysis,pure Ad-ChR2 was obtained.Visual cortex cells derived from 4 1-day-old clean Long Evans rats were primary cultured with serum-free culture media and infected by AdChR2.When expressing green fluorescencc,those cells received the stimulated of blue light with 460 nm.Patch clamp technique was applied to record an action potential. Results After purification and concentration,the titer of AdhCHR2 reached 7.9×1010 PFU/ml.Twenty-four hours after transfect of Ad-ChR2,HEK293 cell membrane showed the green fluorescence for the recombinant plasmid with green fluorescence protein under the inversed fluorescence microscope.The HEK293 cells change their shape from flat to round 13 days after transfected.The primary cultured visual cortex cells exhibited the green fluorescence 3-5 days after infected by Ad-ChR2.The action potentials evoked by blue light stimulation were recorded with patch clamp on those cells expressing green fluorescence. Conclusions Ad-ChR2 expressing vector is constructed successfully in this study.It is verified that Ad-ChR2 expressing vector can infect visual cortex cells with visual function.This result is very important for visual plasticity study.