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国家自然科学基金(81070749)

作品数:6 被引量:5H指数:2
相关作者:阴正勤侯文生姚军平杨红刘慧更多>>
相关机构:第三军医大学西南医院重庆大学更多>>
发文基金:国家自然科学基金重庆市科技攻关计划更多>>
相关领域:医药卫生生物学更多>>

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负载紫红质通道蛋白2重组腺病毒的包装及功能鉴定
2012年
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DFl2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pBHGloxAEI,3Cre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LongEvans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460hill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7.9×10^10PFU/ml,HEK293细胞转染Ad—ChR224h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,460nm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。
姚军平侯文生王浩翁传煌阴正勤
关键词:腺病毒膜片钳
Optogenetics:a novel optical manipulation tool for medical investigation被引量:2
2012年
·Optogenetics is a new and rapidly evolving gene and neuroengineering technology that allows optical control of specific populations of neurons without affecting other neurons in the brain at high temporal and spatial resolution. By heterologous expression of the light-sensitive membrane proteins, cell type-specific depolarization or hyperpolarization can be optically induced on a millisecond time scale. Optogenetics has the higher selectivity and specificity compared to traditional electrophysiological techniques and pharmaceutical methods. It has been a novel promising tool for medical research. Because of easy handling, high temporal and spatial precision, optogenetics has been applied to many aspects of nervous system research, such as tactual neural circuit, visual neural circuit, auditory neural circuit and olfactory neural circuit, as well as research of some neurological diseases. The review highlights the recent advances of optogenetics in medical study.
Jun-Ping Yao,Zheng-Qin Yin
关键词:PROTEINSOPTOGENETICSMANIPULATIONMEDICAL
Perineuronal nets increase inhibitory GABAergic currents during the critical period in rats
2013年
AIM: To investigate inhibitory γ-aminobutyric acid (GABA) ergic postsynaptic currents (IPSCs) and postsynaptic currents (PSCs) in layer IV of the rat visual cortex during the critical period and when plasticity was extended through dissolution of the perineuronal nets (PNNs). METHODS: We employed 24 normal Long-Evans rats to study GABA A-PSC characteristics of neurons within layer IV of the visual cortex during development. The animals were divided into six groups of four rats according to ages at recording: PW3 (P21 -23d), PW4 (P28 -30d), PW5 (P35-37d), PW6 (P42-44d), PW7 (P49-51d), and PW8 (56-58d). An additional 24 chondroitin sulfate proteoglycan (CSPG) degradation rats (also Long-Evans) were generated by making a pattern of injections of chondroitinase ABC (chABC) into the visual cortex 1 week prior to recording at PW3, PW4, PW5, PW6, PW7, and PW8. Immunohistochemistry was used to identify the effect of chABC injection on CSPGs. PSCs were detected with whole-cell patch recordings, and GABA A receptor-mediated IPSCs were pharmacologically isolated. RESULTS: IPSC peak current showed a strong rise in the age-matched control group, peaked at PW5 and were maintained at a roughly constant value thereafter. Although there was a small increase in peak current for the chABC group with age, the peak currents continued to decrease with the delayed highest value at PW6, resulting in significantly different week-by-week com-parison with normal development. IPSC decay time continued to increase until PW7 in the control group, while those in the chABC group were maintained at astable level after an initial increase at PW4. Compared with normal rats, the decay times recorded in the chABC rats were always shorter, which differed significantly at each age. We did not observe any differences in IPSC properties between the age-matched control and penicillinase (P-ase) group. However, the change in IPSCs after chABC treatment was not reflected in the total PSCs or in basic membrane properties in layer IV of the rat visual corte
Hui LiuPeng-Fen GaoHai-Wei XuMing-Ming LiuTao YuJun-Ping YaoZheng-Qin Yin
关键词:POSTSYNAPTICCURRENTSCHONDROITINASECHONDROITINPROTEOGLYCANSWHOLE-CELL
眼球震颤的诊断与治疗研究进展被引量:2
2012年
眼球震颤是常见的眼球运动障碍疾病,临床诊治困难。随着近年眼球运动记录方法的进步和药物、手术及基因治疗的研究进展,为临床该病的诊断和治疗提供了更多的选择,也为眼球震颤患者带来新的希望。本文就该病诊断与治疗的最新研究进展进行了综述。
杨红阴正勤
关键词:眼球震颤
A new measure of nystagmus acuity
2014年
AIM: To construct a new visual acuity measuring function for congenital nystagmus(CN) patients by studying the relationships between acuity,velocities and positions of the eye. ·METHODS: After assessing the relationship between acuity,movement velocities and positions of the eye separately,a new function,which we call the automated nystagmus acuity function(ANAF),was constructed to measure the visual acuity of CN patients. Using a high-speed digital video system working at 500 frames per second,each eye was calibrated during monocular fixation. Twenty-six recorded nystagmus data were selected randomly. Using nystagmus waveforms,the best vision position(foveation period) and visual acuity were analyzed in three groups of subjects,and then all outputs were compared with the well-known expanded nystagmus acuity function(NAFX) and ANAF. Standard descriptive statistics were used to summarize the outputs of the two programs. ·RESULTS: Foveation periods were brief intervals in the CN waveform when the image was on or near the fovea and eye velocity was relatively slow. Results showed good visual acuity happened during the period when velocity was low and the eye position was near the zero position,which fitted the foveation periods. The data analyzed with NAFX and ANAF had a correlation coefficient of 0.934276,with an average error of-0.00973. · CONCLUSION: The results from ANAF and NAFX analyses showed no significant difference. The NAFX manually identifies foveation eye positions and produces accurate measurements. The ANAF,however,can be calculated simply using the factors eye position andvelocity,and it automatically calculates the ANAF without the need to manually identify foveation eye positions.
Jun-Ping YaoZheng TaiZheng-Qin Yin
关键词:NYSTAGMUSVISIONACUITYNYSTAGMUSACUITYMOVEMENTMEASUREMENTS
新生Long Evans大鼠初级视皮层神经元的培养和鉴定被引量:1
2012年
目的建立新生Long Evans大鼠视皮层神经元原代培养方法。方法采用钝性分离1d龄Long Evans大鼠双侧视皮层,2.5g·L-1胰蛋白酶消化法获得细胞悬液,在无血清培养基中进行培养,7~14d后行免疫组织化学及膜片钳鉴定。结果用该方法培养的视皮层神经细胞存活率高,生长状态良好,存活可达1个月左右,β-III tubulin免疫组织化学染色阳性神经元占培养细胞中的80%以上,膜片钳成功记录了180个细胞,静息膜电位在-50~-70mV,存在内向、外向电流,脉冲恒流刺激可诱发多个动作电位。结论本研究所采用的分离及原代培养方法是一种简单、高效的视皮层神经元培养方法。
姚军平侯文生刘慧陈丽峰阴正勤
关键词:视皮层神经元培养
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