脑囊尾蚴病脑脊液差异凝胶电泳方法的建立

中国寄生虫学与寄生虫病杂志 ›› 2008, Vol. 26 ›› Issue (3): 4-178.

脑囊尾蚴病脑脊液差异凝胶电泳方法的建立

李静宜¹，田小军¹，黄勇²，杨艳君²，马巧荣²，薛燕萍^{1 *}

1 北京热带医学研究所，首都医科大学附属北京友谊医院，北京 100050；2 山东省寄生虫病防治所，济宁 272033

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-06-30 发布日期:2008-06-30

Establishment of Two-dimensional Differential Gel Electrophoresis Using Cerebrospinal Fluid from Neurocysticercosis Patients

LI Jing-yi¹，TIAN Xiao-jun¹，HUANG Yong²，YANG Yan-jun²，MA Qiao-rong²，XUE Yan-ping^{1 *}

1 Beijing Tropical Medicine Research Institute, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; 2 Shandong Institute of Parasitic Diseases, Jining 272033, China

Received:1900-01-01 Revised:1900-01-01 Online:2008-06-30 Published:2008-06-30

摘要/Abstract

摘要： 目的建立脑脊液差异凝胶电泳方法，以获得高分辨率的荧光差异双向电泳图谱。方法取确诊的4例脑囊尾蚴病患者脑脊液和4位健康人脑脊液，分别用冰丙酮沉淀法除盐提取蛋白，均等量混合后为脑囊尾蚴病组与健康人对照组，两组总蛋白等量混合为内标组。将各组蛋白分别经花青染料2（Cy2）、Cy3和Cy5标记，再进行单向十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和双向差异凝胶电泳（2-D DIGE），分别在波长488 nm、532 nm和633 nm激发光下扫描，所呈图像分别用ImageQuant和DeCyder 5.0图像分析软件进行蛋白表达差异分析。用DeCyder中的胶内差异分析（DIA）模块对2-D DIGE扫描图像进行蛋白质点检测和含量分析。用DeCyder中的生物学差异分析（BVA）模块分析两组蛋白表达的生物学差异。结果 ImageQuant分析结果表明，所有脑脊液全蛋白样品均能被Cy2、Cy3和Cy5标记，荧光强度随蛋白含量的上升而增强。DIA分析显示，胶1和胶2分别检测到896和894个蛋白质点。胶2与胶1进行匹配，蛋白质点匹配率达90%。BVA分析发现，在脑囊尾蚴病组与健康人组脑脊液蛋白共有表达量变化超过两倍的差异蛋白质点55个，其中在脑囊尾蚴病组中表达上调2倍的有47个蛋白质点，下调2倍以上的有8个蛋白质点。结论以脑囊尾蚴病患者脑脊液建立的差异凝胶电泳方法，获得的2-D DIGE图谱，图像清晰，分辨率高，为脑囊尾蚴病蛋白质组学研究奠定了基础。

关键词: 脑囊尾蚴病, 脑脊液, 蛋白质组, 双向差异凝胶电泳

Abstract: Objective To establish the method of two-dimensional differential gel electrophoresis and obtain high resolution 2D images from cerebrospinal fluid （CSF） of patients with neurocysticercosis. Methods CSF samples were collected from four patients diagnosed as neurocysticercosis clinically and by ELISA, computed tomography （CT） or magnetic resonance imaging（MRI）, and from four healthy subjects without neurological disorders. The CSF samples were precipitated with cold acetone, then pooled by equal amount as patients and controls. The internal standard comprised equal amounts of proteins extracted from both groups. Internal standard，and proteins from the two groups were labeled prior to electrophoresis with spectrally resolvable fluorescent dyes，cyanin dye2（Cy2），Cy3 and Cy5. Sodium dodecylsulfonate polyacrylamide gel chromatography （SDS-PAGE） and two-dimensional differential in-gel electrophoresis （2-D DIGE） of labeled samples were then run. The differential expressed proteins showed in the images of SDS-PAGE and 2-D DIGE gels scanned with 488 nm，532 nm and 633 nm wavelength laser were analyzed by ImageQuant and DeCyde 5.0 respectively. Spot detection and quantification was performed for the differential in-gel analysis （DIA） module of DeCyder. Biological variation analysis （BVA） module of DeCyder was matched gel 1 and gel 2 images to provide data on differential protein expression levels between the two groups. Results The ImageQuant result displayed that the CSF protein was compatible with the dye，and the difference of protein amount was revealed by the difference of fluorescence intensity. DIA indicated that there were 896 and 894 protein dots on gel 1 and gel 2 respectively, and 90% of them were matched each other. BVA showed that there were 55 protein spots with different expressional level between neurocysticercosis and control groups. Protein spots with two-fold increase or decrease were 47 and 8 respectively in neurocysticercosis patients compared with healthy controls. Conclusion The method of 2-D DIGE has been established with high-resolution images for the examination of cerebrospinal fluid, providing a foundation for fur-ther study of neurocysticercosis comparative proteomics.

Key words: Neurocysticercosis, Cerebrospinal fluid, Proteomics, Differential gel electrophoresi

李静宜;田小军;黄勇;杨艳君;马巧荣;薛燕萍. 脑囊尾蚴病脑脊液差异凝胶电泳方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2008, 26(3): 4-178.

LIJing-yi;TIANXiao-jun;HUANGYong;YANGYan-jun;MAQiao-rong;XUEYan-ping*. Establishment of Two-dimensional Differential Gel Electrophoresis Using Cerebrospinal Fluid from Neurocysticercosis Patients[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2008, 26(3): 4-178.

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