建立人肾小管上皮细胞蛋白质组双向电泳的分离体系

中国组织工程研究与临床康复第 14 卷 第 24 期 2010–06–11 出版Vol.14, No.24Journal of Clinical Rehabilitative Tissue Engineering Research June 11, 2010建立人肾小管上皮细胞蛋白质组双向电泳的分离体系★◆张春燕1，刘友平1，杨 烨1，龚 舒2，李 洪1，2Establishment of two-dimensional electrophoresis separation system for proteomes of human kidney tubular epithelial cellsZhang Chun-yan1, Liu You-ping1, Yang Ye1, Gong Shu2, Li Hong1, 2Abstract1Department of Biochemistry, 2Key Laboratory of Sichuan Higher Education Institute for Human Disease Cell Signal and Regulation, Luzhou Medical College, Luzhou 646000, Sichuan Province, China Zhang Chun-yan★, Master, Teaching assistant, Department of Biochemistry, Luzhou Medical College, Luzhou 646000, Sichuan Province, China zcy_2004_116@tom. com. Correspondence to: Li Hong, Professor, Master’s supervisor, Department of Biochemistry, Key Laboratory of Sichuan Higher Education Institute for Human Disease Cell Signal and Regulation, Luzhou Medical College, Luzhou 646000, Sichuan Province, China lihong7188@ Received: 2010-01-12 Accepted: 2010-02-21BACKGROUND: Two-dimensional electrophoresis (2-DE) separation technology is one of core technologies for proteomics studies. However, the resolution of 2-DE map is highly altered due to different experimental conditions. Therefore, a 2-DE map with high resolution can be obtained via the optimization of key experimental conditions for a definite proteome sample. OBJECTIVE: To establish an effective separation system of 2-DE for the proteome of HK-2 human renal tubular epithelial cells. METHODS: HK-2 human renal tubular epithelial cells were cultured routinely. The proteins were extracted from HK-2 cells after lysing, and separated by 2-DE on the standard experimental condition with an optimization for each important experimental parameter. Electrophoresis parameters recommended by Bio-Rad Corporation were adjusted adequately. In the process of silver staining, a sufficient washing operation ensured a weak background of 2-DE maps. The gray densities and numbers of protein spots, resolution and contaminating strands in 2-DE maps were determined. RESULTS AND CONCLUSION: A 2-DE separation system for proteome of human renal tubular epithelial cells was successfully established. The optimized experimental conditions were selected finally as follows: The lysis buffer containing 1% TBP, 4% CHAPS, 0.2% Bio-Lyte, 40 mmol/L Tris, 8 mol/L urea, 2 mol/L thiourea was applied to extract proteins from cells, and the passive rehydration proposal of samples was used to help the separation of large molecular weight proteins on the pH 4-7 IPG gels. Electrophoresis parameters recommended by Bio-Rad Corporation were adjusted adequately. In the process of silver staining, a sufficient washing operation ensured a weak background of 2-DE maps. Under the optimized conditions, 2-DE maps with high resolution have been reproducibly obtained. Zhang CY, Liu YP, Yang Y, Gong S, Li H. Establishment of two-dimensional electrophoresis separation system for proteomes of human kidney tubular epithelial cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2010;14(24): 4416-4420. [ ]摘要背景：双向电泳分离技术是蛋白质组学研究的核心技术之一，但蛋白质样品的分离效果受各种实验条件的影响较大。

因此， 针对不同来源的蛋白样品进行实验条件的优化可获得具有较高分辨率的双向电泳图谱。

目的：拟建立优化的人肾小管上皮细胞株蛋白质组双向电泳分离体系。

方法：常规培养人肾小管上皮细胞株 HK-2 细胞并裂解提取全蛋白，按标准条件对蛋白质进行双向电泳分离，并对各个关键 因素进行优化。

等电聚焦采用缓慢升压模式，电泳参数根据 Bio-Rad 公司的预设方案进行调整。

改良硝酸银法进行蛋白质斑 点染色。

采集电泳图谱并分析双向电泳图谱中蛋白斑点的数量、图像分辨率及背景条纹的变化。

结果与结论：通过对实验条件的筛选和优化，成功建立了具有较高的分辨率和重复性的人肾小管上皮细胞蛋白质组双向电泳 分离体系。

其中，优化后的裂解液配方成分为 1% TBP，4%CHAPS，0.2% Bio-Lyte，40 mmol/L Tris，8 mol/L 尿素，2 mol/L 硫脲；采用 pH 4~7 的 IPG 胶条；上样方式选择被动的水化上样。

等电聚焦过程中使用预设的缓慢升压模式，充分聚焦后选 用合适的电压模式进行 SDS-PAGE 电泳，然后采用改良硝酸银法进行染色，最终获得了满意的蛋白质组双向电泳图谱。

关键词：双向电泳；蛋白质组；人肾小管上皮细胞；优化；分离体系 doi:10.3969/j.issn.1673-8225.2010.24.011 张春燕，刘友平，杨烨，龚舒，李洪. 建立人肾小管上皮细胞蛋白质组双向电泳的分离体系[J].中国组织工程研究与临床康复， 2010，14(24):4416-4420. [ ]4416P.O. Box 1200, Shenyang110004张春燕，等. 建立人肾小管上皮细胞蛋白质组双向电泳的分离体系1 泸州医学院生物 化学教研室， 四川 省泸州市 2 646000； 人类疾 病细胞信号与调 控四川省高校重 点实验室， 四川省 泸州市 646000实验方法： 0 引言 双向电泳作为蛋白质组研究的三大核心 技术之一，是目前分离复杂蛋白质组分最常用 的工具 。

双向电泳/质谱(2-DE/MS)蛋白质组 解决方案能够在整体水平上，大规模、快速地 鉴定复杂蛋白样品。

该方案先通过双向电泳图 谱寻找差异蛋白质，再用质谱确认差异蛋白质 的精细分子量。

双向电泳实验周期长、 步骤多、 操作性强，如样品处理、上样方式、等电聚焦 和染色等各个环节均影响双向电泳的分离效 果[2-4] [1]样品制备：人肾小管上皮细胞株(HK-2)传代后， 采用含体积分数10%胎牛血清的高糖DMEM 培养液，于体积分数5%CO2培养箱，37 ℃常规 培养。

生长两三天至80%左右密度，弃去培养 液，生理盐水洗涤3次，3个培养瓶中分别加入 细胞裂解液A(40 mmol/L DTT，4%CHAPS， 0.2%Bio-Lyte， mmol/L Tris， mol/L尿素)、 40 8 细胞裂解液B(40 mmol/L DTT，4%CHAPS， 0.2%Bio-Lyte，40 mmol/L Tris，8 mol/L尿素， 2 mol/L硫脲)、细胞裂解液C (1%TBP，4% CHAPS， 0.2%Bio-Lyte， mmol/L Tris， mol/L 40 8 尿素， mol/L硫脲)， ℃振荡30 min。