一氧化氮作用前后角质形成细胞差异蛋白的筛选与分析

发布时间：2018-02-03 21:37

本文关键词： NO HaCaT细胞人角质形成细胞双向电泳质谱分析　出处：《大连医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:研究一氧化氮(NO)对体外培养的永生化角质形成细胞(HaCaT细胞)及人原代培养的角质形成细胞(KC)蛋白质表达的影响,在蛋白水平上寻找NO作用的靶位。进一步探讨NO在银屑病发病中的地位和作用。方法:体外培养HaCaT细胞及人原代KC,通过CCK-8法检测不同浓度外源性NO供体S-亚硝基谷胱甘肽(GSNO)作用后,在不同时间点的细胞增殖率,绘制浓度与细胞增值率曲线,筛选最佳GSNO浓度。向处于指数增长期的HaCaT细胞及人原代KC中加入最佳浓度的GSNO进行刺激。对照组不予处理。加药处理的细胞和对照组用细胞裂解液分别进行充分裂解,提取细胞全蛋白。应用2-D纯化试剂盒进行蛋白纯化,蛋白定量。纯化后的蛋白中加入上样缓冲液制成样本,首先进行第一向等电聚焦电泳,胶条的平衡,再进行第二向聚丙烯酰胺凝胶电泳。电泳完成的凝胶进行硝酸银染色,图像扫描,用PD-QUEST8.0软件进行分析,筛选出加入GSNO前后的差异点,切下进行质谱分析,对蛋白质种类进行鉴定。结果: (1)应用CCK-8法,绘制GSNO浓度与细胞增值率曲线显示,随着GSNO浓度的增加,细胞增殖呈下降趋势,与时间无显著关系。(2)HaCaT细胞组及人KC组进行双向电泳后出点良好;加入GSNO前、后HaCaT细胞组平均蛋白质点数分别为482±31,,495±28,组内匹配率达82.1%和81.9%;加入GSNO前、后人KC组平均蛋白质点数分别为543±27和529±29,匹配率为80.8%和81.4%。(3)差异蛋白点数总共为29个,其中,2个点仅在加入GSNO后表达;14个点在加入GSNO后高表达;13个点在加入GSNO后地表达或消失;进行质谱分析,检测到17种已知蛋白,3种未知蛋白。结论:(1)该研究建立了NO作用前、后HaCaT细胞及人KC的双向电泳图谱,发现二者之间存在差异表达蛋白; (2)本研究通过NO作用前后HaCaT细胞及人KC的双向电泳及质谱分析,发现抑制素、角蛋白、肌动蛋白结合蛋白等17种差异表达的蛋白; (3)NO通过调节多种靶向蛋白的表达,调节KC增殖周期、分化或凋亡,介导早期银屑病的炎性反应。
[Abstract]:Objective: to study the effects of nitric oxide (no) on the protein expression of immortalized keratinocytes (HaCaT cells) and human primary cultured keratinocytes (KC) in vitro. To explore the role of no in the pathogenesis of psoriasis. Methods: HaCaT cells and human KC cells were cultured in vitro. The effects of different concentrations of exogenous no donor, Snitroglutathione (Snitroglutathione), were detected by CCK-8 assay. At different time points, the cell proliferation rate, concentration and cell proliferation rate curve were drawn. Select the best concentration of GSNO. Add the best concentration of GSNO to the HaCaT cells in exponential growth stage and human KC. The control group is not treated. The cells treated with drugs are used in the control group. The cell lytic solution was fully lysed respectively. The whole cell protein was extracted. The protein was purified by 2-D purification kit and the protein was quantification.After the purified protein, the sample was prepared by adding the sample buffer solution, and the first isoelectric focusing electrophoresis was carried out, and the equilibrium of the rubber strip was carried out. Then the second polyacrylamide gel electrophoresis was carried out. The gel was stained with silver nitrate, scanned by image and analyzed by PD-QUEST8.0 software. The difference points before and after the addition of GSNO were screened, and the protein species were identified by mass spectrometry. Results: using CCK-8 method, the curves of GSNO concentration and cell proliferation rate showed that the proliferation of cells decreased with the increase of GSNO concentration. There was no significant relationship between the time and the time. The exit point of HaCaT cell group and human KC group were good after two dimensional electrophoresis. Before and after the addition of GSNO, the average protein number of HaCaT cells was 482 卤31, 495 卤28, and the matching rates were 82.1% and 81.9 respectively. Before the addition of GSNO, the average protein points in KC group were 543 卤27 and 529 卤29, respectively, and the matching rates were 80.8% and 81.4, respectively. Among them, 2 dots were expressed only after the addition of GSNO. 14 dots were overexpressed after adding GSNO. 13 dots were expressed or disappeared after adding GSNO. 17 known proteins and 3 unknown proteins were detected by mass spectrometry. Conclusion 1) Two-dimensional electrophoretic patterns of HaCaT cells and human KC before and after no treatment were established in this study. It was found that there were differentially expressed proteins between them. In this study, 17 differentially expressed proteins, such as inhibin, keratin and actin binding protein, were found by two-dimensional electrophoresis and mass spectrometry analysis of HaCaT cells and human KC before and after no treatment. No mediates the inflammatory response of early psoriasis by regulating the expression of many target proteins, regulating the proliferation cycle, differentiation or apoptosis of KC.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.63

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