高糖对大鼠肾小管上皮细胞差异蛋白组学研究

发布时间：2018-12-20 07:29

【摘要】：目的本实验通过对高糖作用下大鼠肾小管上皮细胞蛋白质组学动态变化的观察，了解高糖环境对大鼠肾小管上皮细胞蛋白质成分的影响，从而希望找到糖尿病肾病发生发展的机制。方法1将肾小管上皮细胞分别置于含正常糖浓度组（含糖5.5mM）、高糖组（含糖30mM）的培养基中进行体外培养，分别在8小时、72小时、25天后分别提取细胞总蛋白。2将分离的总蛋白运用双向电泳技术进行分离。3分离后的凝胶应用Image Master2D Platinum6.0软件对其2-DE图像进行分析，通过统计学方法评价双向凝胶电泳结果的重复性和可靠性，运用方差不对齐的t检验获得P值，P0.05并且该点的volume%值变化超出2倍作为标准，找出差异蛋白点。4通过质谱技术（MALDI-TOF, MALDI-TOF-TOF, LC-MS-MS）对找出的差异蛋白点进行鉴定，然后对鉴定出的差异蛋白进行生物信息学分析。5利用MASCOT数据库进行检索，其差异蛋白点的判定标准是以得分大于60，并且覆盖序列大于15%，测量与预测分子量之间相差小于30%，测得的等电点与预测等电点相差小于2.0。最后再利用PUBMED查询差异蛋白的功能。结果1双向电泳凝胶平均获得了蛋白点956.33±21.37个，832个点在所有胶上均出现，匹配率为87%。2运用双向电泳技术分析高糖及正常培养基的肾小管上皮细胞的蛋白组学，发现8个差异蛋白点经过矫正后（P0.05）通过质谱鉴定，它们分别是：膜突蛋白（moesin），膜联蛋白A4（annexin A4），电压依赖性阴离子通道蛋白2（voltage-dependent anion-selective channel protein2VDAC2），磷酸变位酶1（phosphoglycerate mutase1），细胞核酸结合蛋白2（cellular nucleic acid-bindingprotein isoform2），基质细胞衍生因子2-类蛋白1前体（stromal cell-derived factor2-like protein1precursor，SDF2L1），，加帽蛋白，凝溶胶蛋白样，异构型CRA_b（capping protein, gelsolin-like, isoform CRA_b），核苷二磷酸激酶A（nucleosidediphosphate kinase A）。结论通过对蛋白组学的研究，找出了8种差异蛋白，提示这些蛋白的上调或下调与高糖环境密切相关，为糖尿病肾病的机制研究提供了新的思路。
[Abstract]:Objective to investigate the effect of high glucose on the protein composition of renal tubular epithelial cells in rats by observing the dynamic changes of proteomics in renal tubular epithelial cells induced by high glucose. Therefore, we hope to find the mechanism of diabetic nephropathy. Methods 1 Renal tubular epithelial cells were cultured in vitro in normal glucose concentration group (5.5mM) and high glucose group (30mM) in vitro for 8 hours and 72 hours, respectively. After 25 days, the total proteins were extracted. 2 the total proteins were separated by two dimensional electrophoresis. 3 the 2-DE images were analyzed by Image Master2D Platinum6.0 software. The repeatability and reliability of the results of two-dimensional gel electrophoresis were evaluated by statistical method. P value was obtained by using the t test of variance misalignment, and the change of volume% value of this point was more than 2 times as the standard. The differential protein spots were identified by mass spectrometry (MALDI-TOF, MALDI-TOF-TOF, LC-MS-MS). Then the identified differential protein was analyzed by bioinformatics. 5 the MASCOT database was used to retrieve the differential protein points. The criteria for determining the differential protein points were more than 60 and the coverage sequence was greater than 15. The difference between the measured and predicted molecular weight is less than 30 and the difference between the measured isoelectric point and the predicted isoelectric point is less than 2.0. Finally, PUBMED was used to query the function of differential protein. Results (1) the average protein points were 956.33 卤21.37 and 832 spots were found on all the colloids. The matching rate was 87.2 the proteomics of renal tubular epithelial cells with high glucose and normal culture medium was analyzed by two-dimensional electrophoresis. It was found that eight differential protein spots were identified by mass spectrometry after correction (P0.05). They were: membrane process protein (moesin), binding protein A4 (annexin A4), voltage-dependent anion channel protein 2 (voltage-dependent anion-selective channel protein2VDAC2). Phosphatase 1 (phosphoglycerate mutase1), cell nucleic acid binding protein-2 (cellular nucleic acid-bindingprotein isoform2, stromal cell derived factor-2-class protein-1 precursor (stromal cell-derived factor2-like protein1precursor,SDF2L1), capsin, coagulant protein-like, Isomerized CRA_b (capping protein, gelsolin-like, isoform CRA_b, nucleoside diphosphate kinase A (nucleosidediphosphate kinase A). Conclusion through the study of proteomics, eight different proteins were found, which suggested that the up-regulation or down-regulation of these proteins was closely related to the high glucose environment, which provided a new idea for the study of the mechanism of diabetic nephropathy.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.6

【参考文献】