【摘要】：目的本實驗通過對高糖作用下大鼠腎小管上皮細(xì)胞蛋白質(zhì)組學(xué)動態(tài)變化的觀察，了解高糖環(huán)境對大鼠腎小管上皮細(xì)胞蛋白質(zhì)成分的影響，從而希望找到糖尿病腎病發(fā)生發(fā)展的機制。 方法1將腎小管上皮細(xì)胞分別置于含正常糖濃度組（含糖5.5mM）、高糖組（含糖30mM）的培養(yǎng)基中進(jìn)行體外培養(yǎng)，分別在8小時、72小時、25天后分別提取細(xì)胞總蛋白。2將分離的總蛋白運用雙向電泳技術(shù)進(jìn)行分離。3分離后的凝膠應(yīng)用Image Master2D Platinum6.0軟件對其2-DE圖像進(jìn)行分析，通過統(tǒng)計學(xué)方法評價雙向凝膠電泳結(jié)果的重復(fù)性和可靠性，運用方差不對齊的t檢驗獲得P值，P0.05并且該點的volume%值變化超出2倍作為標(biāo)準(zhǔn)，找出差異蛋白點。4通過質(zhì)譜技術(shù)（MALDI-TOF, MALDI-TOF-TOF, LC-MS-MS）對找出的差異蛋白點進(jìn)行鑒定，然后對鑒定出的差異蛋白進(jìn)行生物信息學(xué)分析。5利用MASCOT數(shù)據(jù)庫進(jìn)行檢索，其差異蛋白點的判定標(biāo)準(zhǔn)是以得分大于60，并且覆蓋序列大于15%，測量與預(yù)測分子量之間相差小于30%，測得的等電點與預(yù)測等電點相差小于2.0。最后再利用PUBMED查詢差異蛋白的功能。 結(jié)果1雙向電泳凝膠平均獲得了蛋白點956.33±21.37個，832個點在所有膠上均出現(xiàn)，匹配率為87%。2運用雙向電泳技術(shù)分析高糖及正常培養(yǎng)基的腎小管上皮細(xì)胞的蛋白組學(xué)，發(fā)現(xiàn)8個差異蛋白點經(jīng)過矯正后（P0.05）通過質(zhì)譜鑒定，它們分別是：膜突蛋白（moesin），膜聯(lián)蛋白A4（annexin A4），電壓依賴性陰離子通道蛋白2（voltage-dependent anion-selective channel protein2VDAC2），磷酸變位酶1（phosphoglycerate mutase1），細(xì)胞核酸結(jié)合蛋白2（cellular nucleic acid-bindingprotein isoform2），基質(zhì)細(xì)胞衍生因子2-類蛋白1前體（stromal cell-derived factor2-like protein1precursor，SDF2L1），，加帽蛋白，凝溶膠蛋白樣，異構(gòu)型CRA_b（capping protein, gelsolin-like, isoform CRA_b），核苷二磷酸激酶A（nucleosidediphosphate kinase A）。 結(jié)論通過對蛋白組學(xué)的研究，找出了8種差異蛋白，提示這些蛋白的上調(diào)或下調(diào)與高糖環(huán)境密切相關(guān)，為糖尿病腎病的機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 鹵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.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692.6

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