采用蛋白质组分析高浓度葡萄糖对人腹膜间皮细胞的影响

Effects of high glucose on human peritoneal mesothelial cells:a proteomic analysis

目的 研究腹透液中葡萄糖对人腹膜间皮细胞(HPMCs)分泌蛋白质影响,并采 用蛋白质组学方法分析其对腹膜纤维化和腹膜功能的影响。方法 采用胰蛋白酶消化法从人腹 膜组织中分离HPMCs,第3代用于实验。实验分组:对照组(F12培养基)和高糖组(F12培养基+ 4％葡萄糖),分别观察24 h和48 h。经二维凝胶电泳(2-DE)分离人腹膜间皮细胞总蛋白,每组 重复3次,PDQuest图象分析软件比较对照组和高糖组平均凝胶,求差异蛋白质。采用基质辅助 激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定部分差异蛋白质。结果 获得12块经2-DE 分离的分离胶,每组3块。通过PDQuest图象分析软件发现24 h高糖组与对照组比较有27种蛋 白质表达有差异(P<0．05);48 h高糖组与对照组比较有50种蛋白质的表达有差异(P<0．05), 表达上调有22个蛋白质斑点,表达下调有55个蛋白质斑点;对其中23个差异蛋白质斑点进行 了肽质指纹图分析,鉴定出23种蛋白质,其中有2个点为同一蛋白质。这些蛋白质涉及到高糖 对HPMCs的细胞骨架、糖代谢、黏附、细胞因子、抗氧化剂、表面活性剂等物质的调节。结论 高 糖导致腹膜纤维化是多因素、多途径,要防止腹膜透析腹膜纤维化保护腹膜透析效能,最好避免 过多使用高糖透析液。

Objective To study the effects of high glucose dialysate on proteins secreted by human peritoneal mesothelial cells （HPMCs）, and to analyze the effects of proteins on peritoneal fibrosis and peritoneal functions by proteome methods. Methods HPMCs were isolated from human peritoneal tissues through trypsinizadon, and the third generation was used in the experiment. The cells were divided into the control group （in F12 culture only） and the high glucose group （in F12 culture with 4% glucose）. They were observed for 24 hours and 48 hours respectively. Total proteins of HPMCs were isolated repeatedly three times in each group by two-dimensional gel electrophoresis （2-DE）. PDQuest picture analysis software was used to differentiate protein in the average gel between the control group and the high-glucose group. Matrix assisted laser desorption/ isonization time of flying mass spectrometry （MALDI-TOF-MS） was adopted to identify some of the differential proteins found. Results Twelve gels were produced by 2-DE, with three in each group. Through application of the PDQuest picture analysis software, 27 protein spots showed differential expression （P〈 0.05） in comparison of the average gel between the 24-hour control group and the 24-hour high-glucose group, and 50 protein spots showed differential expression （P 〈 0.05） in comparison of the average gel between the 48-hour control group and the 48-hour high-glucose group.Among them, 22 protein spots indicated up-regulated expression, and 50 indicated down-regulated expression. Twenty-three of the differential protein spots were analyzed by peptide mass finger （PMF）. Two spots were the same protein. These proteins included cofidin 1, aldose reducase, glyceraldehyde-3-phosphate dehydrogenase, interleukin 1 family number 8 （ILIF8）, galectin 1, manganese superoxide dismutase （Mn-SOD）, surfactant, and so on. They were involved in the regulation of cytoskelete, signal transduction, glycose metabolism, cytokine, adhesion, surfactant, antioxygen, and so on. Conclusions Multi-factors lead to peritoneal fibrosis induced by high glucose. To protect peritoneal functions from peritoneal fibrosis in peritoneal dialysis, it is suggested to avoid the use of high glucose dialysate.