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目的 :探讨己糖胺通路 (HBP)在高糖引起系膜细胞功能改变中的作用。 方法 :利用体外培养的人肾小球系膜细胞 ,流式细胞术分析细胞体积、细胞周期和纤维连接蛋白 (FN)的含量 ,以3H 胸腺嘧啶的掺入反映细胞的增生。采用夹心ELISA法和RT PCR检测转化生长因子 β1(TGF β1)蛋白质和mRNA的表达。采用比色法测定HBP限速酶—谷氨酰胺 :6 磷酸果糖转氨酶 (GFAT)的活性。 结果 :HBP激活剂 葡萄糖胺能够诱导系膜细胞肥大、增生抑制和FN合成增多 ,并增加TGF β1的表达。高糖能增加GFAT的活性 ,高糖促进TGF β1表达的作用能够被GFAT的抑制剂—重氮丝氨酸拮抗。 结论 :HBP的过度活化 ,可能通过上调TGF β1的表达 ,从而介导了高糖对系膜细胞的损伤。
Objective: To investigate the role of hexosamine pathway (HBP) in the changes of mesangial cells induced by high glucose. Methods: Human glomerular mesangial cells cultured in vitro were used to analyze the cell volume, cell cycle and the content of fibronectin (FN) by flow cytometry. 3H thymidine incorporation was used to detect the cell proliferation. Sandwich ELISA and RT PCR were used to detect the protein and mRNA expression of TGF-β1. The colorimetric method was used to determine the activity of HBP rate-limiting enzyme glutamine-6-phosphate transglutaminase (GFAT). Results: HBP activator, glucosamine, could induce mesangial cell hypertrophy, hyperplasia inhibition and increase of FN synthesis, and increase the expression of TGFβ1. High glucose can increase the activity of GFAT. The effect of high glucose on TGFβ1 expression can be antagonized by diazoxide, an inhibitor of GFAT. Conclusion: Overexpression of HBP may mediate the injury of mesangial cells by up-regulating the expression of TGF-β1.