蛋白激酶C在高糖诱导近端小管上皮细胞细胞外基质及转化生长因子β1高表达中的作用

Role of protein kinase C on high glucose-induced extracellular matrix and transforming growth factor β1 expression in renal proximal tubular epithelial cells

目的 探讨高糖作用下近端肾小管上皮细胞蛋白激酶Ｃ（ＰＫＣ）活性的变化，以及ＰＫＣ激活对近端肾小管上皮细胞细胞外基质（ＥＣＭ）及转化生长因子β１（ＴＧＦ－β１）表达的调控作用。方法 采用 ＬＬＣ－ＰＫ１细胞株，将细胞分为正常对照组 ＮＧ（５．５ｍｍｏｌ／Ｌ Ｄ－葡萄糖）、高糖组 ＨＧ（２５ ｍｍｏｌ／ＬＤ－葡萄糖）、高渗组 ＨＭ（２５ ｍｍｏｌ／Ｌ甘露醇）、ＮＧ＋ＰＫＣ抑制剂（ＰＫＣＩ）组（１０μｍｏｌ／Ｌ ｃｈｅｌｅｒｙｔｈｒｉｎｅｃｈｌｏｒｉｄｅ）、ＨＧ＋ＰＫＣＩ组、ＨＭ＋ＰＫＣＩ组。分别检测各组细胞 ＰＫＣ活性，并运用原位杂交和免疫细胞化学法检测各组Ⅳ胶原（ＣｏｌⅣ）、纤连蛋白（ＦＮ）及ＴＧＦ－β１ｍＲＮＡ和蛋白表达。结果 ＨＧ组细胞胞膜ＰＫＣ活性较 ＮＧ组升高 ３．３倍。ＨＧ组细胞 ＣｏｌⅣ、ＦＮ及 ＴＧＦ－β１ｍＲＮＡ和蛋白水平均较 ＮＧ组显著升高，ＨＭ组无此变化。高糖导致的ＥＣＭ和ＴＧＦ－β１高表达可被ＰＫＣ抑制剂ｃｈｅｌｅｒｙｔｈｒｉｎｅ ｃｈｌｏｒｉｄｅ所阻断。结论 由高糖诱导的近端小管上皮细胞ＥＣＭ和ＴＧＦ－β１高表达是通过激活ＰＫＣ通路所介导。

Objective To investigate the rolu of PK( activation on high glucose-induced ECM and TGF-βl expression in renal proximal tubular epithelial cells. Methods LLC-PK1 cells were divided into six groups: normal glucose group(NG, 5. 5 mmol/L D-glucose), high glucose group(HG. 25 mmol/L B-glucose), high mannitol group(HM, 25 mmol/L mannitol), NG + PKC inhibitor(10μmol/L chelerythrine chloride), HG * PKCI and HM * PKCI. PKC activity was detected. Expression of ColⅣ, FN and TGF-β1 was examined by in situ hybridization and immunocytochemistry(ABC) techniques. Results Compared with NG group, PKC activity increased significantly in HG group(3. 3 times); All of Co1Ⅳ, FN and TGF-β1 gene and protein expression enhanced singnificantly in HG group. All of these three gene expression downregulated, and ColⅣ and TGF-β1 syntheses were also significantly blocked by PKC inhibitor chelerythrine chloride. Conclusion High expression of ECM and TGF-β1 induced by high glucose may be mediated via the activation of PKC signal transduction pathway.