megsin基因转染对高糖环境中系膜细胞胞外调节蛋白激酶表达的影响

目的 观察megsin基因转染对高糖环境中小鼠肾小球系膜细胞血小板源性生长因子BB（PDGF-BB）、磷酸化胞外调节蛋白激酶1/2（pERK1/2）、转化生长因子β1（TGF-β1）的表达及Ⅳ型胶原水平的影响；探讨megsin基因对细胞外信号调节激酶（ERK）通路的作用。 方法 将小鼠肾小球系膜细胞分为7组：低糖组（A组，5.5 mmol/L）、高糖组（B组，30 mmol/L）、高糖+空质粒组（C组）、高糖+megsin质粒组（D组）、高糖+megsin质粒+ERK通路抑制剂组（E组）、高糖+ megsin siRNA组（F组）和低糖+甘露醇组（24.5 mmol/L甘露醇，G组）。分别在培养12、24、48 h后，采用Western印迹法测定各组系膜细胞megsin、PDGF-BB、pERK1/2、TGF-β1的表达，放射免疫测定法（RIA）测定各组细胞上清液中Ⅳ型胶原浓度。 结果 与A组相比，高糖刺激可上调系膜细胞megsin、PDGF-BB、pERK1/2、TGF-β1的表达及上清液Ⅳ型胶原的水平（均P ＜ 0.05），且有一定的时间依赖性。转染megsin基因可进一步上调高糖环境下系膜细胞PDGF-BB、pERK1/2、TGF-β1、Ⅳ型胶原的表达（均P ＜ 0.05）。应用ERK通路抑制剂后，与D组相比系膜细胞megsin 和PDGF-BB的表达无明显变化（均P ＞ 0.05），而pERK1/2、TGF-β1及Ⅳ型胶原的表达则明显降低（均P ＜ 0.05）。转染megsin siRNA对系膜细胞megsin基因进行干扰后，系膜细胞PDGF-BB、pERK1/2、TGF-β1的表达及Ⅳ型胶原的含量较B组下调（均P ＜ 0.05）。 结论 高糖环境下，转染megsin基因可使小鼠系膜细胞megsin、PDGF-BB表达增高，其可能部分通过ERK通路使TGF-β1表达上调及Ⅳ型胶原合成与分泌增加。megsin siRNA干扰可使上述指标下调，megsin基因可能在糖尿病肾病发病中起重要作用。

Impact of megsin gene transfection on the expression of extracellular signal-regulated protein kinase under high concentration of glucose

objective To observe the effects of megsin gene transfection on glomerular mesangial cells(GMCs)and the expression of platelet-derived growth factor-BB(PDGF-BB),phosphorylated extracellular signal-regulated protein kinase(pERK1/2),transforming growth factor β1 (TGF-β1)and type Ⅳ collagen under high concentration of glucose,and to investigate the impact of megsin gene transfection on the extracellular signal-regulated protein kinase (ERK)siginal pathway. Methods Cultured mesangial cells were divided into seven groups:low glucose group (group A,5.5 mmol/L),high glucose group(group B,30 mmol/L),high glucose plus empty vector group (group C), high glucose plus megsin expression plasmid group (group D), high glucose plus megsin expression plasmids plus U0126 group (group E), high glucose plus megsin siRNA expression plasmids group (group F) and low glucose plus mannitol (24.5 mmol/L,group G). The expressions of megsin, PDGF-BB, pERK1/2, TGF-β1, as well as type Ⅳ collagen in GMCs of each group were detected by Western blotting, after being cultured for 12, 24 and 48 hours respectively. The concentration of type Ⅳ collagen in cell supernatant was measured by radioimmunochemistry. Results Compared with group A, the expression of megsin was increased in GMCs under high glucose medium, with an increase of PDGF-BB, pERK1/2, TGF-β1 in GMCs and type Ⅳ collagen in the supernatants (P＜0.05, respectively). The expression of above indices was in time-dependant manner. The over expression of megsin in exposure to high up-regulated the expression of PDGF-BB, pERK1/2, TGF-β1 and type Ⅳ collagen(P＜0.05, respectively). Compared with group D, the application of U0126 (pERK1/2 inhibitor) had no significant effect on the expression of megsin and PDGF-BB(P＞0.05, respectively). However, the expression of pERK 1/2,TGF-β1 and type Ⅳ collagen were obviously decreased (P＜0.05, respectively). Dowa-regulated expression of megsin by siRNA transfection decreased the expression of PDGF-BB, pERK1/2, TGFβ1 and type Ⅳ collagen(all P＜0.05). Conclusions The expression of megsin and PDGF-BB in GMCs with transfected megsin gene in high glucose medium is increased, possibly in a way of activating ERK signal pathway to some extent that boosts both the expression of TGF-β1 and the production of type Ⅳ collagen. The transfection of megsin siRNA inhibits the expression of obove indices, which probably contributes to the development of diabetic nephropathy.