Gremlin基因转染对高糖环境中系膜细胞增殖的影响

Effect of Gremlin gene transfetion on mesangial cell proliferation under high concentration of glucose

摘要:

目的：观察体外转染Gremlin质粒或Gremlin siRNA质粒对小鼠肾小球系膜细胞（mouse glomerular mesangial cells，MMCs）溴脱氧尿苷（bromodeoxyuridine，BrdU）及Ⅳ型胶原表达的影响。方法：将MMCs分为7组：正常对照组（NG，5.5 mmol/L）、正常糖+甘露醇组（NG+M，24.5 mmol/L甘露醇）、正常糖+空质粒组（NG+V）、正常糖+Gremlin质粒组（NG+P）/正常糖+Gremlin siRNA质粒组（NG+gremlin si）、高糖组（HG，30 mmol/L）、高糖+空质粒组（HG+V）以及高糖+Gremlin质粒组（HG+P）/高糖+Grem－lin siRNA质粒组（HG+gremlin si）。在高糖刺激24 h后，应用ELISA法测定BrdU表达，放免法测定Ⅳ型胶原浓度。结果：与NG组相比，NG+P组和HG 组BrdU表达增强及Ⅳ型胶原浓度增加（均P=0.000）；与HG组相比，HG+P组BrdU表达进一步增强及Ⅳ型胶原浓度进一步上调（均P=0.000）；然而，Gremlin siRNA质粒转染可抑制高糖诱导的BrdU表达增强及Ⅳ型胶原浓度增加（均P=0.000）。结论：Gremlin基因促进MMCs增殖和Ⅳ型胶原聚集；Gremlin基因可能在糖尿病肾病发病中起到重要作用。

Abstract:

Objective：To observe the effects of transfection of Gremlin plasmid and Gremlin siRNA plasmid in vitro on expression of mouse mesangial cells（MMCs），bromodeoxyuridine（BrdU） and collagen Ⅳ. Methods：Cultured MMCs were divided into 7 groups：normal glucose group（NG，5.5 mmol/L），normal glucose plus mannitol group（NG+M，24.5 mmol/L mannitol），normal glucose plus emp－ty vector group（NG+V），normal glucose plus Gremlin expression plasmid group（NG+P）/normal glucose plus Gremlin siRNA plas－mid group（NG+Gremlin si），high glucose group（HG，30 mmol/L），high glucose plus empty vector group（HG+V） and high glucose plus Gremlin expression plasmid group（HG+P）/high glucose plus Gremlin siRNA plasmid group（HG+Gremlin si）. Cell proliferation was examined using BrdU ELISA and accumulation of collagen Ⅳ was measured using radioimmunoassay at 24 h after high glucose stimulation. Results：Expression of BrdU and collagen Ⅳ in the supernatant was increased in NG+P and HG group compared with that in NG group（all P=0.000）. Expression of BrdU and collagen Ⅳ was up-regulated in HG+P group than in HG group（all P=0.000）. Inhibition of Gremlin with Gremlin siRNA plasmid reversed HG-induced abnormalities（increase in BrdU expression and collagen Ⅳ concentration）（all P=0.000）. Conclusions：Gremlin induces cell proliferation and accumulation of collagen Ⅳ in MMCs.Gremlin plays an important role in the development of diabetic nephropathy.