高糖和MBL共培养的人肾小球内皮细胞替代途径补体激活发病机制探讨

目的采用高糖和甘露聚糖结合凝集素（MBL）共同培养人肾小球内皮细胞（HRGEC）,探讨糖尿病肾病是否存在替代途径补体激活发病机制。方法体外培养正常HRGEC,随机分为单纯高糖组和高糖＋MBL组,两组分别用30mmol/L D-葡萄糖培养72h后,加入等量30%MBL缺乏人血清,高糖＋MBL组另外加入10μg/mL MBL,继续培养4h。采用流式细胞技术观察细胞表面MBL-C 3bBb的沉积,采用免疫荧光染色观察补体激活产物膜攻击复合物（MAC）的沉积。结果流式细胞技术证实,高糖＋MBL组细胞表面有明显的MBL和C 3bBb共沉积,免疫荧光染色显示该组细胞表面有明显MAC沉积,单纯高糖组无上述现象。结论高糖和MBL共同存在时发生MBL补体途径激活,并启动补体的替代途径,DN发病可能与上述两条补体途径激活有关。

Preliminary exploration of alternative complement activation pathway in high glucose and MBL co-cultured human renal glomerular endothelial cells

Objective To investigate if there is an alternative complement pathway activation in high glucose and mannose binding lectin(MBL) co-cultured human renal glomerular endothelial cells.Methods Normal HRGEC cultured in vitro were divided randomly into two groups: single high glucose group and high glucose+MBL group.After 72 hours cultured with 30mmol/L D-glucose,equivalent volume of 30% MBL deficiency human serum was added into two groups,10μg/mL MBL was only added into high glucose+MBL group,cells were cultured for another 4 hours.Flow cytometry was used to detect MBL and C3bBb,and immunofluorescence was used to detect Menbrane Attacks Complex(MAC)on cell surface.Results Compared with single high glucose group,Flow cytometry confirmed obvious MBL and C3bBb co-deposition,and immunofluorescence confirmed obvious MAC deposition on cell surface in high glucose+MBL group.Conclusion High glucose together with MBL can bring MBL pathway activation and an alternative complement pathway activation,adn DN may associated with these two complement pathways.