反义cubilin RNA对白蛋白刺激肾小管上皮细胞分泌趋化因子的影响

目的观察反义cubilinRNA对白蛋白刺激人近端肾小管上皮细胞(HK-2)表达单核细胞趋化蛋白(MCP)-1、RANTES的影响。方法应用基因克隆技术构建反义及正义cubilin真核表达载体(pcDNA-ACUB、pcDNA-CUB),酶切鉴定及序列测定证实插入序列是否正确。脂质体DOTAP将其转染至HK-2细胞,流式细胞仪(FCM)检测反义cubilinRNA对HK-2细胞重吸收白蛋白的抑制作用。细胞转染72h后给予高浓度白蛋白(10g/L)刺激24h,细胞ELISA、Western印迹法检测HK-2细胞表达MCP-1、RANTES的变化。结果pcDNA-ACUB转染组白蛋白摄取率较DOTAP组显著降低(P<0.01)。与DOTAP组比较,pcDNA-ACUB转染组HK-2细胞MCP-1、RANTES的表达显著降低(P<0.001)。结论反义cubilinRNA可抑制肾小管上皮细胞对白蛋白的摄取,并能拮抗白蛋白刺激肾小管上皮细胞趋化因子的表达,提示cubilin在白蛋白介导的肾小管上皮细胞分泌趋化因子中起重要作用。

Influence of antisense cubilin RNA on albumin-induced expression of chemokines in human renal proximal tubular epithelial cells

Objective To investigate the role of cubilin on albumin-induced e xp ression of monocyte chemoattractant protein 1(MCP-1) and regulated upon activat ion normal T-cell expressed and secreted (RANTES) in human renal proximal tubul ar epithelial cells (HK-2). Methods The sense and antisense cubilin recombinant eukaryotic expression vectors (pcDNA-CUB and pcDNA-ACUB) were constructed usi ng gene recombining techniques and identified by restriction endonucleases diges ting and DNA sequencing. Both vectors as well as pcDNA3.1( ) vector were respec tively transfected into HK-2 cells with lipofectin DOTAP. Flow cytometer (FCM) was applied to determine the inhibitive effect of antisense cubilin RNA on album in uptake of HK-2 cells. Posterior to cell transfection for 72 h, high concentr ation of albumin (10 g/L) was used to stimulate the cells for 24 h. The expressi on of MCP-1 and RANTES in HK-2 cells was measured by ELISA and Western blot.Re sults Compared with DOTAP group, the albumin uptake was significantly lowered(P< 0 01) and both MCP-1 and RANTES expression of HK-2 cells decreased significa ntly in pcDNA-ACUB trasnfection group(P< 0 001). Conclusions Antisense cubilin RNA can inhibit albumin uptake of HK-2 cells and suppress the albumin-induced up-regulation of MCP-1 and RANTES expression in the HK-2 cells. This indicat es that cubilin may play an important role in albumin-induced chemokines expres sion in renal proximal tubular epithelial cells.