针对结缔组织生长因子的siRNA对高糖诱导人肾小管上皮细胞肥大的影响

Effects of small interfering RNA targeting connective tissue growth factor on high glucose-induced human tubular epithelial hypertrophy

目的观测人肾小管上皮细胞转染针对结缔组织生长因子(CTGF)的siRNA后对高糖诱导细胞肥大的影响。方法细胞分6组培养:正常对照组(培养基含D-葡萄糖1g/L),等渗对照组(培养基含D-葡萄糖1g/L、甘露醇3.5g/L),高糖组(培养基含D-葡萄糖4.5g/L),高糖+空白对照组(细胞转染空质粒后培养于高糖培养基中),高糖+阴性对照组(细胞转染含无关序列的质粒后培养于高糖培养基中),高糖+干扰组(细胞转染针对CTGF的siRNA表达质粒后培养于高糖培养基中)。收集各组培养至24、48、96h的细胞,以实时PCR检测CTGF mRNA水平;Western blotting检测CTGF蛋白水平;MTT法测定细胞增殖活力;流式细胞仪测定细胞周期分布;考马氏亮蓝法测定细胞总蛋白含量。结果高糖刺激可上调HK-2细胞的CTGF mRNA及蛋白水平,使停留于G1期的细胞比例升高,增殖活力受抑制,细胞肥大指标胞内总蛋白含量增加;而通过特异性siRNA抑制CTGF mRNA表达后,细胞的CTGF蛋白表达随时间延长进行性降低,细胞增殖活力增强,更多的细胞由G1期进入S期,细胞内总蛋白含量降低。结论证实了CTGF是高糖诱导HK-2细胞肥大的重要介质。针对CTGF的siRNA能明显改善高糖诱导的肾小管上皮细胞肥大,为进一步寻找DN防治的靶点提供了新的实验依据。

Objective To observe the effect oftransfection with small interfering RNA （siRNA） targeting connective tissue growth factor （CTGF） on human tubular epithelial hypertrophy induced by high glucose. Methods HK-2 cells were cultured in DMEM/F12 medium containing 1 g/L glucose （normal control group）, 4.5 g/L glucose （high glucose group）, or 1 g/L glucose ＋ 3.5 g/L mannitol （iso-osmolar control group）. The cells were transfected with pGenesil-l, pGenesil/neg, or pGenesil/siRNA-CTGF and then cultured in DMEM/F12 medium containing 4.5 g/L glucose as the high glucose ＋ blank control group, high glucose ＋negative control group and high glucose＋ interference group, respectively. After cell culture for 24, 48 and 96 h, the cells were collected to detect the mRNA and protein levels of CTGF by real-time PCR and Western blotting, respectively. The proliferative activities of the cells were evaluated with MTT assay, and the total cellular protein contents were determined with Bradford method. Flow cytometry was employed to analyzed the cell cycle changes. Results High-glucose significantly up-regulated the CTGF mRNA and protein levels in HK-2 cells. The cell proliferation was inhibited after high-glucose exposure with increased cell percentage in G1 phase and total cellular protein content suggesting cellular hypertrophy. Transfection with siRNA targeting CTGF significantly inhibited high glucose-induced up-regulation of CTGF rnRNA and protein and promoted the cell proliferation, resulting also increased cells in S phase and lowered total cellular protein contents. Conclusion CTGF is an important mediator of high glucose-induced tubular epithelial hypertrophy, and transfection with siRNA targeting CTGF can alleviate the hypertrophy, suggesting the potential value of CTGF-targeted treatment in the management of diabetic neohropathv.