CAJ | 학술논문

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1 g/L),等渗对照组(培养基含D-葡萄糖1 g,L、甘露醇3.5 g/L),高糖组(培养基含D-葡萄糖4.5 g/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、96 h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CTGFmRNA及蛋白水平,使停留于G_1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G_1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.
목적 관측인신소관상피세포전염침대결체조직생장인자(CTGF)적siRNA후대고당유도세포비대적영향.방법 세포분6조배양:정상대조조(배양기함D-포도당1 g/L),등삼대조조(배양기함D-포도당1 g,L、감로순3.5 g/L),고당조(배양기함D-포도당4.5 g/L),고당+공백대조조(세포전염공질립후배양우고당배양기중),고당+음성대조조(세포전염함무관서렬적질립후배양우고당배양기중),고당+간우조(세포전염침대CTGF적siRNA표체질립후배양우고당배양기중).수집각조배양지24、48、96 h적세포,이실시PCR검측CTGF mRNA수평;Western blotting검측CTGF단백수평;MTT법측정세포증식활력;류식세포의측정세포주기분포;고마씨량람법측정세포총단백함량.결과 고당자격가상조HK-2세포적CTGFmRNA급단백수평,사정류우G_1기적세포비례승고,증식활력수억제,세포비대지표포내총단백함량증가:이통과특이성siRNA억제CTGF mRNA표체후,세포적CTGF단백표체수시간연장진행성강저.세포증식활력증강,경다적세포유G_1기진입S기,세포내총단백함량강저.결론 증실료CTGF시고당유도HK-2세포비대적중요개질.침대CTGF적siRNA능명현개선고당유도적신소관상피세포비대,위진일보심조DN방치적파점제공료신적실험의거.
Objective To observe the effect of transfection 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-1, 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 G, phase and total cellular protein content suggesting cellular hypertrophy. Transfection with siRNA targeting CTGF significantly inhibited high glucose-induced up-regulation of CTGF mRNA 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 nephropathy.