高糖刺激对大鼠近端肾小管上皮细胞脂联素受体表达的影响

发布时间：2018-07-16 13:29

【摘要】：目的:观察体外高糖环境下大鼠近端肾小管上皮细胞脂联素受体基因及其蛋白表达变化。方法:正常大鼠近端肾小管上皮细胞细胞株(NRK-52E)常规培养于含10%灭活新生胎牛血清的低糖DMEM培养液传代培养,待细胞生长至亚融合状态时,更换为无胎牛血清低糖DMEM培养基培养24h使其同步化,将NRK-52E细胞分为5组:A组为正常对照组,低糖DMEM完全培养液(含D-葡萄糖5.5mmol/L);B组为高糖刺激12h组:高糖DMEM完全培养液(含D-葡萄糖25mmol/L)刺激12h;C组为高糖刺激24h组:高糖DMEM完全培养液刺激24h;D组为高糖刺激48h组:高糖DMEM完全培养液刺激48h;E组为高糖刺激72h组:高糖DMEM完全培养液刺激72h。然后分别提取各组总RNA和总蛋白,分别用RT-PCR检测各组目的基因AdipoR 1/2mRNA的表达,Western印迹法检测各组目的蛋白AdipoR 1/2蛋白的表达。结果:脂联素受体1(AdipoR1)和脂联素受体2(AdipoR2)在NRK-52E细胞中均有表达;在高糖刺激情况下,随着刺激时间的延长,目的基因AdipoR 1/2mRNA的表达均有先增高后降低的趋势,在刺激24h时升高到最高点,和对照组相比有统计学意义(P0.05);与正常对照组相比,高糖刺激后各时间点AdipoR 1/2蛋白的表达有上升的趋势,但差异无统计学意义(P0.05)。结论:NRK-52E细胞AdipoR1/2蛋白的表达不受高糖刺激的影响;高糖影响NRK-52E细胞AdipoR1/2mRNA的表达,并随着刺激时间的延长表现出先增高后降低。高糖通过影响脂联素受体基因的表达从而影响脂联素发挥其生物学作用,以致影响大鼠近端肾小管上皮细胞的功能。
[Abstract]:Aim: to observe the changes of adiponectin receptor gene and its protein expression in proximal tubular epithelial cells of rats under high glucose environment in vitro. Methods: normal proximal tubular epithelial cell line (NRK-52E) was cultured in low glucose DMEM medium containing 10% inactivated fetal bovine serum. The NRK-52E cells were divided into 5 groups (n = 5) as normal control group (n = 5), and cultured in the medium containing fetal bovine serum low glucose DMEM for 24 h, and NRK-52E cells were divided into 5 groups as normal control group. Low glucose DMEM complete culture medium (containing DMEM 5.5 mmol / L) group B was treated with high glucose stimulation for 12 h, high glucose DMEM complete culture medium (including D- glucose 25 mmol / L) for 12 h and high glucose stimulation group for 24 h; high glucose DMEM complete culture medium for 24 h; high glucose group for 48 h; Group C for 12 h; Group C for 24 h; Group D for 48 h; Group D for 24 h; High glucose DMEM complete culture medium was stimulated with high glucose for 72 h and high glucose DMEM for 72 h. The expression of AdipoR 1 / 2 mRNA was detected by RT-PCR and the expression of AdipoR 1 / 2 protein was detected by Western blot. Results: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) were expressed in NRK-52E cells. Compared with the control group, the expression of AdipoR 1 / 2 protein increased at 24 hours after high glucose stimulation (P0.05), but the difference was not statistically significant (P0.05). Conclusion the expression of AdipoR1 / 2 protein in NRK-52E cells was not affected by high glucose stimulation, but the expression of AdipoR1 / 2 mRNA in NRK-52E cells was increased first and then decreased with the prolongation of stimulation time. High glucose affects the biological function of adiponectin by affecting the expression of adiponectin receptor gene, thus affecting the function of proximal tubular epithelial cells in rats.
【作者单位】：武汉大学中南医院肾内科;
【基金】：国家自然科学基金资助项目(编号:163411)
【分类号】：R363

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