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

Effects of High Levels Glucose on the Expression of Adiponectin Receptors in Rat Renal Proximal Tubular Cells

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(P<0.05);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(P>0.05)。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。

To observe the effect of high glucose stimulation on the expression of adiponectin receptor（AdipoR） mRNA and protein in rat proximal tubular epithelial cells in vitro. Methods： NRK-52E cells were cultured in the low glucose DMEM containing 10% heat-inactivated newborn fetal bovine serum. When the cells reached to confluence, we cultured them in the serum-free DMEM for 24 hours. Then the NRK-52E cells were divided into five groups according to the dif- ferent treatment. Cells in the control group were cultured in DMEM low-level glucose solution containing D-glucose 5.5 mmol/L. All other groups used high-level glucose solutions as culture medium which contained D-glucose 25 mmol/L to culture the cells for 12 h, 24 h, 48 h and 72 hseparately. Adipo R 1/2 mRNA expression was detected by RT-PCR and the protein expression was detected by Western blotting. Results： AdipoR 1 and 2 were both expressed in rat renal prox- imal tubular epithelial cells. Under high glucose condition, AdipoR 1/2 mRNA expression in creased first and then decreased, and it reached the peak at 24 hours. The differences of AdipoR 1/2 mRNA expression between high glucose groups and the control group were statistically sig- nificant （P（0.05）. The expression of AdipoR 1/2 protein in the high glucose group increased too, but compared with the control group, there was no significant difference statistically （P~ 0.05）. Conclusion： The expression of AdipoR mRNA in rat renal proximal tubular epithelial cells was affected by high glucose concentration and increased first and then decreased with the stimu- lation. AdipoR protein expression was not influenced by high glucose in NRK-52E cell. Hyper glyeemia has an influence on the biological role of the adiponectin through affecting the expression of adiponectin receptor gene, thus can affect the functions of rat renal proximal tubular epithelial cells.