24p3/NGAL介导铁转运通过抑制肾小管上皮细胞凋亡减少急性肾损伤

24p3 Protein Reduces the Acute Renal Injury in Rats by Mediating Iron Transport

目的:阐明24p3/NGAL介导铁转运对急性肾损伤肾小管上皮细胞的保护作用。方法:用分子克隆的方法构建Pc DNA3.1/24p3质粒,采用脂质体转染的方法瞬时转染小鼠胚胎成纤维(NIH/3T3)细胞;实时荧光定量PCR及ELISA验证24p3在NIH/3T3细胞中的表达,以未转染质粒的空白组和转染空载体质粒组作为对照,RT-PCR检测各组细胞24p3mRNA水平,ELISA检测各组细胞培养上清中24p3水平,收集各组上清液;用氯化钴缺氧诱导小鼠肾小管上皮(TCMK-1)细胞,用收集的NIH/3T3细胞上清液在正常铁离子浓度下培养,流式细胞仪检测各组TCMK-1细胞凋亡情况。结果:转染Pc DNA3.1/24p3质粒后的NIH/3T3细胞24p3mRNA和蛋白表达水平明显增加,证实转染成功;与对照组相比,加入重组质粒转染组上清液的TCMK-1组凋亡细胞数量明显减少(P<0.05)。结论:24p3/NGAL介导的铁转运通过抑制肾小管上皮细胞的凋亡,减少急性肾损伤。

Objective： Investigate the protective effect of 24p3 / NGAL gene in renal function recovery of acute renal injury by mediating iron transport. Methods： Pc DNA3. 1 /24p3 plasmid was constructed by molecular cloning method,24p3 was transiently transfected into NIH /3T3 cells. Real time fluorescence quantitative PCR and ELISA were used to verify the expression of 24p3 in NIH /3T3 cells,and the blank group and the balnk plasmid group were used as control groups,PCR was used to detect 24p3 mRNA level in NIH /3T3 cells. ELISA was used to detect the expression of 24p3 in cell culture supernatant. The supernatant of each group were collected which used to culture the hypoxia induced TCMK- 1 cells in normal iron concentration. The cell apoptosis was detected by Flow cytometry. Results： the expression level of 24p3 mRNA and 24p3 protein in NIH /3T3 cells transfected with Pc DNA3. 1 /24p3 plasmid were significantly increased,and significantly reducing TCMK- 1 cells which cultured with supernatant of Pc DNA3. 1 /24p3 plasmid transfection group apoptosis compared with control groups（ P 〈0. 05）. Conclusion： 24p3 / NGAL protein has important protective effect on renal function recovery of acute renal injury by inhibiting the apoptosis of cells with mediating iron transport.