An in vitro model of simulated ischemia-reperfusion injury established by using primary cultured mouse renal tubular epithelial cells / 中华器官移植杂志

ABSTRACT

Objective By using primary cultured mouse renal tubular epithelial cells (TECs) to develop an in vitro model of simulated ischemia-reperfusion (IR) injury.Methods The outer medulla of C57BL/6J mouse kidney was flushed and primary cultured after digestion in type Ⅰ collagenase,and then immunocytochemical staining was used to verify TECs.Primary cultured TECs were immersed in mineral oil to simulate the ischemic process,and 60 min later the whole culture medium was added to simulate reperfusion process.The cells were collected and RAN was extracted at indicated time points after medium replacement.The expression of TNF-α,IL-1β and IL-6 was detected by using real-time fluorescence quantitative RT-PCR. The culture supernatants were collected at 24 h after medium replacement for detection of the expression of cytokine protein by using ELISA.Results Primary cultured TECs were identified by cobblestone-shaped morphology and then verified by cytokeratin 18 (CK18) staining.In TECs of IR group after medium replacement the mRNA expression of TNF-α,IL-1β and IL-6 was higher than in control group.The expression of TNF-α after medium replacement was increased to a peak level at 0.5 h,about (24.45 ±6.51) times (P＜0.01 ) higher than the control group,and gradually declined thereafter.The mRNA expression of IL-1β after medium replacement kept an increasing tendency,about ( 15.27 ± 4.29) times (P＜0.05) higher than the control group at 6 h,and that of IL-6 after medium replacement was increased to a peak level at 3 h,about ( 11.19 ±4.55) times (P＜0.01) higher than the control group. In the IR group at 24 h after medium replacement,the protein expression of NF-α,IL-1β and IL-6 in the supernatants was significantly higher than in the control group.Conclusion High purity of primary cultured TECs was achieved from the outer medulla of mouse kidney by separation and digestion.The in vitro model of simulated IR in primary cultured mouse renal TECs was successfully created using paraffin oil.