应用原代培养的小鼠肾小管上皮细胞建立体外模拟缺血再灌注损伤模型

An in vitro model of simulated ischemia-reperfusion injury established by using primary cultured mouse renal tubular epithelial cells

目的原代培养小鼠。肾小管上皮细胞（TEC），建立小鼠TEC体外模拟缺血再灌注（IR）的模型。方法切取C57BL／6J小鼠肾脏外侧髓质，用I型胶原酶消化后进行原代培养，采用免疫组织化学法进行鉴定。将贴壁生长的TEC用石蜡油覆盖模拟缺血过程，60min后更换全培养基模拟再灌注过程。在更换培养基后不同时间点收集细胞，抽提RNA，采用实时荧光定量聚合酶链反应检测肿瘤坏死因子-α（TNF-α）、白细胞介素1β（IL-1β）和IL-6mRNA的表达；在再灌注后24h收集细胞培养上清液，采用酶联免疫吸附试验检测各细胞因子蛋白的表达。结果经过原代培养的小鼠TEC呈上皮细胞特有的“铺路石”状，细胞胞浆内高表达细胞骨架蛋白18。与对照组相比，IR组TEC在更换培养基后TNF-α、IL-1β和IL-6的转录水平均明显增高。TNF-αmRNA表达量在更换培养基后0．5h达峰，约为对照组的（24．45±6．51）倍（P〈0．01），随后逐渐下降；在更换培养基后IL-113mRNA表达量呈逐渐上升的趋势，更换培养基后6h约为对照组的（15．27±4．29）倍（P〈0．05）；IL-6mRNA表达量在更换培养基后3、h达峰，约为对照组的（11．19±4．55）倍（P〈0．01）。与对照组相比，1R组TEC在更换培养基后24h，上清液中TNF-α、IL-1β和IL-6的蛋白表达均明显增高。结论小鼠肾脏外侧髓质经分离、消化后可获得高纯度的原代培养TEC；应用石蜡油可以成功建立小鼠TEC体外模拟IR的模型。

Objective By using primary cultured mouse renal tubular epithelial cells （TECs） to develop an in vitro model of simulated ischemia-repeffusion （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 rain 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 IL6 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.