磷酸铵镁对羊肾小管上皮细胞的损伤作用

Damage of magnesium ammonium phosphate on sheep renal tubular epithelial cells

为了研究磷酸铵镁(MAP)对羊肾小管上皮细胞(RTECs)的损伤作用,取20份结石羊尿沉渣进行镜检,并观察肾脏组织病理变化;其中在14份尿沉渣中观察到磷酸铵镁晶体,肾组织结构严重受损,RTECs变性坏死。观察尿结石羊症状并采集样品进行实验室检测。胶原酶组织块消化法分离培养羊RTECs,角蛋白18进行鉴定。MAP分别处理羊RTECs 12,24和48 h,CCK-8法检测羊RTECs的活力,Western blot法检测羊RTECs NF-κB信号通路的表达及Caspase-3、Bcl-2和Bax的水平,ELISA法检测细胞上清中IL-6和TNF-α的水平及LDH表达量。结果显示,尿结石羊尿液pH值比健康羊高,血液和尿液镁离子和磷离子水平也明显升高。胶原酶消化能获得较多且完整的肾小管节段,12 d左右细胞融合达80%~90%,呈典型的铺路石状,纯化后95%以上细胞CK-18鉴定为阳性。MAP能显著降低羊RTECs的活力,使IL-6和TNF-α的表达增加,细胞LDH含量增多。MAP组羊RTECs p-IκBα和p-NF-κB p65蛋白相对表达量增加,凋亡蛋白Caspase-3的表达明显增加,Bcl-2/Bax比值降低。综上所述,MAP能导致羊RTECs炎症反应与损伤,激活NF-κB通路,诱导细胞的凋亡。

In order to study the damage of magnesium ammonium phosphate(MAP)on renal tubular epithelial cells(RTECs).Twenty urinary sediment samples were examined by microscope,and renal tissue were taken to observe the histopathological changes.MAP crystals were observed in 14 urine sediment samples.The renal tissue structure was damaged seriously and RTECs were degenerated and necrotic.The clinical symptoms of urinary calculi in sheep were observed and the samples were collected for laboratory test.The sheep RTECs were isolated and cultured by collagenase digestion and identified by cytokeratin 18.The sheep RTECs were treated with MAP for 12,24 and 48 h respectively,the activity of sheep RTECs was detected by CCK-8 method.The expression of NF-κB signal pathway and the levels of Caspase-3,Bcl-2 and Bax in sheep RTECs were also detected by Western blot.The expressions of IL-6 and TNF-αin supernatants were detected by ELISA.The level of LDH was also detected.The results showed that the urine pH value of the stone sheep was higher than that of the healthy sheep,and the levels of magnesium and phosphorus in blood and urine increased significantly.A large number of renal tubular segments could be obtained by collagenase digestion.With 12 d culture,the cells were 80%-90%confluence and showed typical paving stone shape.More than 95%cells were identified by CK-18 as positive after purification.MAP could reduce the activity significantly and promote the expressions of IL-6and TNF in sheep RTECs.The content of LDH also increased.Western blot showed that the relative protein expressions of p-p65 and p-IκBαof sheep RTECs increased with MAP treatment.The expression of Caspase-3increased significantly,and Bcl-2/Bax ratio decreased.In conclusion,MAP can cause inflammatory response and injury in sheep RTECs,activate NF-κB pathway and induce apoptosis.