基于MDCK细胞单层的药物早期肾毒性检测初探

Preliminary Research on Early Detection of Drug Nephrotoxicity Based on MDCK Cells Monolayer

目的:初步探索基于犬肾小管上皮细胞(Madin-Darby canine kidney,MDCK)细胞单层的药物早期肾毒性检测方法。方法:将MDCK细胞培养于微孔滤膜上,待其形成完整的细胞单层。通过MTT试验分别选择对MDCK细胞没有明显抑制及具有明显抑制的不同质量浓度作用于细胞单层,24 h后进行荧光素钠透过性试验以及细胞单层两侧分泌的碱性磷酸酶(alkaline phosphatase,ALP)、γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,γ-GT)和乳酸脱氢酶(lactate dehydrogenase,LDH)活性检测,同时用扫描电镜观察细胞单层结构。结果:选择3.13,6.25,12.5 mg.L-13种质量浓度的马兜铃酸(aristolochic acid,AA)进行毒性检测,其对MDCK细胞生长的抑制率分别为-0.93%,0.93%和24.30%。AA 3.13 mg.L-1没有对细胞单层的完整性造成破坏,荧光素钠不能透过细胞单层,而AA 6.25,12.5 mg.L-1在与细胞作用30 min后,能明显增加荧光素钠的透过(P<0.05,P<0.1),其中12.5 mg.L-1造成的荧光素钠透过率增加更多。各组细胞单层两侧ALP,γ-GT的分泌没有明显改变,但12.5 mg.L-1能使LDH的分泌显著增加(P<0.05)。扫描电镜观察显示,AA 6.25,12.5 mg.L-1组细胞单层结构被破坏,表面出现大量小球状物体,细胞单层破损而变得不完整,有不同程度的微孔滤膜露出。结论:基于MDCK细胞单层,在AA对细胞生长没有明显抑制的浓度(6.25 mg.L-1),可以检测到药物对细胞的毒性损伤,提示建立在微孔滤膜上的MDCK细胞单层体系可以用于药物肾毒性的早期检测。

Objective： To study a detection method for drug early nephrotoxicity based on Madin-Darby canine kidney（MDCK） cells monolayer.Method： MDCK cells were cultured on microporous membrane to form a complete monolayer.Then the cells were treated with different drug concentration which had obviously inhibition on MDCK or not selected by MTT test.After 24 h,the cells were detected with fluorescein sodium permeability test and investigated by scanning electron microscope.At the same time,the activity of alkaline phosphatase（ALP）,γ-glutamyl transpeptidase（γ-GT） and lactate dehydrogenase（LDH） that secreted by both sides of the cell layer were also detected.Result： We choose three concentration of aristolochic acid（AA） to detect it’s toxicity,they were 3.13,6.25,12.5 mg.L-1.And their inhibition on MDCK cell were-0.93%,0.93% and 24.30%.AA 3.13 mg.L-1 could not disrupt the integrity of cell monolayer,and fluorescein sodium also can not penetrate the monolayer.AA 6.25,12.5 mg.L-1 could obviously increase the permeation of fluorescein sodium after 30 min（P ＆lt;0.05,P ＆lt;0.1）,especially for AA 12.5 mg.L-1.In either side of the cell layer,there showed no difference with ALP or γ-GT secretion.However,AA 12.5 mg.L-1 could increase LDH leakage on both sides of monolayer evidently（P ＆lt; 0.05）.In SEM detection,cells treated with AA 6.25,12.5 mg.L-1 emerged a large number of small spherical objects in surface,and the monolayer became incomplete.Conclusion： Based on MDCK monolayer,AA toxicity can be detected at the concentration which has no obviously inhibition on cells.It suggests that MDCK monolayer system established on microporous membrane could be used for early detection of drug nephrotoxicity.