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目的:研究马兜铃酸Ⅰ(AAⅠ)致肾小管上皮细胞(LLC-PK1细胞)DNA损伤和对细胞周期的影响,并探讨其与P53途径的关系。 方法:不同浓度的AAⅠ纯品(80、160、320、640和1280ng/ml)体外刺激LLC-PK1细胞24h,采用单细胞凝胶电泳和流式细胞仪观察AAⅠ对LLC-PK1细胞DNA损伤和细胞周期的影响。用免疫荧光染色和流式细胞仪检测小管上皮细胞P53蛋白的表达。 结果:单细胞凝胶电泳实验发现 AAⅠ浓度为160,320,640和1280ng/ml时能导致LLC-PK1细胞产生彗星拖尾现象,且剂量越大,拖尾越明显,尾长和尾部荧光值与对照组比较有明显差异(P<0.05或P<0.01)。此剂量的AAI使LLC-PK1细胞在G2/M期的比例明显增高,且剂量越大,增高越明显,与对照组比较有明显差异(P<0.05或P<0.01)。各组小管上皮细胞P53蛋白的表达无明显差异(P>0.05)。 结论:AAⅠ可导致LLC-PK1细胞DNA损伤,使细胞周期阻滞在G2/M期,这可能是马兜铃酸肾毒性损伤后肾小管上皮细胞再生修复能力差和形成泌尿道肿瘤的机制之一。AAⅠ对LLC-PK1细胞DNA损伤和细胞周期阻滞作用是非P53依赖的,具体机制有待进一步研究。
OBJECTIVE: To study the effect of aristolochic acid Ⅰ (AA Ⅰ) on DNA damage and cell cycle in renal tubular epithelial cells (LLC-PK1) and to explore its relationship with P53 pathway. METHODS: LLC-PK1 cells were stimulated in vitro with various concentrations of AA Ⅰ (80, 160, 320, 640 and 1280 ng / ml) for 24 h. The DNA damage and the DNA fragmentation of LLC-PK1 cells were detected by single cell gel electrophoresis and flow cytometry Effect of cell cycle. The expression of P53 protein in tubular epithelial cells was detected by immunofluorescence staining and flow cytometry. Results: The results of single cell gel electrophoresis showed that the concentration of AI at 160, 320, 640 and 1280 ng / ml caused LLC-PK1 cells to produce tailing phenomenon, and the larger the dose, the more obvious tailing, tail length and tail fluorescence values compared with the control group There was a significant difference (P <0.05 or P <0.01). The dose of AAI significantly increased the proportion of LLC-PK1 cells in G2 / M phase, and the greater the dose, the more obvious the increase was, compared with the control group (P <0.05 or P <0.01). There was no significant difference in the expression of P53 protein in each group of tubular epithelial cells (P> 0.05). CONCLUSION: AAⅠ can induce DNA damage in LLC-PK1 cells and block the cell cycle in G2 / M phase, which may be the mechanism of renal tubular epithelial cell regeneration and repair and the formation of urothelial tumors after aristolochic acid nephrotoxicity one. AA Ⅰ on LLC-PK1 cells DNA damage and cell cycle arrest is non-P53-dependent, the specific mechanism to be further studied.