Abstract: Objective　To explore the pathological mechanism of nanobacteria（NB） on damage of human tubular epithelial HK-2 cells and the subsequent stone formation.Methods　NB was isolated from the urine of patients with kidney stones but without medication or surgery admitted to the Department of Urology Surgery in the First Affiliated Hospital of Medical College，Shihezi University from October 2016 to October 2017.The subjects were divided into control group（fetal bovine serum，1640 medium + HK-2 cells），NB group（NB culture，fetal bovine serum，1640 medium + HK-2 cells），calcium oxalate monohydrate（COM） group（5 mmol/L COM suspension，fetal bovine serum，1640 medium + HK-2 cells），nanograde hydroxuapatite（nHAP） group（300 mg/L nHAP，fetal bovine serum，1640 medium + HK-2 cells） and tetracycline（TET+NB） group（5 mg/L tetracycline，NB culture，fetal bovine serum，1640 medium + HK-2 cells）.The cell morphology was observed via hematoxylin-eosin（HE） staining and transmission election microscope（TEM） 6，12，and 24 hours after co-culture.The activities of Na+/K+ ATPase and Ca2+/Mg2+ ATPase were detected after ultrasonically smashing cells.Lactate dehydrogenase（LDH），hydrogen peroxide（H2O2） and malondialdehyde（MDA） in culture were detected.Laser scanning confocal microscope（LSCM） was used to observe the crystal adhesion to cells.Results　As shown by the observations of HE staining and TEM，the degree of cell damage in nHAP and TET+NB groups were significantly lower than that of NB group.The activities of Na+/K+ ATPase and Ca2+/Mg2+ ATPase in NB group and COM group were lower than those in the control group and the content of H2O2 in NB group and COM group was higher than that in the control group 12，and 24 hours after co-culture（P<0.05）.The activities of Na+/K+ ATPase and Ca2+/Mg2+ ATPase in TET+NB group were higher than those in the NB group 12，and 24 hours after co-culture（P<0.05）.The content of MDA in NB group and COM group was significantly higher than that in control group（P<0.05），while the content of MDA in TET+NB group was significantly lower than that in NB group 6，12，and 24 hours after incubation（P<0.05）.The content of LDH in COM group was higher than that in control group（P<0.05），while the content of LDH in NB group and TET+NB group was significantly lower than that in COM group（P<0.05）.Conclusion　NB may damage HK-2 cells by inducing lipid peroxidation.The degree of cell damage and the amount of crystal adhesion are positively related to the incubation time of NB.Tetracycline showed potential to inhibit this process and reduce crystal adhesion.

Key words: Kidney calculi, Nanobacteria, HK-2 cell

摘要： 目的　探究纳米细菌（NB）损伤人肾小管上皮细胞（HK-2细胞）并导致晶体滞留的机制。方法　于2016年10月—2017年10月在石河子大学医学院第一附属医院泌尿外科收集临床确诊肾结石且未应用药物或手术治疗患者的尿液培养NB。实验分为5组：对照组（胎牛血清、1640培养液+HK-2细胞）、NB组（NB菌液、胎牛血清、1640培养液+HK-2细胞）、一水草酸钙（COM）组（5 mmol/L COM悬液、胎牛血清、1640培养液+HK-2细胞）、纳米级羟基磷灰石（nHAP）组（300 mg/L nHAP、胎牛血清、1640培养液+HK-2细胞）和四环素干扰组（5 mg/L四环素、NB菌液、胎牛血清、1640培养液+HK-2细胞）。共同培养6、12、24 h后，采用光学显微镜及透射电子显微镜观察HK-2细胞的形态学变化；超声粉碎细胞后检测Na+/K+ ATP酶和Ca2+/Mg2+ ATP酶活性；检测培养液中乳酸脱氢酶（LDH）、过氧化氢（H2O2）和丙二醛（MDA）含量；用激光扫描共聚焦显微镜观察加入COM晶体后各组细胞的晶体黏附情况。结果　苏木素-伊红（HE）染色和透射电子显微镜结果可见，nHAP组和四环素干扰组HK-2细胞的损伤程度明显低于NB组。共同培养12、24 h后，NB组和COM组Na+/K+ ATP酶和Ca2+/Mg2+ ATP酶活性均低于对照组，H2O2含量均高于对照组（P<0.05），nHAP组Ca2+/Mg2+ ATP酶活性均低于对照组（P<0.05），四环素干扰组Na+/K+ ATP酶和Ca2+/Mg2+ ATP酶活性均高于NB组（P<0.05）。共同培养6、12、24 h后，NB组和COM组MDA含量均高于对照组（P<0.05），四环素干扰组MDA含量均低于NB组（P<0.05），COM组LDH含量均高于对照组（P<0.05），nHAP组、四环素干扰组LDH含量均低于COM组（P<0.05）。结论　NB可能通过诱导脂质过氧化反应损伤HK-2细胞，损伤程度和晶体黏附量与NB作用时间呈正比。四环素可以抑制NB对HK-2细胞的损伤并减少损伤后的晶体滞留。

关键词: 肾结石, 纳米细菌, HK-2细胞