纳米细菌在肾结石形成过程中的作用研究

Investigation on the role of nanobacteria in the formation of renal calculi

目的探究纳米细菌(NB)损伤人肾小管上皮细胞HK-2并导致晶体滞留的机制。方法应用肾结石患者尿液培养的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)、COM组(一水草酸钙,COM悬液、胎牛血清、1640培养液+HK-2细胞)、nHAP组(纳米级羟基磷灰石,nHAP、胎牛血清、1640培养液+HK-2细胞)和四环素干扰组(TET+NB,四环素、NB菌液、胎牛血清、1640培养液+HK-2细胞)。将HK-2细胞分别与各组作用物共培养6、12、24 h后,用光学显微镜观察HK-2细胞的形态学变化;超声粉碎细胞后检测Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性;检测培养液中乳酸脱氢酶(LDH)、过氧化氢(H_(2)O_(2))和丙二醛(MDA)含量。结果HE结果可见,nHAP组和四环素干扰组中HK-2细胞的损伤程度明显低于NB组(P<0.05)。共同培养12、24 h后,NB组和COM组Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-ATP酶活性均低于对照组,H_(2)O_(2)含量均高于对照组(P<0.05),nHAP组Ca^(2+)-Mg^(2+)-ATP酶活性均低于对照组(P<0.05),四环素干扰组Na^(+)-K^(+)-ATP酶和Ca^(2+)-Mg^(2+)-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HK-2细胞损伤的原因可能是NB通过诱导脂质过氧化反应造成的,NB作用时间越长,损伤程度越重。四环素在一定程度上可以阻止NB对HK-2细胞的损伤。

Objective To explore the mechanism of nanobacteria(NB)to damage human renal tubular epithelial HK-2 cells and lead to crystal retention.Methods The urine of patients with renal calculi was applied to culture NB.The experiment included five groups:the control group(fetal bovine serum,1640 culture medium+HK-2 cells),the NB group(NB bacterial solution,fetal bovine serum,1640 culture medium+HK-2 cells),the COM group(calcium oxalate monohydrate,i.e.,COM suspension,fetal bovine serum,1640 culture medium+HK-2 cells),the nHAP group(nano-hydroxyapatite,i.e.,nHAP,fetal bovine serum,1640 culture medium+HK-2 cells)and the tetracycline(TET)interference group(TET+NB,tetracycline,NB bacterial solution,fetal bovine serum,1640 culture medium+HK-2 cells).HK-2 cells were co-cultured with each group of agents for 6 h,12 h and 24 h,respectively,and then the morphological changes of HK-2 cells were observed by optical microscope.The activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase were detected after ultrasonic comminution to break cells.The contents of lactate dehydrogenase(LDH),hydrogen peroxide(H_(2)O_(2))and malondialdehyde(MDA)in the culture medium were detected.Results HE results showed that the damage degree of HK-2 cells in nHAP group and tetracycline interference group was significantly lower than that in NB group(P<0.05).After co-culture for 12 and 24 h,the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase in NB group and COM group were lower than those in control group,and the content of H_(2)O_(2) was higher than that in control group(P<0.05).The activities of Ca^(2+)-Mg^(2+)-ATPase in the nHAP group were lower than those in the control group(P<0.05),and the activities of Na^(+)-K^(+)-ATPase and Ca^(2+)-Mg^(2+)-ATPase in the tetracycline interference group were higher than those in the NB group(P<0.05).After 6,12,and 24 h of co-culture,the MDA content in the NB group and the COM group were higher than that in the control group(P<0.05),and the MDA content in the tetracycline interference group was lower than that in the NB group(P<0.05);the LDH content in the COM group was higher than that in the control group(P<0.05),the LDH content in the nHAP group and the tetracycline interference group were lower than that in the COM group(P<0.05).Conclusion The damage of HK-2 cells may be caused by NB-induced lipid peroxidation.The longer the action time of NB,the more serious the damage.Tetracycline can prevent NB from damaging HK-2 cells to a certain extent.