探究纳米细菌损害HK-2细胞后的指标变化

Changes of Indexes in HK-2 Cells Injury Induced by Nanobacteria

目的:通过培养纳米细菌,探究纳米细菌(nanobacteria,NB)损伤人肾小管上皮HK-2后Na^(+)/K^(+)ATP酶等指标的改变。方法:选取HK-2细胞,随机分为五组:NB组,一水草酸钙(Calcium oxalate monohydrate,COM)组,纳米级羟基磷灰石(Nanograde hydroxuapatite,nHAP)组,四环素干扰组和空白对照组,在不同组中进行培养HK-2细胞,共培养6h,12h和24h时形态学观察;先将乳酸脱氢酶(LDH)、过氧化氢(H_(2)O_(2))以及丙二醛(MDA)的含量检测完之后,再经超声粉碎机粉碎HK-2细胞,最后检测各组培养液中ATP酶(Ca^(2+)/Mg^(2+)ATP酶和Na^(+)/K^(+)ATP酶)的活性。结果:经HE可见,同空白对照组相比,NB组HK-2细胞在实验6h时,可见部分细胞胞体膨胀增大,胞核松散,核膜显示不清,刷状缘结构排列紊乱,随着时间的推移,细胞遭到严重破坏,数目减少。COM组HK-2细胞情况与NB组相似,程度略重。NB组HK-2细胞的数量明显多于nHAP组和四环素干扰组。同空白对照组相比,在6h,12h,24h测得COM组中两种ATP酶活性最低,其次为NB组。nHAP组中两种ATP酶活性明显低于NB组,NB组中的两种ATP酶活性均低于四环素干扰组(P<0.05)。与空白对照组相比,COM组中H_(2)O_(2)含量和MDA含量最高,在6h时间点上,LDH含量低于对照组,NB组LDH、H_(2)O_(2)和MDA的含量高于对照组,但H_(2)O_(2)和MDA的含量低于COM组。四环素干扰组中H_(2)O_(2)的含量和MDA的含量明显低于NB组(P<0.05),nHAP组中H_(2)O_(2)和MDA的含量无明显变化(P>0.05)。结论:HK-2细胞损伤的机制或许由NB诱导脂质过氧化反应造成,也可能联合Na^(+)/K^(+)ATP酶、Ca^(2+)/Mg^(2+)ATP酶活性及LDH、H_(2)O_(2)、MDA的含量来判断其是否受到纳米细菌感染;HK-2细胞被NB作用的时间越长,其HK-2细胞损伤程度越重,晶体黏附量越多;四环素或许可以缓解NB对HK-2细胞的伤害。

Objective:To investigate the changes of Na^(+)/K^(+) ATPase and other indexes after the injury of human renal tubular epithelium HK-2 by nanobacteria(NB)by culture of nanobacteria.Methods:HK-2 cells were selected and randomly divided into 5 groups:HK-2 cells were cultured in the NB group,Calcium oxalate monohydrate(COM)group,Nanograde hydroxuapatite(nHAP)group,tetracycline interference group and blank control group.Morphology was observed at 6h,12h and 24h after co-culture.After the contents of lactate dehydrogenase(LDH),hydrogen peroxide(H_(2)O_(2))and malondialdehyde(MDA)were detected,then HK-2 cells were crushed by ultrasonic grinder.Finally,the activities of ATPase(Ca^(2+)/Mg^(2+) ATPase and Na^(+)/K^(+) ATPase)in culture medium of each group were detected.Results:It showed that,compared with the blank control group,HK-2 cells in NB group had swelling and enlargement of some cells,loose nuclei,unclear nuclear membrane,and disordered arrangement of brush border structure at 6h after the experiment.As time went by,cells were seriously damaged and the number of cells decreased.The condition of HK-2 cells in COM group was similar to that in NB group,and the degree was slightly worse.The number of HK-2 cells in NB group was significantly higher than that in nHAP group and tetracycline interference group(as seen by electron microscope).Compared with the blank control group,the two ATPase activities were the lowest in the COM group at 6h,12h and 24h,followed by the NB group.The two ATPase activities in nHAP group were significantly lower than those in NB group,and the two ATPase activities in NB group were lower than those in tetracycline interference group(P<0.05).Compared with the blank control group,the contents of H_(2)O_(2)and MDA were the highest in the COM group.At the time point of 6h,LDH content was lower than that in the control group.The contents of LDH,H_(2)O_(2)and MDA in the NB group were higher than that in the control group,but the contents of H_(2)O_(2)and MDA were lower than that in the COM group.The contents of H_(2)O_(2)and MDA in tetracycline interference group were significantly lower than those in NB group(P<0.05),but there were no significant changes in the contents of H_(2)O_(2)and MDA in nHAP group(P>0.05).Conclusions:The damage mechanism of HK-2 cells may be caused by lipid peroxidation induced by Nb,and it is also possible to determine whether HK-2 cells are infected by nanobacteria by combining the activities of Na^(+)/K^(+) ATPase,Ca^(2+)/Mg^(2+) ATPase and the contents of LDH,H_(2)O_(2)and MDA.The longer HK-2 cells were treated by NB,the more severe the damage degree of HK-2 cells was and the more the crystal adhesion amount was.The experimental results showed that tetracycline may alleviate the damage of NB to HK-2 cells.