纳米细菌对肾小管上皮细胞的损伤及晶体滞留的影响  

Effect of nanobacteria on cell damage and crystal retention in renal tubular epithelial cells

作　　者：于澄钒[1] 黄晓波[1] 陈亮[1] 许清泉[1] 胡卫国[1] 王晓峰[1] 

机构地区：[1]北京大学人民医院泌尿外科,北京100034

出　　处：《北京大学学报（医学版）》2010年第4期436-442,共7页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(40772026)资助~~

摘　　要：目的:研究纳米细菌(nanobacteria,NB)对HK-2细胞的损伤作用及机制,并探讨损伤后细胞与一水草酸钙(calcium oxalate monohydrate,COM)晶体粘附性的变化。方法:应用肾结石患者血清培养NB,免疫组化法对NB进行鉴定。实验分为4组:空白对照组、NB组、纳米级羟基磷灰石(nanograde hydroxuapatite,nHAP)组和COM组。HK-2细胞与各组作用物共同培养12、24h后进行形态学观察;培养6、12、24h后取培养液进行过氧化氢(H2O2)、LDH、MDA含量测定,消化细胞并超声粉碎后进行Na+/K+及Ca2+/Mg2+ATP酶活性的测定;培养6、12、24h后各组分别加入COM晶体,利用异硫氰酸荧光素-鬼笔环肽进行细胞荧光染色,于激光共聚焦显微镜下观察细胞与COM晶体的粘附情况。结果:NB组、nHAP组12h后出现细胞形态改变,刷状缘排列紊乱,胞浆、胞核松散,空泡形成;24h后线粒体肿胀,部分细胞核膜溶解、核仁消失,NB组核膜溶解细胞数量多于nHAP组。NB组12、24h H2O2含量高于空白对照组、nHAP组,6、24h MDA含量高于空白对照组、nHAP组,但NB组各时间点LDH含量与空白对照组、nHAP组无明显差异。Na+/K+ATP酶活性在12h后,NB组、nHAP组低于空白对照组,24h后,仅NB组低于空白对照组;Ca2+/Mg2+ATP酶活性在12h后,NB组、nHAP组低于空白对照组,24h后仅NB组低于空白对照组。12h后,NB组粘附COM晶体数量较前增多,而COM组还可见到部分晶体已进入细胞内;24h后,NB组与COM组晶体粘附情况与12h相近。空白对照组与nHAP组未观察到细胞与显著数量的COM晶体粘附。结论:NB对共同培养的HK-2细胞造成损伤,损伤程度随作用时间加长而加重,并强于nHAP,损伤过程中有脂质过氧化反应参与。NB损伤细胞后,细胞的晶体粘附性明显增强,随损伤作用时间的延长,细胞与一水草酸钙晶体的粘附量也会相应地增加;nHAP损伤细胞后未对细胞的晶体粘附性产生显著影响。Objective：To study the damage of nanobacteria on HK-2 cells,the possible principles,the effect of crystals（COM） adhering to HK-2 cells after the damage.Methods：Four groups were chosen for the study：control group,NB group,nHAP group and COM group.Morphological changes of the HK-2 cells were observed after HE stain and with TEM after 12 hours and 24 hours.Meanwhile,the levels of H2O2,LDH,MDA and ATPases were surveyed after 6 hours,12 hours and 24 hours,respectively.And 6,12,and 24 hours later,COM crystals were mixed into the culture fluids of each group.Then phalloidin-FITC was used to finish fluorescent staining of the cells.At last,the adhering effects of each group with the laser scanning confocal fluorescence microscope were observed and contrasted.Results：After HE stain and with TEM：in NB and nHAP group,the shape of the cells changed,brush borders were arranged in disorder,vacuoles formed in the kytoplasms,the mitochondria became swelled up,the karyotheca dissolved and the nucleolus disappeared in some cells.After 24 hours,in NB group,the number of the cells in which the karyotheca dissolved was more than that in nHAP group.After 12 and 24 hours,the level of H2O2 in NB group was higher than that in control group and nHAP group;After 6 and 24 hours,the level of MDA in NB group was higher than that in control group and nHAP group;At each time point,there was no significant difference in the level of LDH between control group,nHAP group and NB group;After 12 hours,the activities of Na＋/K＋ ATPases in NB group and nHAP group were lower than those in control group.And after 24 hours,the activity of Na＋/K＋ ATPases in NB group was lower than that in control group;After 12 and 24 hours,the activities of Ca2＋ /Mg2＋ ATPases in NB group was lower than those in control group.After 12 hours,the activity of Ca2＋/Mg2＋ ATPases in nHAP group was lower than that in control group.The observation with the laser scanning confocal fluorescence microscope：after 12 hours,showed that the number of the cr

关 键 词：肾结石 纳米细菌 肾小管 羟基磷灰石类 

分 类 号：R692.4[医药卫生—泌尿科学]