納米細菌在腎結(jié)石形成過程中的作用研究

楊恒 錢彪 鄭麗英 王勤章 郝志強 王敬珅 汪淵 李永樂 譚明輝 劉志立

[摘要]目的探究納米細菌（NB）損傷人腎小管上皮細胞 HK-2并導(dǎo)致晶體滯留的機制。方法應(yīng)用腎結(jié)石患者尿液培養(yǎng)的 NB，實驗分為5組：對照組（胎牛血清、1640培養(yǎng)液+HK-2細胞）、NB 組（NB 菌液、胎牛血清、1640培養(yǎng)液+HK-2細胞）、COM 組（一水草酸鈣， COM 懸液、胎牛血清、1640培養(yǎng)液+HK-2細胞）、nHAP組（納米級羥基磷灰石，nHAP、胎牛血清、1640培養(yǎng)液+HK-2細胞）和四環(huán)素干擾組（TET+NB，四環(huán)素、NB 菌液、胎牛血清、1640培養(yǎng)液+HK-2細胞）。將 HK-2細胞分別與各組作用物共培養(yǎng)6、12、24 h 后，用光學(xué)顯微鏡觀察 HK-2細胞的形態(tài)學(xué)變化;超聲粉碎細胞后檢測 Na+-K+-ATP 酶和 Ca2+-Mg2+-ATP 酶活性;檢測培養(yǎng)液中乳酸脫氫酶（LDH）、過氧化氫（H2O2）和丙二醛（MDA）含量。結(jié)果 HE 結(jié)果可見，nHAP組和四環(huán)素干擾組中 HK-2細胞的損傷程度明顯低于 NB 組（P <0.05）。共同培養(yǎng)12、24 h 后， NB 組和 COM 組 Na+-K+-ATP 酶和 Ca2+-Mg2+-ATP 酶活性均低于對照組，H2O2含量均高于對照組（P <0.05），nHAP組 Ca2+-Mg2+-ATP 酶活性均低于對照組（P <0.05），四環(huán)素干擾組 Na+-K -ATP 酶和 Ca+2+-Mg2+-ATP 酶活性均高于 NB 組（P <0.05）。共同培養(yǎng)6、12、24 h 后， NB 組和 COM 組 MDA 含量均高于對照組（P <0.05），四環(huán)素干擾組 MDA 含量均低于 NB 組（P <0.05）; COM 組 LDH 含量均高于對照組（P <0.05），nHAP組、四環(huán)素干擾組 LDH 含量均低于 COM 組（P <0.05）。結(jié)論 HK-2細胞損傷的原因可能是 NB 通過誘導(dǎo)脂質(zhì)過氧化反應(yīng)造成的， NB 作用時間越長，損傷程度越重。四環(huán)素在一定程度上可以阻止 NB 對 HK-2細胞的損傷。[關(guān)鍵詞]腎結(jié)石;納米細菌; HK-2細胞;脂質(zhì)過氧化;四環(huán)素

[中圖分類號] R692.4? [文獻標識碼] A?? [文章編號]2095-0616（2022）07-0021-05

Investigation on the role of nanobacteria in the formation ofrenal calculi

YANG HengQIANbiao??? ZHENG Liying??? WANG QinzhangHAO? Zhiqiang1，2WANG? Jingshen1，2????? WANG? Yuan1，2????? LI? Yongle1，2????? TAN? Minghui1，2LIU? Zhili

1. Department of Urology， First Affiliated Hospital， School of Medcine， Shihezi University， Xinjiang， Shihezi 832000， China;2. First Affiliated Hospital of Gannan Medical University， Jiangxi， Ganzhou 341000， China

[Abstract] 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 Ca2+-Mg2+-ATPase were detected after ultrasonic comminution to break cells. The contents of lactate dehydrogenase （LDH）， hydrogen peroxide （H2O2） 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 NBgroup （P <0.05）.After co-culture for 12 and 24 h， the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in NB group and COM group were lower than those in control group， and the content of H2O2 was higher than that in control group （P <0.05）. The activities of Ca2+-Mg2+-ATPase in the nHAP group werelower than those in the control group （P <0.05）， and the activities of Na+-K+-ATPase and Ca2+-Mg2+- 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.

[Key words] Renal calculi; Nanobacteria; HK-2 cells; Lipid peroxidation; Tetracycline

腎結(jié)石在泌尿系結(jié)石中最常見，而腎結(jié)石中以草酸鈣結(jié)石最為常見[1]，在《黃帝內(nèi)經(jīng)》中稱之為石淋或者砂淋。目前對于該疾病的診斷和治療已經(jīng)……