CAJ | 학술논문

糖基化终产物(AGEs)在糖尿病肾病的发生发展过程中起着重要的作用.但目前其作用机制还不太清楚.通过体外乳鼠肾脏细胞的原代培养,探讨AGEs对肾细胞的损伤作用及可能的作用机制.取出生3天的SD大鼠的乳鼠肾脏进行体外原代细胞培养,并取传代到4-6代的细胞进行实验研究.分别用不同浓度的AGEs(0、1.2、2.5、5、10、20 mg/ml),不同的作用时间(6、12、18、24 h)作用于体外培养的肾细胞,用MTT法检测AGEs对肾细胞的增殖情况,用酶试剂盒法检测AGEs对肾细胞培养液中乳酸脱氢酶(LDH)、β-N-乙酰氨基葡萄糖苷酶(NAG)的含量,以及肾细胞内还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的含量.实验结果表明随着AGEs作用肾细胞时间的延长和浓度的增加,细胞存活率、细胞内GSH含量和SOD活性均逐渐下降,而细胞培养液中LDH和NAG的含量则逐渐升高,与正常培养的对照组细胞相比差异非常显著(P＜0.001),并且AGEs对细胞的作用与其浓度和作用时间呈显著的量效关系.实验结果说明AGEs对原代培养的肾细胞有明显的损伤作用,并随着AGEs作用浓度的增加和作用时间的延长对肾细胞的损伤越来越严重,实验结果也表明.肾细胞对AGEs的作用很敏感,其损伤细胞的途径和作用机制可能是由于改变了肾细胞膜的通透性和降低肾细胞抗氧化能力,该实验研究也进一步提示了AGEs是导致糖尿病肾脏并发症发生的重要原因之一.
당기화종산물(AGEs)재당뇨병신병적발생발전과정중기착중요적작용.단목전기작용궤제환불태청초.통과체외유서신장세포적원대배양,탐토AGEs대신세포적손상작용급가능적작용궤제.취출생3천적SD대서적유서신장진행체외원대세포배양,병취전대도4-6대적세포진행실험연구.분별용불동농도적AGEs(0、1.2、2.5、5、10、20 mg/ml),불동적작용시간(6、12、18、24 h)작용우체외배양적신세포,용MTT법검측AGEs대신세포적증식정황,용매시제합법검측AGEs대신세포배양액중유산탈경매(LDH)、β-N-을선안기포도당감매(NAG)적함량,이급신세포내환원형곡광감태(GSH)화초양화물기화매(SOD)적함량.실험결과표명수착AGEs작용신세포시간적연장화농도적증가,세포존활솔、세포내GSH함량화SOD활성균축점하강,이세포배양액중LDH화NAG적함량칙축점승고,여정상배양적대조조세포상비차이비상현저(P＜0.001),병차AGEs대세포적작용여기농도화작용시간정현저적량효관계.실험결과설명AGEs대원대배양적신세포유명현적손상작용,병수착AGEs작용농도적증가화작용시간적연장대신세포적손상월래월엄중,실험결과야표명.신세포대AGEs적작용흔민감,기손상세포적도경화작용궤제가능시유우개변료신세포막적통투성화강저신세포항양화능력,해실험연구야진일보제시료AGEs시도치당뇨병신장병발증발생적중요원인지일.
The advanced glycation end products (AGEs) may play an important adverse role in process of diabetic nephropathy; however, the pathological mechanism can not be explained. This study aimed to investigate the effect of AGEs on primary cultured neonatal rat kidney cells and discussed the functional mechanism. The kidney cells were isolated from 3-day-old rats for in vitro primary culture, and the 4-6th generations of the cells culture were treated with AGEs at different concentrations (0, 1.2, 2.5, 5, 10, 20 mg/ml) and different times (6, 12, 18, 24 h). Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) method, and the enzyme-linked assay kit evaluated the extracellular concentration alteration of lactate dehydrogenase (LDH) and N-acetyl-β-Dglucosaminidase (NAG), and the intracellular concentration alteration of reduced glutathione (GSH) and superoxide dismutase (SOD) influenced by AGEs. The results suggest that, along with higher concentration and longer action time of AGEs, the cell livability, the intracellular concentration of GSH, and the SOD activity are gradually decreased, however, the concentrations of LDH and NAG in culture solution are significantly increased (P＜0.001), compared with the control group. There is a significant concentration-effect relationship between the concentration and action time of AGEs. Our findings support that AGEs can significantly damage primary cultivated kidney cells, Moreover, the effect of AGEs on kidney cell is dose and time-dependent. Therefore, we conclude that kidney cells are sensitive to AGEs and the changes of cell permeability and antioxidant capacity induced by AGEs might be linked to the pathogenesis of diabetic nephropathy.