CAJ | 학술논문

目的：探讨高糖对原代肾小球足细胞表达 MST1的影响。方法采用健康 SD 大鼠取肾组织，经过梯度离心后获取细胞悬液，培养原代肾小球足细胞，分别采用流式细胞术、免疫荧光染色验证原代细胞性质；将原代足细胞分为正常培养组（5 mmol／L）及高糖培养组（25 mmol／L）分别培养，以培养后12、24、48、72 h 为观察时间点。采用间接免疫荧光染色及 Western 印迹检测 caspase-3及MST1的表达。多组比较采用单因素方差分析，组间比较采用 q 检验。结果（1）采用健康 SD 大鼠培养可以获得稳定的原代细胞，间接免疫荧光染色显示细胞稳定表达 Nephrin、Wilms 肿瘤基因1（WT1），流式细胞计数检测显示93．18％的原代细胞稳定表达 Nephrin 蛋白。（2）免疫荧光染色显示正常培养组足细胞可见少量 MST1表达，主要位于细胞浆；高糖培养组，MST1表达有所增强，48 h 时开始出现较多细胞核表达。（3）Western 印迹检测结果显示，正常培养组足细胞可见低浓度的Caspase-3及 MST1表达，高糖培养组 Caspase-3及 MST1表达上调，差异有统计学意义（F ＝84．989、312．407，P ＜0．001）；高糖培养组48 h时 MST1可见亚带表达（MST1裂解片段），随造模时间延长表达增加。结论MST1信号通路异常可能参与糖尿病肾病的发生。
목적：탐토고당대원대신소구족세포표체 MST1적영향。방법채용건강 SD 대서취신조직，경과제도리심후획취세포현액，배양원대신소구족세포，분별채용류식세포술、면역형광염색험증원대세포성질；장원대족세포분위정상배양조（5 mmol／L）급고당배양조（25 mmol／L）분별배양，이배양후12、24、48、72 h 위관찰시간점。채용간접면역형광염색급 Western 인적검측 caspase-3급MST1적표체。다조비교채용단인소방차분석，조간비교채용 q 검험。결과（1）채용건강 SD 대서배양가이획득은정적원대세포，간접면역형광염색현시세포은정표체 Nephrin、Wilms 종류기인1（WT1），류식세포계수검측현시93．18％적원대세포은정표체 Nephrin 단백。（2）면역형광염색현시정상배양조족세포가견소량 MST1표체，주요위우세포장；고당배양조，MST1표체유소증강，48 h 시개시출현교다세포핵표체。（3）Western 인적검측결과현시，정상배양조족세포가견저농도적Caspase-3급 MST1표체，고당배양조 Caspase-3급 MST1표체상조，차이유통계학의의（F ＝84．989、312．407，P ＜0．001）；고당배양조48 h시 MST1가견아대표체（MST1렬해편단），수조모시간연장표체증가。결론MST1신호통로이상가능삼여당뇨병신병적발생。
Objective To investigate the effect of high glucose on MST1 (mammalian sterile 20-like kinase 1)expression in primary glomerular podocytes.Methods Primary podocytes cultured from healthy SD rat kidneys after gradient centrifugation were divided into normal group (cultured with glucose of 5 mmol/L)and high glucose group (cultured with glucose of 25 mmol/L).The cell properties were verified with flow cytometry and immunofluorescence.The time points for observation were after 12,24,48,and 72 hours of cell culture.And immunofluorescence and Western blotting method were used for detecting MST1 and caspase-3 expression.Multiple-group comparison was performed with single factor analysis of variance, while two groups were compared with Q test.Results (1)It was shown by flow cytometry that 93.18% of the podocytes cultured expressed nephrin protein,suggesting that stable culture of primary podocytes was successful.Besides,indirect immunofluorescence staining showed that the podocytes stably expressed nephrin and WT-1 proteins.(2)Immunofluorescence staining showed a small amount of MST1 expression in podocytes of the normal group mainly locating in the cytoplasm,while the expression of MST1 increased in the high glucose group,and more nuclear expression began to appear after 48 h of treatment.(3)Western blotting showed that the levels of caspase-3 and MST1 were significantly higher in the high glucose group than in the normal group (F =84.989,312.407,P <0.001 ).Furthermore,after 48 h of treatment, cleaved form of MST1 was observed in the high glucose group,which also increased with the time of treatment.Conclusions The dysfunction of MST1 pathway may be involved in the pathogenesis of diabetic nephropathy.