坏死性凋亡对肾小管上皮细胞...的影响及聚乙二醇的干预作用_万洋.pdf

海军军医大学学报2023 年 4 月第 44 卷第 4 期http:/Academic Journal of Naval Medical University,Apr.2023,Vol.44,No.4 394 论 著 收稿日期 2022-12-13 接受日期 2023-03-07基金项目 国家自然科学基金面上项目（8197032203）Supported by General Program of National Natural Science Foundation of China(8197032203).作者简介 万 洋，硕士生 E-mail:*通信作者（Corresponding author）.Tel:021-31161721,E-mail:坏死性凋亡对肾小管上皮细胞晶体黏附的影响及聚乙二醇的干预作用万 洋，彭泳涵，高小峰*海军军医大学（第二军医大学）第一附属医院泌尿外科，上海 200433摘要 目的 考察坏死性凋亡抑制剂及分子量为 4 000 的聚乙二醇（PEG-4000）对小鼠肾小管上皮细胞 TCMK-1 表面一水草酸钙（COM）晶体黏附沉积的影响。方法 分别用 400、800 g/mL COM 作用于 TCMK-1 细胞，或先用受体相互作用的丝氨酸/苏氨酸蛋白激酶（RIPK）3 抑制剂 GSK-872 预处理后再加入 400、800 g/mL COM 处理TCMK-1 细胞，37 孵育 12 h 后在倒置相差显微镜下观察细胞表面晶体黏附情况，用 CCK-8 法检测细胞增殖活性，2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）探针法检测细胞氧化应激水平，蛋白质印迹法检测坏死性凋亡相关蛋白 RIPK1、RIPK3、磷酸化混合谱系激酶结构域样蛋白（p-MLKL）的表达，电感耦合等离子体发射光谱法（ICP）检测细胞表面晶体黏附量。将TCMK-1 细胞分为 3 组，分别用 800 g/mL COM、先用PEG-4000 溶液再加入 800 g/mL COM、先用 800 g/mL COM再加入PEG-4000 溶液处理细胞，37 孵育 12 h 后在倒置相差显微镜下观察细胞表面晶体黏附情况，CCK-8 法检测细胞增殖活性、DCFH-DA 探针法检测细胞氧化应激水平，ICP 检测细胞表面晶体黏附量。结果 400 g/mL COM 作用时，与 COM 处理组相比，GSK-872 预处理组中 TCMK-1 细胞晶体黏附量减少、细胞增殖活性增强（P0.05）、氧化应激水平降低（P0.05）；800 g/mL COM 作用时，与 COM 处理组相比，GSK-872 预处理组中TCMK-1 细胞晶体黏附量无明显变化、细胞增殖活性增强（P0.05）、氧化应激水平降低（P0.05）、RIPK3 和 p-MLKL 表达减少（P0.05）。与 COM 处理组相比，PEG-4000 预处理组 TCMK-1 细胞晶体黏附量明显减少、细胞增殖活性增强、氧化应激水平降低（P均0.05），而后加入 PEG-4000 组与 COM 处理组相比晶体黏附量、细胞增殖活性、氧化应激水平均无明显变化（P均0.05）。结论 用 GSK-872 抑制坏死性凋亡可以一定程度减少 COM在细胞表面黏附沉积，但在较高的晶体负荷下晶体可在细胞表面聚集形成不定型沉淀。在培养基中使用 PEG-4000 预处理能够使 COM 微晶粒在悬液中保持悬浮稳定，减少晶体聚集沉积及细胞黏附和细胞氧化应激损伤；但充分接触 COM晶体后的 TCMK-1 细胞再加入 PEG-4000 不能逆转晶体细胞黏附聚集沉淀。关键词 肾小管上皮细胞；草酸钙结石；坏死性凋亡；GSK-872；聚乙二醇中图分类号 R 692.4文献标志码 A文章编号 2097-1338(2023)04-0394-08Effect of necroptosis on crystal adhesion of renal tubular epithelial cells and the intervention of polyethylene glycolWAN Yang,PENG Yong-han,GAO Xiao-feng*Department of Urology,The First Affiliated Hospital of Naval Medical University(Second Military Medical University),Shanghai 200433,China Abstract Objective To explore the effect of necroptosis inhibitor and polyethylene glycol with a molecular weight of 4 000(PEG-4000)on the adhesion and deposition of calcium oxalate monohydrate(COM)on the surface of transformed C3H mouse kidney 1(TCMK-1)cells.Methods TCMK-1 cells were separately treated with 400 or 800 g/mL COM or pretreated with receptor-interacting serine/threonine-protein kinase(RIPK)3 inhibitor GSK-872 before 400 or 800 g/mL COM treatment.After incubation at 37 for 12 h,the crystal adhesion of TCMK-1 cells was observed under the phase inverted microscope.The cell proliferation activity was detected by cell counting kit 8(CCK-8)method,the cell oxidative stress level was detected by 2,7-dichlorodihydrofluorescein diacetate(DCFH-DA)probe method,the expression of necroptosis-related proteins RIPK1,RIPK3,and phospho-mixed lineage kinase domain-like protein(p-MLKL)was detected by Western blotting,and the crystal adhesion of TCMK-1 cells was detected by inductively coupled plasma emission spectroscopy(ICP).TCMK-1 cells were divided into 3 groups and separately treated with 800 g/mL COM,PEG-4000 solution followed by 800 g/mL COM,or 800 g/mL COM followed by PEG-4000 solution.After incubation at 37 for 12 h,the crystal adhesion of TCMK-1 cells was observed under the phase inverted microscope.The cell proliferation activity was detected by CCK-8 method,the oxidative stress level was detected by DCFH-DA probe method,and the surface crystal DOI：10.16781/j.CN31-2187/R.20220929 395 肾结石是一种易复发性疾病，发病的结石成分主要是钙结石，以草酸钙最为常见1。研究表明，草酸钙结石的重要触发事件在于磷酸钙晶体在肾髓质间质组织中形成，蚀穿肾乳头上皮形成典型的Randall斑，草酸钙晶体可能沉积于该病灶顶部，附着于肾乳头形成结石2。肾结石的外科治疗通常只处理位于集合系统内的结石3，理论上肾髓质内的钙斑可能继续生长并促进残留结石或晶体在肾乳头上皮处黏附和聚集4-6。目前尚不明确影响晶体在肾小管上皮细胞黏附聚集的靶点及药物。研究表明，肾小管细胞损伤是促进草酸钙晶体在细胞表面黏附沉积的重要因素7，损伤引起的细胞膜结构及蛋白表达变化会促进草酸钙晶体在肾小管上皮细胞上的黏附沉积8。晶体沉积诱导的细胞毒性涉及受体相互作用的丝氨酸/苏氨酸 蛋白激酶（receptor-interacting serine/threonine-protein kinase，RIPK）3-混合谱系激酶结构域样蛋白（mixed lineage kinase domain-like，MLKL）介导的坏死性凋亡9。坏死性凋亡是程序性死亡模式的一种，其信号通路可通过RIPK3 等进行调节，有望通过调节肾组织细胞坏死性凋亡、减轻细胞损伤来干预肾脏的草酸钙晶体沉积10。然而Mulay等11通过构建坏死性凋亡相关的肿瘤坏死因子受体（tumour necrosis factor receptor，Tnfr）1 基因敲除和Tnfr1/Tnfr2 基因双敲除小鼠的肾脏晶体沉积模型，发现其肾损伤指标低于对照组，但其肾脏晶体沉积并无差异。尿液中草酸钙微晶体的聚集是导致晶体快速增大和结石形成的关键因素12-13。从化学角度adhesion was detected by ICP method.Results At 400 g/mL of COM,compared with the COM treated group,the GSK-872 pretreatment group showed a decrease in TCMK-1 cell crystal adhesion,an increase in cell proliferation activity(P0.05),and a decrease in oxidative stress level(P0.05).At 800 g/mL of COM,the crystal adhesion of TCMK-1 cells in the GSK-872 pretreated group had no significant changes compared with the COM treated group,while the cell proliferation activity was increased(P0.05),the oxidative stress level was decreased(P0.05),and the expression of RIPK3 and p-MLKL was decreased(both P0.05).Compared with the COM treated group,the crystal adhesion of TCMK-1 cells in the PEG-4000 pretreated group was significantly decreased,with enhanced cell proliferation activity and decreased oxidative stress level(all P 0.05).There were no significant changes in crystal adhesion,cell proliferation activity