| 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 μg/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 μg/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 μg/mL COM、先用PEG-4000溶液再加入800 μg/mL COM、先用800 μg/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 400μg/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（P＜0.05）、氧化应激水平降低（P＜0.05）；800 μg/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（P＜0.05）、氧化应激水平降低（P＜0.05）、RIPK3和p-MLKL表达减少（P＜0.05）。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 |
| DOI：10.16781/j.CN31-2187/R.20220929 |
| 投稿时间：2022-12-13修订日期：2023-03-07 |
| 基金项目:国家自然科学基金面上项目(8197032203). |
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| Effect of necroptosis on crystal adhesion of renal tubular epithelial cells and the intervention of polyethylene glycol |
| WAN 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
*Corresponding author) |
| 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 μg/mL COM or pretreated with receptor-interacting serine/threonine-protein kinase (RIPK) 3 inhibitor GSK-872 before 400 or 800 μg/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 μg/mL COM, PEG-4000 solution followed by 800 μg/mL COM, or 800 μg/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 adhesion was detected by ICP method. Results At 400 μg/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 (P＜0.05), and a decrease in oxidative stress level (P＜0.05). At 800 μg/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 (P＜0.05), the oxidative stress level was decreased (P＜0.05), and the expression of RIPK3 and p-MLKL was decreased (both P＜0.05). 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 or oxidative stress levels between the PEG-4000 post-added group and the COM treated group (all P＞0.05). Conclusion Inhibition of necroptosis by GSK-872 can reduce the adhesion deposition of COM in cells to a certain extent, but the crystals can aggregate on the cell surface to form amorphous precipitates under high crystal load. The pre-application with PEG-4000 in the culture medium can maintain the suspension stability of COM micrograins in suspension, with less accumulation, adhesion, deposition of crystals and less oxidative stress damage of cells. Addition of PEG-4000 to TCMK-1 cells exposed to COM could not reverse the crystal adhesion, aggregation or deposition. |
| Key words: renal tubular epithelial cells calcium oxalate stones necroptosis GSK-872 polyethylene glycol |
