草酸诱导HK-2细胞核苷酸结合寡聚化结构域样受体3炎症小体的激活在草酸钙肾结石形成中的作用及机制  

The mechanism of oxalate - induced nucleotide binding oligomerization domain - like receptor pro- tein 3 inflammasome activation contributing to the formation of calcium oxalate stone in HK - 2 cells

作　　者：齐士勇[1] 何振[1] 徐勇[1] 张志宏[1] 

机构地区：[1]天津医科大学第二医院泌尿外科天津市泌尿外科研究所,300211

出　　处：《中华实验外科杂志》2017年第10期1635-1639,共5页Chinese Journal of Experimental Surgery

基　　金：天津市卫生局科技基金项目（2015KZ102）

摘　　要：目的探讨高浓度草酸作用于人肾小管上皮细胞HK-2后核苷酸结合寡聚化结构域样受体3（NLRP3）炎症小体在草酸钙肾结石形成过程中的激活作用机制。 方法通过测定细胞培养基中乳酸脱氢酶（LDH）的含量、4’，6-二脒基-2-苯基吲哚（DAPI）染色法分析草酸对HK-2细胞的毒性及损伤；使用相差显微镜观察高浓度草酸作用于HK-2细胞后细胞表面晶体黏附；通过Western blot法和实时荧光定量聚合酶链反应（FQ-PCR）分别检测高浓度草酸作用于HK-2细胞后NLRP3、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）及白细胞介素-1β（IL-1β）蛋白和mRNA表达量的影响，同时检测高浓度草酸作用于HK-2细胞后对透明质酸合酶1（HAS1）、透明质酸合酶2（HAS2）、透明质酸合酶3（HAS3）、骨桥蛋白（OPN）以及CD44 mRNA表达的影响，并采用二氯荧光黄二乙酸酯（DCFH-DA）检测活性氧自由基（ROS）；Western blot和FQ-PCR法分别检测使用ROS抑制剂N-乙酰基半胱氨酸（NAC）预处理HK-2细胞后高浓度草酸作用于细胞后NLRP3的蛋白及mRNA的表达水平变化；使用RNA干扰技术，将NLRP3-小干扰RNA（siRNA）和阴性对照（NC）转染HK-2细胞后，相差显微镜观察晶体黏附变化，FQ-PCR法分别检测HAS1、HAS2、HAS3、OPN以及CD44 mRNA表达水平变化。 结果使用不同浓度的草酸处理HK-2细胞24 h，测定LDH含量结果显示当草酸浓度为0.8 mmol/L时，细胞培养基中LDH开始比草酸浓度为0.6 mmol/L时显著增加[（528.37±25.65）比（300.55±17.18） mmol/L，P＝0.001]，DAPI染色法显示不同浓度草酸侧细胞数量分别为：55.67±3.30、52.33±6.13、52.33±2.05、58.00±9.09、50.00±1.63、47.33±2.05、33.67±3.30、20.33±2.05，当草酸浓度为1.0 mmol/L时，细胞数量开始比草酸浓度为0.8 mmol/L时明显减少（33.67±4.04比47.33±2.52，P＝0.008）；相差显微镜观察高浓度草酸＋一水草酸钙（COM）作用于HK-2�To observe the mechanism of nucleotide binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome to the formation of calcium oxalate stone after exposure to high concentrate of oxalate. Methods The human kidney proximal tubular epithelial HK-2 cells were cultured and stimulated with different concentrations of soluble oxalate cells. Lactate dehydrogenase （LDH） released assays from cell culture medium and 4’, 6-diamidino-2-phenylindole （DAPI） staining were used to determine the effect of cellular toxicity and injury after exposure to oxalate. All the cells of oxalate group and control group were incubated with calcium oxalate monohydrate （COM） crystals for 24 h, and cell surface adhesion of crystals were observed using an inverted phase-contrast microscope. Western blotting and real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） were applied to detect protein level and mRNA quantity of NLRP3, cysteinyl aspartate-specific protease-1 （Caspase-1）, and interleukin-1β （IL-1β） from HK-2 cell lysates after exposure to oxalate. FQ-PCR was applied to analyse mRNA quantity of hyaluronan synthase 1 （HAS1）, hyaluronan synthase 2 （HAS2）, hyaluronan synthase 3 （HAS3）, osteopontin （OPN） and CD44 from HK-2 cell after exposure to oxalate. Intracellular reactive oxygen species （ROS） generation was estimated by the method of using 2’, 7’-dichlorofluorescein diacetate （DCFH-DA） as per constructor’s protocol after exposure to oxalate. Thereafter, N-acetylcysteine （NAC）, a scavenger of ROS was pre-treated with HK-2 cells for 2 h, and Western blotting and FQ-PCR were applied to detect protein level and mRNA quantity of NLRP3 gene. HK-2 cells were transfected by NLRP3-small interfering RNA （siRNA）. After oxalate stimulation, an inverted phase-contrast microscope was used to detect the number of the binding COM crystals on the surface of the cells, and the levers of HAS1, HAS2, HAS3, OPN and CD44 mRNA through FQ-PCR. Results Expos

关 键 词：核苷酸结合寡聚化结构域样受体3炎症小体 草酸 活性氧自由基 草酸钙 结石 HK-2 

分 类 号：R692.4[医药卫生—泌尿科学]