二水草酸钙晶体尺寸对非洲绿猴肾上皮细胞的损伤差异比较

doi:10.15541/jim20150332

摘要/Abstract

摘要：

采用扫描电子显微镜、激光扫描共聚焦显微镜、电感耦合等离子体发射光谱仪、流式细胞仪、酶标仪、细胞活性分析试剂盒和乳酸脱氢酶试剂盒(LDH)等方法比较研究了尺寸分别约为700 nm和15 μm的二水草酸钙(COD)晶体对非洲绿猴肾上皮细胞(Vero)损伤的差异。结果显示这两种不同尺寸的COD晶体都能引起Vero活力下降、LDH释放量升高以及碘化丙啶染色增强, 并表达带负电荷的骨桥蛋白, 说明它们对Vero都具有损伤作用, 且700 nm COD对Vero的细胞毒性及在细胞表面的粘附量均大于15 μm COD晶体。本研究从尺寸减小后COD的晶面变化、表面电荷变化、表面粘附位点、细胞与COD晶体间氢键作用等方面解释了亚微米COD毒性显著增强的原因。

关键词: 晶体粘附, 尺寸效应, 细胞毒性, 草酸钙

Abstract:

Cytotoxcity differences of submicron (700 nm) and micron (15 μm) calcium oxalate dihydrate (COD) on African green monkey kidney epithelial (Vero) cells were investigated by scanning electron microscope, laser scanning confocal microscope, inductively coupled plasma emission spectrometer, flow cytometry, microplate reader, cell proliferation assay kit and lactate dehydrogenase (LDH) release assay kit. The results revealed that both submicron and micron COD could decrease cell viability, increase LDH release amount, enhance propidium iodide staining, and up-regulate negatively-charged osteopontin expression, which indicated that both submicron and micron COD crystals could damage Vero cells. Submicron COD exhibited higher cytotoxicity and more adhesion amount than micron COD. The higher cytoxicity of submicron COD was discussed from change of crystal face, surface charge, surface adhesion sites, and hydrogen bonding between cells and crystals.

Key words: crystal adhesion, size effect, cytotoxicity, calcium oxalate

中图分类号:

TQ174

甘琼枝, 张冲宇, 欧阳健明. 二水草酸钙晶体尺寸对非洲绿猴肾上皮细胞的损伤差异比较[J]. 无机材料学报, 2016, 31(3): 317-323.

GAN Qiong-Zhi, ZHANG Chong-Yu, OUYANG Jian-Ming. Effect of Crystal Size of Calcium Oxalate Dihydrate on Cytotoxicity of African Green Monkey Kidney Epithelial Cells[J]. Journal of Inorganic Materials, 2016, 31(3): 317-323.

图/表 7

图1 亚微米/微米COD晶体的XRD图谱(a, b)和SEM照片(c, d)

Fig. 1 XRD patterns (a, b) and SEM images (c, d) of submicron/micron COD crystalsScale bar in (c) is 1 μm, in (d) 10 μm

图2 亚微米/微米COD晶体对Vero细胞活力(a)和LDH释放量(b)的影响

Fig. 2 Changes in cell viability (a) and LDH release amount (b) of Vero cells after exposure to submicron/micron COD crystals

图3 亚微米/微米COD与正常Vero作用6 h后经PI染色的结果

Fig. 3 PI staining results of Vero cells after exposure to submicron/micron COD crystals for 6 h(a) Control group; (b) Micron COD crystals treated group; (c) Submicron COD crystals treated group. Crystal concentration: 200 μg/mL. Magnification×600

图4 Vero与亚微米/微米的COD作用6 h后的ROS水平

Fig. 4 Intracellular ROS level of Vero cells after exposure to submicron/micron COD crystals for 6 h(a) Control group; (b) Micron COD crystals treated group; (c) Submicron COD crystals treated group

图5 正常Vero与不同尺寸COD作用6 h后的OPN表达

Fig. 5 OPN expression in Vero cells after exposure to submicron/micron COD crystals for 6 h(a) Control group; (b) Micron COD crystals treated group; (c) Submicron COD crystals treated group Green color represents OPN

图6 Vero与亚微米/微米COD粘附后的SEM照片

Fig. 6 SEM images of submicron/micron COD crystals adhered to Vero cells for 6 h(a) Control group; (b) Micron COD crystals treated group; (c) Submicron COD crystals treated group Crystal concentration: 200 μg/mL

图7 随着COD尺寸减小, COD晶体的形貌和(100)、(101)晶面变化模型图

Fig. 7 Schematic representation of morphology and crystal face (100) and (101) variation of COD crystals with decreased size

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