CuInS2/ZnS量子点对人卵颗粒细胞的毒性效应与机制

目的: 体外原代培养人卵颗粒细胞,检测CuInS₂/ZnS量子点的细胞毒性效应并分析其分子机制。方法: 采用透明质酸酶消化收集人超排卵外的颗粒细胞,进行体外原代培养,设立PBS对照组和低(1 μg/mL)、中(10 μg/mL)、高(100 μg/mL) CuInS₂/ZnS量子点实验组,利用CCK-8法检测量子点处理后颗粒细胞活力的变化并计算半数抑制浓度(IC₅₀);采用Annexin V/PI双染色法流式细胞术检测颗粒细胞凋亡情况;采用不同试剂盒分别检测性激素孕酮(P)和雌二醇(E2)的释放以及氧化应激相关指标活性氧(ROS)、丙二醛(MDA)、总抗氧化能力(T-AOC)、总超氧化物歧化酶(T-SOD)及谷胱甘肽还原酶(GSH)活性的变化。结果: 体外原代培养人卵颗粒细胞生长良好,在体外培养72 h后开始自发凋亡并黄素化。采用CuInS₂/ZnS量子点处理后48 h 的IC₅₀为66.72 μg/mL。与PBS对照组比较,高剂量量子点处理组明显抑制细胞活力(P<0.01),诱导细胞凋亡(P<0.01),增加颗粒细胞P和E2的释放(P<0.05或P<0.01),中、高剂量量子点处理后颗粒细胞中ROS水平显著增加(P<0.05或P<0.01),颗粒细胞内的MDA、T-AOC及T-SOD活性均显著降低(P<0.01),高剂量量子点作用后细胞内的GSH活性显著升高(P<0.01)。结论: 本研究成功进行了人卵颗粒细胞系的体外原代培养,发现10 μg/mL 剂量以上CuInS₂/ZnS 量子点可以通过侵入颗粒细胞引起氧化损伤,促进细胞凋亡引发毒性效应,为CuInS₂/ZnS量子点在临床应用中的生物安全性评估提供了有价值的信息。

Cytotoxic effects of CuInS2/ZnS quantum dots on human ovarian granulosa cells

OBJECTIVE: To culture human granulosa cells in vitro,detect the cytotoxic effect of CuInS₂/ZnS quantum dots and analyze its molecular mechanism. METHODS: Hyaluronidase was used to remove granulosa cells outside of human superovulation,and primary cultures were carried out. Cells were treated with medium (10 μg/mL) and high (100 μg/mL) CuInS₂/ZnS quantum dot doses. Changes of cell viability after treatment were detected by the CCK-8 method and the median lethal dose (IC₅₀) was calculated. Apoptosis was detected by flow cytometry with the Annexin V/PI double staining method;and release and oxidation of sex hormones were detected by kits stress changes. RESULTS: Human granulosa cells were successfully cultured in vitro. The IC₅₀of CuInS₂/ZnS quantum dots was 66.72 μg/mL. Compared with the PBS control group,cell viability was significantly inhibited and apoptosis was induced in the high- dose group (P<0.01). In the two treatment groups,release of progesterone (P) and estradiol (E2) were obviously increased (P<0.05 or P<0.01),levels of reactive oxygen species (ROS) in granulosa cells were significantly elevated (P<0.05 or P<0.01),and malondialdehyde (MDA),total antioxidative capacity (T-AOC) and total superoxide dismutase (T-SOD) activity were significantly decreased (P<0.01) compare with the control group. In addition, intracellular glutathione(GSH) activity was remarkedly increased in the high-dose treated group (P<0.01). CONCLUSION: Human granulosa cell lines were successfully cultured in vitro,and CuInS₂/ZnS quantum dots above 10 μg/mL induced oxidative damage by invading granulosa cells and promoted apoptosis. Our results provide valuable information for the biosafety assessment of CuInS₂/ZnS quantum dots in clinical applications.