纳米硒、亚硒酸钠对大骨节病关节软骨细胞凋亡和活性氧的影响

Effects of selenium nanoparticles and sodium selenite on apoptosis and reactive oxygen species in chondrocytes from articular cartilage in patients with Kashin-Beck disease

目的比较纳米硒（Nano－Se）、亚硒酸钠（Na2SeO３）对体外培养的大骨节病关节软骨细胞凋亡和活性氧的影响，为大骨节病的药物治疗提供科学依据。方法依据《大骨节病临床诊断》标准（WS／T207—2010），收集8例进行I、Ⅱ度大骨节病膝关节置换病例的关节软骨。分离软骨组织，得到软骨细胞。分别给予０、25、50、100、200、300、400、500μg／LNano．Se和Na2SeＯ3培养液培养5d，采用四氮唑蓝（MTr）法检测软骨细胞存活率，以Nano．Se和Na２SeO３干预后的最高存活率对应的浓度与时间作为后续实验条件。取软骨细胞，分为对照组、Na２eO３组、Nano－Se组，每组8例，以上述得到的最佳实验浓度和时间进行培养，采用流式细胞术测定法测定软骨细胞凋亡率及活性氧荧光强度。结果用Nano-Se、Na2SeO3干预后，软骨细胞最高存活率对应的最佳浓度分别为100、400μg／L，最佳干预时间均为3d。Nano-Se组、Na2seO3组的总凋亡率[（4．67±0．89）％、（7．07±0．25）％]、晚期凋亡率[（1．51±0．48）％、（4．37±0．37）％]均低于对照组[（9．95±0．38）％、（6．93±0．42）％，P均〈0．01]，Nano-Se组的总凋亡率、晚期凋亡率均低于Na2SeO3组（P均〈0．01）。对照组、Na2SeO3组、Nano-Se组的早期凋亡率[（3．02±0．41）％、（2．7±0．46）％、（3．16±0．56）％]组间比较，差异无统计学意义（F=2．11，P=0．35）。Nano-Se组、Na2Se03组软骨细胞活性氧荧光强度[（87．13±6．60）％、（56．04±4．81）％]均低于对照组[（125．17±16．60）％，P〈0．01]，Nano-Se组低于Na2SeO3组（P〈0．01）。结论适宜浓度纳米硒较亚硒酸钠能更有效地缓解大骨节病软骨细胞的氧化应激水平，降低细胞凋亡，为深入研究大骨节病药物治疗提供了新的科学依据。

Objective To compare the effects of selenium nanoparticles （Nano-Se） and sodium selenium （Na2SeO3） on apoptosis and reactive oxygen species （ROS） of articular chondrocytes from patients with Kashin-Beck Disease （KBD） in vitro, and provide a scientific basis for preventing KBD. Methods The subjects with KBD were diagnosed on National Clinical Diagnostic Criteria of KBD （WS/T207-2010）, articular cartilage from 8 patients undertaken joint replacement operation were collected. In vitro, chondrocytes were treated with concentration of 0, 25, 50, 100, 200, 300, 400 and 500 μg/L of Nano-Se and Na2SeO3 for 5 d, respectively. Cell growth was detected by MTT assay, and the highest concentration and time corresponding to the highest survival rate of Nano-Se and Na2SeO3 were used in the following experiment. KBD chondrocytes were treated with Nano-Se and Na2SeO3, and divided into control group, Na2SeO3 group, Nano-Se group according to the randomized design. Each group had 8 cases. The cell apoptosis and ROS were detected by flow cytometry. Results The optimal intervention concentration of Nano-Se and Na2SeO3 was 100 and 400 μg/L, respectively. The optimal intervention time of NanoSe and Na2SeO3 both was 3 days. There was a significant decrease in the total and terminal apoptosis, ROS level of chondrocytes in Nano-Se group [（4.67± 0.89）%, （1.51 ±0.48）%, （56.04 ± 4.81）%] and Na2SeO3 group [（7.07 ± 0.25）%, （4.37 4. 0.37）%, （87.13 ± 6.60）%] compared with those of control group [（9.95±0.38）%, （6.93 ± 0.42）%, （125.17 ± 16.60）%, all P 〈 0.01]. The difference of early apoptotic rate among control group, Na2SeO3 group, NanoSe group [（3.02 ± 0.41）%, （2.7 4. 0.46）%, （3.16 ± 0.56）%] was not statistically significant （F = 2.11, P = 0.35）. Conclusion Appropriate concentration of Nano-Se can significantly decrease oxidative stress of KBD chondrocytes and inhibit apoptosis compared to Na2SeO3.