硒蛋白GPx1在硒拮抗脱氧雪腐镰刀菌烯醇致猪脾脏淋巴细胞毒性中的作用

Deoxynivalenol (DON) is one of the most common mycotoxins in the feed. DON can destroy the immune system in animal and induce immunosuppression, which cause high incidence of disease. The main target organ of DON in immune system is the spleen. DON can cause the apoptosis of the splenic lymphocyte, and inhibit the body's immune function. The damage of DON could be ameliorated by selenium. Selenoprotein GPx1, a cytoplasmic enzyme which widely present in various tissues, has important biological functions. The roles of GPx1 in the detoxification function of selenium are still unknown. In this study, the porcine splenic lymphocytes are cultured in vitro. The GPx1 gene slience cell model and the GPx1 gene overexpression cell model are established using the SiRNA technique and the gene transfection technique, respectively. Different concentrations of Se and DON are added into the culture systems. Flow cytometry, Western blot, real time quantitave RT-PCR, high performance liquid chromatography, and fluorescence labeling are used to detect the viability, the apoptosis, the GPx1 expression, the antioxidant capacities, the free radical contents, the DNA methylation and other indicators of the splenic lymphocytes. The roles of selenoprotein GPx1 in the lymphocytotoxicity of deoxynivalenol in swine and ameliorative effect by selenium would be revealed from the oxidative stress mechanism and the epigenetics mechanism. Our results would provide valuable reference in the human health, the food security, the adsorbent of mycotoxin, and the enviromental protection.

脱氧雪腐镰刀菌烯醇（DON)是饲料中最常见的霉菌毒素，DON最重要和最本质的危害是破坏机体免疫系统造成免疫抑制，引起疾病高发。脾脏是DON作用于免疫系统的主要靶器官，DON可使脾脏淋巴细胞凋亡，进而降低机体免疫功能。硒能够拮抗其毒性作用。硒蛋白GPx1是广泛存在于各组织的细胞质酶且具有重要生物学功能，但其在硒解毒功能中的作用尚不清楚。本项目以体外培养猪脾脏淋巴细胞为实验对象，应用SiRNA技术与基因转染技术建立GPx1基因沉默和蛋白过表达细胞模型，在培养体系中加入硒与DON，应用流式细胞术、免疫印迹、实时定量RT-PCR、高效液相色谱、荧光探针标记等方法，检测细胞活性、细胞凋亡、细胞内GPx1表达、抗氧化功能、自由基含量、DNA甲基化等指标，从氧化应激及表观遗传学方面揭示GPx1在硒拮抗DON致猪脾脏淋巴细胞毒性中的作用。为人类健康、食品安全、饲料去毒剂的研发及环境保护等方面提供参考。

本研究采用原代培养猪脾脏淋巴细胞作为研究对象，探明GPx1在硒拮抗DON致猪脾脏淋巴细胞毒性中的作用。测定了染DON后 48h对猪脾脏淋巴细胞的半数抑制浓度（IC50）为0.82±0.35 μg/mL。通过细胞活力检测确定Na2SeO3的最适浓度为2μmol/L在此基础上设计DON单独染毒剂量水平为0.103μg/mL、0.206μg/mL、0.412μg/mL和0.824μg/mL，Na2SeO3单独作用剂量为2μmol/L，DON和Na2SeO3联合作用的剂量为0.103μg/mL和2μmol/L、0.206μg/mL和2μmol/L、0.412μg/mL和2μmol/L、0.824μg/mL和2μmol/L，分别进行DON、Na2SeO3单独或联合作用对猪脾脏淋巴细胞的毒性损伤和保护作用。.模型分为正常组，运用基因转染技术构建GPx1过表达型猪脾脏淋巴细胞，和应用RNAi技术建立GPx1沉默型猪脾脏淋巴细胞。正式试验分为空白对照，空载，S，D1，D2，D3，D4，SD1，SD2，SD3，SD4共11组，于体外分别培养48h后，分别检测①细胞活力、细胞培养上清液乳酸脱氢酶（LDH）活性，检测细胞抗氧化水平；②实时荧光定量PCR检测GPx1、SOD、凋亡相关基因、甲基化相关酶的mRNA表达水平；③流式细胞仪检测细胞的凋亡率；④ELISA检测部分细胞因子和免疫球蛋白含量及mRNA表达水平。.结果表明DON单独作用于原代猪脾脏淋巴细胞会导致其氧化损伤，硒可对其起到拮抗作用，并且GPx1在硒拮抗DON致猪脾脏淋巴细胞毒性中的作用非常重要。