摘要
本研究的目的在于探讨外源性金属硫蛋白（MT）对热应激下体外培养奶牛淋巴细胞凋亡的影响。将屠宰获取的奶牛脾脏淋巴细胞随机分为4组，每组细胞培养液中MT的终浓度分别为0μg／mL（对照组）、70μg／mL（第1组）、140μg／mL（第Ⅱ组）、210μg／mL（第Ⅲ组），同时置于细胞培养箱（37℃、5％CO2）中培养20h，之后同时置于43℃水浴锅中进行热应激处理1h，再回细胞培养箱中培养3h，然后利用形态学观察、RT—PCR等方法进行各项指标的检测。结果如下，1）通过形态学观察和AO／EB染色观察，表明添加MT可以有效缓解热应激对奶牛淋巴细胞的损伤，但缓解效果随MT浓度的增加而下降；2）第Ⅰ～Ⅲ组的CAT活性、SOD活性和GSH—Px活性均高于对照组（P〈0．01），MDA含量均低于对照组（P〈0．01），表明MT能够有效改善淋巴细胞的抗氧化能力；3）试验Ⅰ～Ⅲ组的Bax基因相对表达量均显著低于对照组（P〈0．01），但试验Ⅰ～Ⅲ组Bcl-2基因的表达量和p50基因的表达量均显著低于对照组（P〈0．01），表明MT可以调节与细胞凋亡相关基因的表达。综上所述，添加MT可以降低奶牛淋巴细胞死亡率，改善奶牛淋巴细胞抗氧化性能，抑制奶牛淋巴细胞凋亡，以添加终浓度为70μg／mL的MT效果最好。
The effect of exogenous MT on lymphocyte apoptosis of the dairy cow was studied in vitro under heat stress. The splenic dairy cow lymphocytes were divided into 4 groups which had final concentrations of MT 0 （control group）, 70 （group Ⅰ ）, 140 （group Ⅱ ） and 210 μg/mL （group Ⅲ）. The cells of each group were cultured in a cell incubator （37℃, 5%CO2）. After 20 h, the cells of each group were put into water bath pot （43℃） for 1 h, and then recultured in a cell incubator （37℃, 5%CO2） for 3 h. Finally, various indexes were tested by observation of morphology and RT--PCR. 1） MT added in cell culture medium reduced the damage of dairy cow lymphoeytes under heat stress, but the effect decreased with an increase in MT concentration. 2） The activities of CAT, SOD, GSH-Px in groups Ⅰ to Ⅲ were higher than those in the control group and the content of MDA were lower. It is suggested that MT could improve antioxidation of lymphoeytes. 3） The relative transcript levels of Bax and p50 in group Ⅰ to Ⅲ were significantly （P〈0.01） lower than those in the control group. The relative transcript level of Bcl-2 in groups Ⅰ to Ⅲ were significantly （P〈0.01） higher than those in the control group. This indicated that MT could regulate the expression of genes related to lymphocyte apoptosis. In conclusion, exogenous MT decreased lymphocyte mortality rate, improved lymphocyte inoxidizability and restrained lymphocyte apoptosis . The best final concentration of MT was 70 μg/mL.
出处
《草业学报》
CSCD
北大核心
2014年第3期215-223,共9页
Acta Prataculturae Sinica
基金
国家自然科学基金项目(31072053
30671516)
湖南省自然科学基金重点项目(11JJ2014)
教育部高等学校博士学科点专项科研基金(博导类)项目(20104320110001)资助
关键词
金属硫蛋白
热应激
淋巴细胞凋亡
细胞培养
奶牛
metallothionein（MT）
heat stress
lymphocyte apoptosis
cell culture
dairy cow
