CAJ | 학술논문

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Ⅰ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20 h，之后同时置于43℃水浴锅中进行热应激处理1 h，再回细胞培养箱中培养3 h，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。
본연구적목적재우탐토외원성금속류단백（MT）대열응격하체외배양내우림파세포조망적영향。장도재획취적내우비장림파세포수궤분위4조，매조세포배양액중MT적종농도분별위0μg/mL（대조조）、70μg/mL（제Ⅰ조）、140μg/mL（제Ⅱ조）、210μg/mL（제Ⅲ조），동시치우세포배양상（37℃、5％CO2）중배양20 h，지후동시치우43℃수욕과중진행열응격처리1 h，재회세포배양상중배양3 h，연후이용형태학관찰、RT-PCR등방법진행각항지표적검측。결과여하，1）통과형태학관찰화AO/EB염색관찰，표명첨가MT가이유효완해열응격대내우림파세포적손상，단완해효과수 MT농도적증가이하강；2）제Ⅰ～Ⅲ조적CAT활성、SOD활성화 GSH-Px활성균고우대조조（P＜0．01），MDA함량균저우대조조（P＜0．01），표명 MT능구유효개선림파세포적항양화능력；3）시험Ⅰ～Ⅲ조적Bax기인상대표체량균현저저우대조조（P＜0．01），단시험Ⅰ～Ⅲ조Bcl-2기인적표체량화p50기인적표체량균현저저우대조조（P＜0．01），표명MT가이조절여세포조망상관기인적표체。종상소술，첨가MT가이강저내우림파세포사망솔，개선내우림파세포항양화성능，억제내우림파세포조망，이첨가종농도위70μg/mL적 MT효과최호。
The effect of exogenous MT on lymphocyte apoptosis of the dairy cow was studied invitro under heat stress.The splenic dairy cow lymphocytes were divided into 4 groups which had final concentrations of MT 0 (control group),70 (groupⅠ),140 (groupⅡ)and 210μg/mL (groupⅢ).The cells of each group were cul-tured 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 lymphocytes 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 lymphocytes.3)The rela-tive 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 lym-phocyte apoptosis.In conclusion,exogenous MT decreased lymphocyte mortality rate,improved lymphocyte inoxidizability and restrained lymphocyte apoptosis .The best final concentration of MT was 70μg/mL.