【作者】 罗佳捷；

【导师】 张彬；

【作者基本信息】 湖南农业大学 ， 生理学， 2013， 博士

【摘要】 热应激对奶牛的生产性能、繁殖性能及免疫性能等方面均有不利影响,探索减少或缓解热应激的途径一直是奶牛饲养业的重大课题之一。为探讨外源性金属硫蛋白(MT)对热应激中国荷斯坦奶牛外周血淋巴细胞凋亡率及细胞周期的影响及其机理,选取20头中国荷斯坦奶牛经产泌乳母牛,随机均分为A、B、C、 D4组,分别按每头0(对照)、4.0、8.0和12.0mg的剂量经颈静脉注射Zn-MT,于试验第1(注射Zn-MT之前)、21、36、51和61d逐头采取血样,测定不同剂量的外源性MT对热应激奶牛血淋巴细胞凋亡率、细胞周期、亚型分布,细胞凋亡及细胞周期调控相关基因、蛋白的表达水平和相关蛋白磷酸化水平的影响。结果表明：1)MT对热应激奶牛外周血淋巴细胞凋亡率、细胞周期和亚群分布的影响补给外源性MT后,B、C两组奶牛的外周血淋巴细胞凋亡率分别比对照组降低了26.63%和24.84%,但未达到显著(P>0.05)水平;B、C两组奶牛处于Go/G1期的淋巴细胞数量略有降低,而处于S期和G2/M期的细胞数量则有一定程度的增加,但增幅和减幅均未达到显著水平(P>0.05)；试验组奶牛血液中的CD3+细胞数量比对照组分别高出了7.02%(P>0.05)、5.45%(P>0.05)和3.85%(P>0.05),而CD4+细胞数量分别比对照组高出31.04%、35.68%和39.34%,均达到显著(P<0.05)水平,CD8+细胞数量和CD4+/CD8+的水平也有明显提高,其中C组奶牛的CD4+/CD8+水平要显著(P<0.05)高于对照组。说明外源性MT能有效降低热应激奶牛的血淋巴细胞凋亡率并改变其细胞周期,还能对T淋巴细胞的亚型分布产生一定影响,且表现出较为明显的剂量效应。2)MT对热应激奶牛外周血淋巴细胞周期及凋亡相关基因表达水平的影响注射外源性MT后,各试验组奶牛外周血淋巴细胞中促凋亡基因cytc、Fas、 Apaf-1和p53的表达水平均有不同程度的降低,其中,B组奶牛cytc基因表达水平的降幅达到显著(P<0.05)水平,D组奶牛Fas基因表达水平的降幅、3组奶牛Apaf-1和p53表达水平的降幅达到极显著(P<0.01)水平；在细胞周期相关基因的表达水平方面,B、C两组奶牛CyclinA、CyclinD和CyclinE基因的表达水平均有不同程度的升高或与对照组持平,其中,CyclinA基因表达水平的增幅达到极显著(P<0.01)水平,而D组奶牛这3种基因的表达水平则呈现降低的趋势；除了C组奶牛CDK6基因表达水平稍有增加外,3个试验组奶牛CDK2、 CDK4和CDK6基因表达水平的增幅均达到显著(P<0.05)或极显著(P<0.01)水平。说明外源性MT能有效降低奶牛外周血淋巴细胞中促凋亡基因的表达水平,并能增加细胞周期正性调节因子基因的表达水平,且表现出较为明显的剂量效应。。3)MT对热应激奶牛外周血淋巴细胞p53、p105蛋白表达水平及p53、p65、p105蛋白磷酸化水平的影响注射外源性MT后,各试验组奶牛p53、p105蛋白的表达水平及p53、p65、p105蛋白的磷酸化水平均出现下降的趋势,其中,B、C两组p53蛋白表达水平及其磷酸化水平分别比对照组降低了35.00%(P<0.01)、32.00%(P<0.01)和18.00%(P<0.01)、12.00%(P<0.01)；3个试验组奶牛p105蛋白表达水平及其磷酸化水平分别比对照组降低了25.00%(P<0.01)、20.00%(P<0.01)、10.00%(P<0.05)和10.00%(P<0.01)、8.00%(P<0.05)、4.00%(P>0.05)；而p65蛋白的磷酸化水平则分别比对照组降低了16.00%(P<0.01)、11.00%(P<0.01)和5.00%(P>0.05)。说明外源性MT能通过抑制促凋亡蛋白的表达及其磷酸化来降低淋巴细胞的凋亡率,且表现出较为明显的剂量效应。综上所述,外源性MT能通过抑制促凋亡基因的表达水平和促凋亡蛋白的表达及其磷酸化水平来降低淋巴细胞的凋亡率,并能通过增加细胞周期正性调节因子的表达水平来推动细胞周期的正常进行；此外,MT还能有效改善淋巴细胞亚型的分布状态,最终增强热应激奶牛机体的免疫功能。在本研究中,以4.0和8.0mg注射量为佳。更多还原

【Abstract】 Heat stress have a negative impact on production performance, reproductive performance and immune performance of dairy cattle, and explore the ways to decrease or relieve heat stress was always one of the major topics of dairy cattle breeding. To research the effect of exogenous metallothionein (MT) on apoptosis rate and cell cycle of lymphocytes in peripheral blood of dairy cattle under heat stress,20lactating Chinese Holstein cows were selected and randomly divided into group A, group B, group C and group D, Zn-MT in the levels of0,4.0,8.0and12.0mg were supplemented by intravenous injection respectively. Blood samples were collected at1st (before injection),21st,36th,51st and61st day, and the apoptosis rate, cell cycle and subsets distribution as well as the levels of cell cycle and apoptosis related genes expression and related protein phosphorylation of lymphocytes in peripheral blood of dairy cattle were determined. The results showed that:1) Effect of MT on cell cycle, apoptosis rate and subsets distribution of lymphocytes in peripheral blood of dairy cattle under heat stressThe apoptosis rate of lymphocytes in group B and C were lower than those in group A by26.63%(P>0.05) and24.84%(P>0.05), respectively. The number of lymphocytes at G0/G1phage in B and C groups were decreased and the number of lymphocytes at S and G2/M phages were tend to increase, but significant differences were not be observed(P>0.05). The number of CD3+in three trial groups were greater than those in group A by7.02%(P>0.05),5.45%(P>0.05) and3.85%(P>0.05), while the number of CD4+in experimental groups were higher than those in control group by31.04%(P<0.05),35.68%(P<0.05) and39.34%(P<0.05), respectively. The number of CD8+and the levels of CD4+/CD8+in trial groups were increased observably, but significant differences(P<0.05) was observed in the levels of CD4+/CD8+between group A and C only. It suggested that exogenous Zn-MT can decrease apoptosis rate, and regulate cell cycle and subsets distribution in dairy cattle in a dose-dependent manner.2) Effect of MT on the expression levels of cell cycle related and apoptosis related genes of lymphocytes in peripheral blood of dairy cattle under heat stressThe expression levels of cytc, Fas, Apaf-1and p53genes of lymphocytes in peripheral blood were decreased after injection of MT, and the expression level of cytc in group B was lower than control group significantly(P<0.05), the expression level of Fas in group D and the expression levels of Apaf-1and p53in group B, C and D were decreased highly significantIy(P<0.01) compared to group A. The expression levels of Cyclin A, Cyclin D and Cyclin E in group B and C were increased or the same as control group, the expression level of Cyclin A in group B and C was great than group A highly significantly(P<0.01), and the trend of decrease was observed in group D. The expression levels of CDK2, CDK4and CDK6in experimental groups were higher than control group significantly(P<0.05) or highly significantly(P<0.01), except CDK6in group C. It suggested that exogenous Zn-MT can decrease the expression level of apoptotic genes and increased the expression level of positive regulator in cell cycle effectively in a dose-dependent manner.3) Effect of MT on the expression levels of p53and p105protein, and phosphoryl-ation of p53, p65and p105proteinThe expression levels of p53and p105protein, and phosphorylation of p53, p65and p105protein were decreased after injection of MT. The expression level and phosphorylation of p53protein in group B and C were lower than control group by35.00%(P<0.01),32.00%(P<0.01) and18.00%(P<0.01),12.00%(P<0.01) respectively. The expression levels and phosphorylation of p105protein in group B, C and D were lower than group A by25.00%(P<0.01),20.00%(P<0.01),10.00%(P<0.05) and10.00%(P<0.01),8.00%(P<0.05),4.00%(P>0.05)respectively. The phosphorylation of p65protein in experimental groups were lower than control group by16.00%(P<0.01),11.00%(P<0.01) and5.00%(P>0.05) respectively. It suggested that exogenous Zn-MT can decrease the apoptosis rate of lymphocytes in peripheral blood by inhibiting the expression levels and phosphorylation of pro-apoptotic proteins, and dose-dependent manner was exerted.In conclusion, exogenous MT can decrease the apoptosis rate of lymphocytes by inhibiting the expression levels of pro-apoptotic genes and proteins as well as the phosphorylation of pro-apoptotic proteins. Exogenous MT also can promote the regular cell cycle by increaseing the expression levels of positive regulator, while improve the subsets distribution of lymphocytes, and finally enhance the immunity of dairy cattle under heat stress. The dosage in4.0and8.0mg were batter than12.0mg in this study.更多还原