【作者】 吴力专；

【导师】 张彬；

【作者基本信息】 湖南农业大学 ， 生理学， 2013， 博士

【摘要】 为了揭示高效抗氧化剂MT(Metallothionein)对热应激泌乳牛淋巴细胞凋亡/坏死的调节机理,本论文首先以中国荷斯坦奶公犊脾淋巴细胞为体外培养对象,采用1h,43℃或正常培养,诱导细胞发生不同程度的凋亡和坏死,同时采用不同终浓度的MT处理,结合流式细胞术和western-blot支术,研究抗氧化剂MT和对细胞凋亡/坏死的影响及其相关信号通路的调节机理,初步了解抗氧化剂MT对热应激细胞的凋亡/坏死的影响及部分与凋亡信号通路相关的活性因子的调节机理；后以20头正处于热应激的中国荷斯坦泌乳牛为试验研究对象,依据不同MT剂量分成：A(0mg/头)、B(4mg/头)、C(8mg/头)和D(16mg/头)4组,静脉注射后采取时间递减法采血(第1次采血为注射MT之前),应用流式细胞术、western-blot和实时定量PCR技术,进行MT对奶牛热应激所致血淋巴细胞凋亡/坏死的影响及调节机理的研究,为生理活性物质和细胞凋亡的理论研究及其在生产优质安全奶产品中的应用提供科学依据和探索新的途径。主要研究结果如下：1MT为5.25～8.25μg/mL时,对正常细胞凋亡有轻微上调作用；低浓度MT(2.25μg/mL)小幅度上调细胞坏死率；MT可提高细胞线粒体膜电位,其中热应激细胞所需MT浓度高(8.25μg/mL),而正常细胞需MT浓度宜适中(3.75～6.75μg/mL),其它浓度则不能充分保护细胞；热应激导致细胞的核转录因子活性亚基p50的磷酸化蛋白表达上调,但抑制活性亚基p65的磷酸化蛋白表达,对非热应激的原代脾淋巴细胞,MT可提高核转录因子亚基p65的活性,p65是细胞中发挥效用的亚基,因此热氧化应激导致NF-κB的活性下降,热应激信号不能很快转入细胞核内；MT具有上调phospho-P53的表达及其活性,调节热应激损伤所致细胞的凋亡,达到其保护作用：MT使用浓度为2.25μ.g/mL,因上调Phosphs-c-jun的表达量而提高AP-1的生成量,而热氧化应激时细胞Phospho-c-jun表达量低而抑制AP-1的生成量。因此,热处理提高牛脾淋巴细胞线粒体膜电位,但同时也导致细胞坏死率增加,而添加MT不仅提高细胞线粒体膜电压,还对热应激导致细胞坏死具有较好的抑制作用,MT保护细胞比热处理更具有现实的意义,同时也初步证明线粒体通路是MT调节细胞凋亡/坏死的信号转导通路之一2注射MT后,在51～60d时,校正标准产奶量B、C和D组均显著性高于A组(P<0.05)；35d时C组(8mg/头)的血淋巴细胞凋亡率显著性低于B、D组(P<0.05), D组(16mg/头)35d时凋亡率达到最大,随后(50-60d)显著性下调(P<0.05)；细胞的坏死率A组50d时较1d时显著下调(P<0.05),C组(8mg/头)35、50d时显著下降(P<0.05),而D组(16mg/头)坏死率呈现下降趋势,且在50、60d时均显著低于注射MT之前的血淋巴细胞坏死率；35d时,A组的线粒体膜电位值(△ψm)最高,但上升速度最快的是B组(4mg/头),在35d时显著高于20d时的△ψm(P<0.05)；50d时A组和B组的△ψm不但不上升,反而下降,其中A组下降的速度更快,35～50d C组(8mg/头)保持基本恒定,60d略有上升,D组(16mg/头)1～35d时逐步上升,50～60d时保持恒定,在60d时,△ψm与注射MT浓度呈正相关；C组(8mg/头)中,在注射后50d,其细胞内NO含量较注射MT之前,显著增加(P<0.05)；60d时,细胞内NO含量较注射MT之前,极显著(P<0.01)增加,同时显著大于注射35d时(P<0.05)。可见,8mg/头的MT注射剂量对细胞内NO含量影响最大；细胞坏死对细胞内NO含量有调节作用,反之则否；因此MT在注射剂量为4m/头时,其抑制动物热应激损伤的作用时间约为35d,注射剂量为8mg/头时,其作用时效则约为50d,而16mg/头时,其作用时间超过60d；说明外源性MT不仅能提高热应激状态动物的体质,还可让泌乳母牛从热应激状态平稳的过渡到热应激损伤修复状态。3C组的HSP70在20d时相对表达量最高,说明MT在动物处于热应激状态可通过促进HSP70的表达而达到保护动物的目的；HSP90-a的表达水平在35d时上调(除B组外),50-60d时,注射MT各试验组均表现为HSP90-a表达水平的增加,但对照组表现出下调趋势,此时动物进入热应激后损伤恢复期。35-50d时,C组Bcl-2的mRNA表达丰度显著高于其他各组(P<0.05),而Bax的mRNA表达量在50d时下降；B组50d时Bcl-2的mRNA表达丰度显著高于其他时间段(P<0.05),16mg/头则Bcl-2的表达受到抑制；35d时的Bax和Bcl-2同时增高,而后进入热应激后损伤恢复期(50d),Bax的基因相对表达量下降,但Bcl-2基因相对表达量增高,因此：mRNA Bcl-2的升高对mRNABax的表达具有抑制作用,证实了MT通过调节Bcl-2与Bax的协同作用而调控细胞凋亡。对照组中,HSP90-a/Bax. Bax/Bcl-2、HSP90-a/Bax之间的相关系数R依次下降,HSP70/Bcl-2、HSP70/Bax之间均无显著相关；因此MT由于上调抗凋亡因子如Bc1-2表达量,使得细胞的生存能力加强,反馈调节热休克蛋白的表达。4对照组50-60d时,c-myc的相对表达丰度值上调,而试验组显著低于对照组c-myc的基因相对表达丰度值(P<0.05)；动物注射MT后,B组,20～50d,p27kipl的相对表达丰度值为注射MT前的0.66～1.09倍,保持较低的表达水平,C组在20～60d时p27kipl的表达基本保持稳定,D组20～60d的p27kipl相对表达丰度值是注射MT之前的1.49～3.05倍,而对照组的p27kipl基因则在20～35d处于高水平相对表达。C组的c-jun和c-fos、Phospho-c-jun和Phospho-c-fos均呈强正线性相关,且线性斜率最大,推测其细胞AP-1的活性最大,促进的下游基因的表达,同时C组Bcl-2、Bax、p27kipl和NF-κBp50等表达量变化幅度也最大；C组在20、50和60d时,mRNA NF-κBp50的表达丰度均值显著大于其他各组(P<0.05),随着离注射MT时间越长,其mRNA NF-κBp50的表达丰度值下降,对照组的值在试验期间,呈现上升态势,因此8mag/头的注射剂量可以激活NF-KBp50的mRNA表达；当MT注射剂量为4m/头时,对NF-κB亚基p65抑制作用约为35d,注射剂量为8m/头的抑制时效为50d,MT为16mg/头时,抑制作用超过60d,因此MT的基因表达调控表现为化学剂量作用,即注射剂量越大,表现出对p65基因表达抑制作用的时间越长,推知,MT对NF-κBp50表达符合“全或无”的规律,在MT注射剂量为8m/头时,表现出促进亚基p50的表达,而对活性亚基p65的基因表达调控表现为化学剂量作用,通过这两种不同的方式分别对NF-κB进行调控,MT通过分别调节c-jun和c-fos的表达来控制AP-1的含量和活性,实现对细胞增殖、生长和凋亡等生理活动进行双向调节。更多还原

【Abstract】 In order to reveal the regulatory mechanisms of MT (metallothionein) which a highly effective antioxidant on apoptosis/necrosis in lactating cows under heat stress, the spleen lymphocytes from neonatal male calf were cultured firstly in37℃or43℃for1hour respectively to get varying degrees of apoptosis and necrosis, and adding different working concentration of MT in culture media, by flow cytometry and western-blot. And then,20Holstein dairy cattle in heat stress were divided into4groups:A (0mg/capita), B (4mg/capita), C (8mg/capita) and D (16mg/capita) by intravenous, were conducted with experiment for blood sampling by the law of diminishing time(the first bleed before intravenous injecting). The samples were determined by flow cytometry, western-blot and real-time PCR. It will provide scientific basis and new ways for applying of physiological activator and studying theoretical apoptosis. The results are as follows:1Heat stress increased cell apoptosis. MT only has inhibitory effect on the apoptosis induced by heat stress, while the inhibitory effect of MT on normal apoptosis is not obvious. The necrocytosis can be promoted by the lower MT when the concentration was2.25ug/ml; High fever can protect mitochondria. And the△Ψm value can be increased more by MT which strengthen cellular tissue because of its anti-apoptosis ability, but the high stress of cell require higher concentration of MT (8.25μg/mL), and the nomal cell require moderate concentration, cell can not be protected enough by too high(MT was8.25ug/ml) or too low (MT was2.25ug/ml) concentration of MT. Fever lead to increase the expression of phospho-p50and inhibit the expression of phospho-p65. While the primary spleen lymphocytes cultured in37℃can be increased expression of phospho-p65who mean to main effectiveness of NF-κB. Therefore, because of thermal oxidative stress resulting in decreasing activity of NF-κB, heat stress signal can not be quickly transferred into the nucleus. Heat stress injury caused by apoptosis could be regulated by MT increasing the expression of phospho-P53in case of its protective function. When the working concetration of MT was2.25μg/mL, the production of AP-1could be improved because of the expression of phospho-c-jun in cells in thermal oxidationstress decreased. Thus the production of AP-1could be inhibited by thermal oxidationstress. Therefore, fever improve mitochondrial membrane potential of bovine spleen lymphocytes, and it also led to increasing necrosis, while adding MT in culture media, not only mitochondrial membrane in cell could be improved, but also the necrosis being resulted in heat stress be inhibited better.It has more practical meaning for adopting adding MT to improve the integrity of cellular mitochondria than high fever, at the same time, it also showed that one of signal transduction pathway which MT can regulate apoptosis, necrosis induced heat stress is mitochondrial pathway.2Effects of exogenous metallothionein (MT) on lymphocytes apoptosis, necrosis and mitochondrial transmembrane potential(△Ψm) of China Holstein cows were studied using20lactating cows who being in heat stress, randomly allocated to groups A, B, C and D. MT was supplemented at0.0,4.0,8.0and16.0mg/capita, respectively, by intravenous injection. The results show that corrected milk yields in groups B, C, D were significant higher than those in group A after being injected MT during51-60d(P<0.05); at35d, the lymphocytes apoptosis rate of group C (8mg/capita) significantly lower than those of groups B and D (P<0.05), the lym Corrected milk yields e in group C (8mg/capita) was decreased significantly at35d and50d (P<0.05); And the necrosis rates in group D (16mg/head) in50,60d were significantly lower than in1d, the mitochondrial membrane potential (△Ψ) in group A reach to the supreme at35d, but the△Ψ in group B was significantly rising in35d (P<0.05), the△Ψ in group A and B was not only gone up, but decreased in50d, the△Ψ in group C was constantly during, in group D (16mg/head) kept rising during1～50d and kept constantly during51-60d, the dose of MT was higher resulted in the△Ψ being higher in60d.In conclusion, the content of NO in group C was increased significantly (P<0.05) in35d or50d and extremely significant (P<0.01) in60d comparing to before cows being injected MT.8mg/capitaof MT appeared the greatest impact for the NO in cell. And the content of NO in cell was regulated by necrosis, but the versa was not. At the same time there were35days for inhibiting heat stress leading to injury in cows for injection MT4mg/capita, and there were50days for8mg/capita,60days for16mg/capita. In conclusion, not only the heath of cows being in heat stress can be improved, but also the animals can be converted heat stress to heat stress damage repaired state by the exogenous MT.3The relative expression of HSP70in group C was the highest, and it mean that MT can protect animal in heat stress through promoting the expression of HSP70; HSP90-α expression was increased in35d(except for group B), during50-60d when the animals were at the state of recovering after heat stress injury, HSP90-a expression was rising in experimental groups and falling in control group; in group C, the gene expression of Bcl-2was creased significantly (P <0.05) during35-50d, and the gene expression of Bax was decreased in50d; in group C, the gene expression of Bcl-2was higher significantly in50d than in the other time(P<0.05), and the gene expression of Bcl-2was supressed for injecting16mg/capita; both the gene expression of Bax and Bcl-2were increased in35d, and the gene expression of Bax was decreased while that of Bcl-2was increased in50d which was the moment of recovering from heat stress injury; in the case of these, the gene expression of Bax was inhibited by the gene expression of Bcl-2being increased, that confirmed MT adjusted apoptosis through the synergistic effect of Bcl-2and Bax. In control group, the correlation coefficient between HSP90-α and Bax, Bax and Bcl-2, HSP90-α and Bax were decrease in turn, and there was no significant correlation between HSP70, Bcl-2and Bax; in conclusion, MT increased the gene expression of anti-apoptotic such as bcl-2which lead to strengthen the viability of cells, and regulated the expression of the heat shock in feedback.4The gene expression of c-myc were rising during50-60d in control group, and was higher significantly than in experimental groups (P<0.05); the relative gene expression of p27kipl in group B maintained at from0.66to1.09times during20-50d as before injecting MT, and kept a low level of gene expression; and the relative gene expression of p27kipl in group C was remained stable; the relative gene expression of p27kipl in group D maintained at from1.49to3.05times during20-60d as before injecting MT; there is a high level relative expression of p27kiplduring20-35d in control group; there was a strong positive linear correlation between c-jun and c-fos, Phospho-c-jun and phospho-c-fos in group C, and the linear slope reached to the maximum, it suggested that the activity of AP-1in cells reached to the maximum, and promoted the expression of downstream genes, furthermore, the range of variation on gene expression also reached the maximum corresponding; the gene expression of NF-κB p50in group C was greater significantly than in the other groups in20,50-60d(P<0.05), along with the more time from injection MT, the gene expression of NF-κ B p50kept decreasing, while that of in group A appeared rising during the experiment; therefore, when the injected dose MT was8mg/capita, the gene expression of NF-κ B p50could be activated; when the injected dose MT was4mg/capita, NF-κ B subunit p65kept being inhibited about35days; and when the injected dose MT was8mg/capita,NF-κ B subunit p65kept being inhibited about50days; and when the injected dose MT was16mg/capita, the time of inhibition on NF-κ B subunit p65were more than60days; in conclusion, the greater the dose of MT, the longer time of inhibition on gene expression of p65. the law on MT regulating gene of NF-κ B p50was "all or none", beause of showing the promotion expression in MT dose to8mg/capita, while the chemical dose affection was appeared in the MT regulating the gene expression of p65, and the two laws consist of the regulatory mechanisms of MT on NF-κ B. By controlling the activity and contents of AP-1respectively through regulating the expression of c-jun and c-fos, MT completed the bidirectional regulation on cell proliferation, growth and apoptosis and other physiological activities.更多还原