论文标题:应激肉牛几种组织中HSPs表达的研究
Studies on the Expression of HSPs in Tissues of the Stressed Cattle
论文作者 杨焕民
论文导师 胡仲明,论文学位 博士,论文专业 基础兽医学
论文单位 中国人民解放军军需大学,点击次数 193,论文页数 129页File Size6044k
2002-05-01论文网 http://www.lw23.com/lunwen_3130607/ 肉牛;应激;HSPs;HSP;mRNA
beef cattle;stress;HSPs;HSPmRNA
肉牛经常受到各种应激源的作用，比如低温、饲养管理、更换饲料、运输等等，会影响到肉牛的健康和生产力，所以应激已成为制约畜牧业发展的重要因素。目前很多学者对应激的研究主要集中在内分泌、能量代谢、消化、免疫等生理和病理生理变化，一般以体内激素浓度的变化作为应激指标。但是绝大多数激素在体内是在不断变化的，甚至某些激素的变化多是瞬间发生的。目前仍然应用血液中糖皮质激素的水平或糖皮质激素与促肾上腺皮质激素的比值作为应激的指标，因此，用此项标准来判断动物应激显然是不科学的。近年来的研究表明：热休克蛋白(HSPs)不仅由高温诱导产生，而且受多种刺激的影响。为此，探讨HSPs是否可以作为肉牛应激的指标确有必要。 本研究对肉牛施以冷应激、运输应激和饲料应激，用Western blot和RT-PCR方法检测肉牛在这些应激状态下骨骼肌、淋巴细胞和瘤胃粘膜乳头内的HSP90、HSP70、HSC70以及HSP70 mRNA和HSC70 mRNA的表达，用无线遥感仪测定了运输应激前后的体温，用酸度计测定饲料应激状态下的瘤胃液pH值。实验数据均用SPSS软件统计分析。结果显示：冷应激肉牛和常温下肉牛背最长肌中均有HSP90、HSP70和HSC70的表达，冷应激肉牛比常温下肉牛背最长肌中HSP70的表达量明显增加(P＜0.05)，而HSP90和HSC70的表达量无显著差异(P＞0.05)，提示HSP70对应激的反应比较敏感。测定运输应激肉牛的体温结果为：肉牛运输前1h平均体温为38.81±0.16℃，运输后0h体温为39.18±0.42℃，运输后1h为39.06±0.34℃。运输后0h、1h比运输前1h体温明显升高(P＜0.01)，体温升高表明运输导致动物发生了应激反应；运输应激后比运输前牛外周血淋巴细胞HSP70 mRNA表达量有显著增加(P＜0.05)，HSC70mRNA在运输前后无显著差异(P＞0.05)，这也提示HSP70 mRNA对运输应激的反应比较敏感：检测运输应激肉牛外周血淋巴细胞中 HSP90、HSP70和 HSC70在运输后 0 h均比运输前 lh表达量显著增加po0．of），HSP90运输后 lh比运输前 lh表达量也明显增加（P＜0．05）。提示运输应激可使HSPS表达量增加。测定饲料应激肉牛的结果为：饲喂50o／o、70o／o精料水平的日粮对外周血淋巴细胞中HSP90的表达有不同程度的影响。饲喂50％的精料日粮比饲喂干草在饲喂后 2 h、4 h HSP90的表达明显增加po0．of）：饲喂30o和 70％的精料日粮在饲喂后 2 h、4 h和 6 h HSC70的表达比饲喂前明显增加（P＜0．of），饲喂 50O精料日粮在饲喂后 6 h比饲喂前 HSC70的表达明显增加（P＜0．of），饲喂30o、50O和70O精料日粮比饲喂干草HSC70的表达量显著增加件0．们，除50％精料在采食后4 h州：饲喂30％、50％和70％精料日粮在饲喂后6 h均比饲喂前外周血淋巴细胞中 HSP70表达明显增加此0．of），饲喂30o、SOO和 70o精料日粮在饲喂后 2 h、4 h和 6 h均比饲喂干草在相同时间外周血淋巴细胞中HSP70表达明显增加 （P＜0．of，除饲喂 50％和 70％精料日粮在饲喂后 4 h）。提示饲料应激可以诱导＊驴go、＊哑7o和＊＊00表达量增加：当日粮中精料达50o／o时，采食后3 h、6 h、9 h牛瘤胃液pH值比采食干草明显降低（P＜0．of人 当日粮中精料达 50o／o、70％时，在采食后 6 h、9 h时 pH值比采食干草和采食30O／O精料在相同时间以值明显降低（P＜0．of人提示肉牛采食精料比例越高，瘤胃液酸度就越低；肉牛采食的日粮精料为50o／o、70％时，瘤胃粘膜乳头中 HSP70表达量明显增加（P＜0．05），HSP90和 HSC70无明显变化必0．05卜 提示＊肥7o比＊S网0和＊s＊7o对饲料应激的刺激更敏感： 用 Western blot和 RTICR方法检测肉牛在冷应激、运输应激和饲料应激状态下骨骼肌、淋巴细胞和瘤胃粘膜乳头内的HSP90、HSP70、HSC70以及HSP70 mRNA和HSC70 mRNA的表达。在冷应激状态下，骨骼肌中 HSP70表达明显增加：运输应激下，淋巴细胞中 HSP70 mRNA明显增2明显增加，同时HSP90、HSP70、HSC70也明显增加；饲料应激可以诱导外周血淋巴细胞中 HSP90、HSP70和 HSC70三种应激蛋白的表达。 以上实验结果证明三种应激可以诱导应激蛋白合成增加。从三种应激蛋白的表达看，以HSP70对应激源的刺激敏感性更高，HSP70可以作为监测肉牛应激与否的指标。
Beef cattle are subjected to a variety of stresses, which can impact the efficiency of production and health of the animal at various stages of the production cycle. These stressors may involve the following: temperature extremes, nutritional deprivation, transport, weaning and vaccination, abrupt diet changes and lactic acidosis. Stress in animal production becomes a main restriction factor for the industry development. At present, the research focuses on understanding metabolic, energetic, endocrine, immune and digestive function as a result of cattle experienced particular types of stress. These have included cold, wind, heat, under-nutrition and aspects of handling and transport stress. Generally, blood levels of glucocorticoids or the ratio of glucocorticoids and adrenocoticotropin hormone (ACTH) have often been used as an index of stress but collection of samples is a sufficient stress to induce elevated glucocorticoid levels. Therefore, blood hormone, although providing useful information about how an animal is reacting to stress, do not appear to indicate either the severity of stress or situations where animal may suffer serious health problem as a result of the stress. Recent years, a group of stress proteins known as heat shock proteins (HSPs) are studied to show many stress may induce HSP besides heat stress. Stress proteins may play a role in aiding protein in repairing damaged cell protein in response to environmental stress. It is necessary to study whether the proteins are used as a potential candidate for use in the assessment of stress in cattle.A controlled environmental temperature in cold stress experiments, 12 cattle werr subdivided evenly into subgroups and assigned to one of the following: cold environment(-18 C ) and warm environmen(18C ).The cattle in cold environment were stressed for 96 h. Blood samples for Western blot were collected from each animal. After three weeks acclimation, 6 beef cattle were stressed to ship 5 h to assess stress responses. Blood samples for western blot and RT-PCR were taken via jugular puncture prior to loading for shipment and after loading. The body temperature was recorded with a thermocouple probe as well as various body surface and environmental temperatures with a hand-held, infra-redthermometer. 6 fistulated steers were used in a cross - over design experiment. The steers were fed the all roughage diet, and had their diet changed from roughage to concentrate diet. In this case, the diet was changed in three steps from all roughage diet: initially to a 30% concentrate diet for 3 days, then to a 50% concentrate diet for 3 days and finally to a 70% concentrate diet for a further 3 days. Blood samples for Western blot were collected via jugular catheters (inserted the previous day) just before feeding and every 2 h, for 6 h after feeding on day zero, day three, day six and day nine. Rumen samples for measuring pH were collected via the rumen cannula before feeding and every 3 h after feeding on day zero, day three, day six and day nine. Rumen papilla samples for Western blot and RT-PCR were taken via the rumen cannula.For all measures, the data were analyzed by SPSS. The results are as follows: the expression of HSP70 in skeletal muscles in cold stresses beef cattle was higher than that of control (PO.01). The body temperature of the transport stresses animal were 38.81 ?.16C just 1 h before loading, 39.18 + 0.42C just 0 h after shipping, 39.06?.34C just 1 h after shipping, respectively. The body temperature of the animal after shipping was higher than that, of the animal before shipping (P<0.01). The expression of HSP70 mRNA was significantly elevated between before and after shipping (P<0.05), HSC70 mRNA was not significant (P>0.05). HSP90, HSP70 and HSC70 in lymphocytes after shipping 0 h were significantly elevated (P<0.01). In diet changes experiment. The expression of HSP90 in lymphocytes for feeding 50% concentrate diet 2 h, 4 h after feeding were significantly higher (P<0.01) than that of animal for feed roughage .The expression of HSC70
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