【作者】 于纪棉；

【导师】 鲍恩东；

【作者基本信息】 南京农业大学 ， 基础兽医， 2009， 博士

【摘要】 我国是一个禽类养殖业大国,禽类的养殖数量居世界前列。然而,由于我国地域辽阔,季节性气候因素对养禽业的影响也不容忽视,如在我国中、南部的大部分地区,每年夏秋季处于高热的时间长达2-3个月之久,对热应激相对敏感的养禽业每年由此而造成的经济损失十分惊人。因此,了解和研究热应激损伤特征和规律以及采取合理的措施以减少热应激损伤对养禽业的危害具有重要意义。热应激是一种全身性的反应,对机体的神经-内分泌、消化吸收、血液循环、泌尿生殖、机体免疫和物质代谢等方面均会产生影响。但热应激反应中也可转录合成一组特殊的热休克蛋白(heat shock proteins, Hsps),这种具有高度保守性的Hsps对维持细胞生存和内环境的稳定起着十分重要的作用。本文的主要目的在于通过对受试鸡进行持续的热应激处理,动态观察和了解热应激肉鸡组织中Hsps的转录和表达水平以及Hsps在组织细胞中的定位等特点,研究Hsps的变化及其规律与肉鸡应激性损伤间的相关,探讨Hsps的组织细胞保护机制。将100只1日龄AA肉鸡随机分成5组,每组20只,置于温控气候室内。为使受试鸡适应新的环境,尽可能降低应激原的影响,所有受试鸡均进行为期30天的适应性饲养。在此阶段,严格按照AA肉鸡的饲养标准进行饲养,并且在整个饲养过程中,受试鸡由专人饲养。待受试鸡经过30天的适应性饲养后,除将对照组鸡仍然置于正常饲养环境(22±1℃)以外,将其余4组鸡的环境温度于20 min内由22±1℃迅速提高到37±1℃进行热应激处理。待热应激分别持续2h、3h、5h和10h后,颈静脉取血,分离血清,检测血清酶CK和GPT的含量。取血后迅速剖杀对照组鸡和热应激处理组鸡,采取受试鸡的心脏、肝脏、肾脏和肌肉组织样本,其中一部分于4%多聚甲醛中固定,用于组织病理学、免疫组织化学和原位杂交研究；另一部分则速冻于液氮,-80℃保存,用于ELISA和荧光定量RT-PCR研究。血清酶含量的检测结果发现,热应激后受试鸡血清CK和GPT均显著升高,并且呈现随着热应激时间的增加而逐渐增加的趋势,提示热应激处理后肉鸡心脏和肝脏出现随应激时间延长而加重的应激性损伤。病理组织学检查结果也同时显示,受试鸡心脏、肝脏、肾脏的实质细胞在热应激过程中表现出以颗粒变性、水泡变性、脂肪变性甚至坏死为特征的急性病理损伤,且实质细胞的这种应激性损伤表现出随着热应激时间的持续而逐渐加重的趋势。提示热应激导致肉鸡组织细胞的病理损伤与所检测的血清酶活性密切相关,所检血清酶活性可以间接反映肉鸡组织细胞的应激性病理损伤及其程度。免疫组织化学检测结果显示,热应激组受试鸡和对照组受试鸡的组织细胞中均有Hsps的表达和分布,但不同的Hsps在细胞中的定位不同。Hsp60主要定位于心脏、肝脏和肾脏实质细胞的胞浆内,而且Hsp60阳性信号在结构比较完整的胞浆区域内有更强的表达,在热应激受试鸡呈现出明显颗粒变性和水泡变性或发生断裂的实质细胞内Hsp60阳性信号表达则明显偏弱,提示组织细胞的应激性损伤与Hsp60的表达存在一定的关联；Hsp70和Hsp90于心脏、肝脏和肾脏实质细胞的胞浆和胞核内均有表达,且核内信号较强,说明Hsp70和Hsp90具有与胞核保护、信息调控的功能。免疫组化结果还显示,Hsp70和Hsp90在心脏、肝脏和肾脏的血管系统中有较强的表达和分布,说明Hsp70和Hsp90与血管的舒缩功能间存在密切的联系,可能与热应激过程中对心血管疾病(如缺血再灌注损伤等)的发生发展有关联。hsp70 mRNA的定位采用原位杂交的方法,结果显示hsp70 mRNA主要定位于心肌细胞、肝细胞和肾小管上皮细胞的胞浆内,而在胞核内却无hsp70 mRNA信号,暗示了Hsp70从胞浆到胞核的穿梭机制。热应激受试鸡组织细胞中Hsps及其相应mRNA的ELISA和荧光定量RT-PCR检测结果显示,肉鸡心脏中Hsp70、Hsp90及其相应的hsp70 mRNA和hsp90 mRNA在热应激2 h后均显著升高,但随着热应激时间的延长又呈现出迅速下降。与此不同的是,hsp60 mRNA在2 h热应激肉鸡心脏中的含量急剧上升(P<0.01),之后便恢复正常水平,而其相应的Hsp60蛋白水平则在热应激持续的整个阶段一直维持着较高的水平(P<0.01)。说明Hsp70、Hsp90和Hsp60在热应激肉鸡组织细胞中呈现不同特征和程度的波动,尤其是Hsp60显示出其在热应激初期作为心肌细胞反映应激损伤的重要分子标志特征,也可能是肉鸡抵抗外界不利环境因素的一组重要保护性蛋白。热应激2 h后,肝脏中Hsp90表现出显著的上升(P<0.01),说明短时间热应激(2 h-3 h)时Hsp90显示出活跃的肝脏保护作用；而在5 h(P<0.01)和10 h(P<0.01)热应激后,肉鸡肝脏中Hsp70的表达量出现了极显著的上升,说明较长时间热应激(5 h-10 h)Hsp70表现出较明显的肝脏保护作用。而整个热应激过程中,Hsp60蛋白在受试鸡肝脏中的含量是逐渐递减的。热应激后肾脏中Hsp60的表达量尽管呈现较大起伏的波动性变化,但与对照组相比没有显著的统计学差异。热应激3 h后,肾脏组织中Hsp70开始显著上升；热应激处理2 h(P<0.01)和10 h(P<0.05)后,肉鸡肾脏组织中Hsp90的含量与对照组相比有显著上升。然而Hsp60在热应激肉鸡肾脏组织中的变化不显著,说明在肾脏组织中Hsp70和Hsp90发挥了重要的保护作用。荧光定量RT-PCR结果显示,hsps mRNA的转录与Hsps蛋白的表达之间无明显规律,说明Hsps从转录到翻译之间存在十分复杂的调控机制,且不同蛋白转录与翻译作用机制是不同的。肉质测定结果表明,与对照组相比,不同时间热应激处理后的肉鸡胸肌pH30min和pH24h均显著下降(P<0.01),且热应激处理5 h后,受试鸡胸肌pH30min和pH24h的下降达到最低值。持续性热应激还能显著提高肉鸡胸肌的汁液损失、压榨损失和煮制损失,尤其在热应激5 h后的损失最为严重。热应激可导致肉鸡胸肌剪切力值增大,使肉鸡胸肌L*值升高,并显著降低肉鸡胸肌a*值(P<0.01)。相关性分析结果表明,热应激导致的肉鸡胸肌pH值下降对持水性、剪切力以及胸肌肉色有较大影响,长时间热应激会导致肉鸡胸肌产生PSE肉。更多还原

【Abstract】 China is one of powerful poultry raising countries. The poultry industry often suffer great economic losses because of durative high temperature last 2-3 months during summer time in the most parts of China. Heat stress response is a systematical response, which has an important effect on nerve and endocrine system, digestion and absorption, blood circulation, urinary and reproductive system, immune system and substance metabolism. When living organisms are exposed to thermal and nonthermal stressors, a group of highly conserved proteins known as heat shock proteins (Hsps) are rapidly synthesized, which play important roles in the protection of cells and stabilization of internal environment. Therefore, it is very important to study the characteristics of the heat stress-induced injury and take reasonable measures to reduce injury in poultry industry. The objective of this study was to investigate the expressions and localizations of Hsps and their corresponding mRNA in the tissues of heat-stressed broilers, and to correlate these Hsps levels with tissue damages at various stressing times.One-day-old male AA broiler chicks (n=100) were housed in large coops (20 birds per coop) and the coops were placed in a controlled-climate chamber. The birds were given a 30-day period to acclimate to their new surroundings and to reduce the influence of environmental stressor. During this period, the broilers were raising under standard conditions by specified person to avoid environmental stress. At 30 days of age, the room temperature was suddenly increased from 22±1℃to 37±1℃within 30 min and the broilers were heat stressed for 0 (control),2,3,5, and 10 h, respectively. On terminating the heat treatment, the plasma samples were obtained from jugular vein for subsequent CK and GPT determination. Following exsanguinations, the birds were manually eviscerated, and the heart, liver, and kidneys were quickly dissected and fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline, for histological analysis, immunohistochemistric analysis and in situ hybridization analysis. Further tissues samples were frozen in liquid nitrogen, and then stored at-80℃for ELISA detection and real time RT-PCR analysis. Enzyme results indicated that the concentrations of plasma CK and GPT increased obviously with heat stressing, implying damages to myocardial cells and hepatic cells were more serious as the time of heat stress prolonged. Histopathological results indicated that parenchymal cells of broiler heart, liver and kidney showed acute granular degeneration, fatty degeneration and even necrosis. The changes of parenchymal cells became more severe as the time of heat exposure prolonged. Enzyme results and histopathological results implied that broiler tissue damages were closely related to the detected plasma enzyme activities. The concentration of plasma enzyme can indirectly reflect stress-induced tissue injury and the degree of injury.Immunohistochemistry (IHC) results indicated that Hsps positive signals were located both in the heat stressed groups and the control group, however, the localization of different Hsps was varied in the three detected tissues. Hsp60 positive signals were observed in the cytoplasm of myocardial cells, hepatic cells and the renal tubular epithelial cells. Hsp60 expression was more prominent in the cytoplasm of intact tissue cells than in that of granular degenerated cells, vacuolar degenerated cells and collapsed cells. This maybe implied an association between tissue damage and the expression of Hsp60 in response to heat stress. Positive Hsp70 and Hsp90 protein signals were detected in the nucleus and cytoplasm of parenchymal cells in broiler heart, liver and kidney, however, the positive signals were stronger in the nucleus than the cytoplasm. This implied that Hsp70 and Hsp90 may effect as the important proteins for nucleus protection and message transmission. Hsp70 and Hsp90 positive signals were also detected in the blood vessel walls of heart, liver and kidney, which implied an close association between Hsp70 and Hsp90 expression and the function of vasoconstriction and vasodilatation. The expression of Hsps in vascular system may play an important role in the development of cardiovascular diseases, such as ischemia-perfusion injury during heat stress process. The localization of hsp70 mRNA in broiler tissues was studied by in situ hybridization method. Hybridization signals for hsp 70 mRNA were detected in the cytoplasm of myocardial cells, hepatic cells and the renal tubular epithelial cells, however, the nucleus of the three examined tissues in broilers exhibited no hsp70 mRNA positive signals. It gave us an implication that Hsp70 can shuttle from cytoplasm to nucleus after heat shock.The levels of Hsps and their mRNA were determined by the methods of ELISA and real time RT-PCR, respectively. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P＜0.01) at 2 h of heat stress but then decreased to normal level, while the level of Hsp60 protein in the heart increased (P＜0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicated that the expression of Hsp70、Hsp90 and Hsp60 showed different characteristics and fluctuations in the three tissues of heat stressed broilers. Especially, Hsp60 in heart, may be important markers reflecting tissue injury at the beginning of heat stress and act as protective proteins in adverse environments. The level of Hsp90 in liver of broilers increased significantly after 2 h heat exposure (P＜0.01), whereas the level of Hsp70 increased significantly after 5 h (P＜0.01) and 10 h (P＜0.05) heat exposure, which implied that Hsp90 acted an important role after short-term heat stress (2 h-3 h), whereas Hsp70 contributed more effect after long-term heat stress (5 h-10 h). However, the level of Hsp60 in the liver tissues decreased significantly during heat exposure. The levels of Hsp60 in the kidney fluctuated after 10 h of heat stressing, however, there was no significant difference between heat stressed groups and the control. After 3 h of heat stress, the levels of Hsp70 in kidney of broilers increased significantly (P＜0.01) compared with the control. Hsp90 levels increased significantly after 2 h (P＜0.01) and 10 h (P＜0.05) of heat stressing. It implied that Hsp70 and Hsp90 played important roles on kidney protection during 10 h of heat exposure.Real time RT-PCR results demonstrated that the transcription of hsps mRNA wasn’t coincide with the translation of Hsps. It implied that the mechanism of hsps mRNA transcription and Hsps translation was so complicated. Different Hsps may have different mechanisms.The results indicated that pH30min and pH24h of pectoralis muscles significantly decreased (P<0.01) after heat stress for varying amounts of time compared with control. The values of pH3omin and pH24h reached their lowest point after 5 h of heat exposure. Broilers exposed to high temperature had a higher drip loss, expressible moisture and cooking loss, especially after 5 h of heat exposure. After heat stress, broilers had a higher shear value, L* value and lower a* value (P＜0.01) compared to control. Correlation analysis indicated that the decrease of pH value may have an important impact on water holding capability, tenderness and meat color of pectoralis muscles after heat exposure. High temperature would lead to PSE meat of broilers pectoralis muscles.更多还原