【作者】 张淼；

【导师】 鲍恩东；

【作者基本信息】 南京农业大学 ， 基础兽医， 2011， 博士

【摘要】 随着中国养猪业集约化程度的不断提高,规模化养猪场猪的转场等所面临的中、长途运输问题越来越普遍,造成的损失也越来越明显。了解和研究运输应激损伤特征和规律以及采取合理的措施减少运输应激造成的危害具有重要意义。热休克蛋白(Heat shock proteins, HSPs)是一组高度保守的蛋白,对维持细胞生存和内环境的稳定起着十分重要的作用。本文主要通过建立运输应激模型来动态观察运输应激过程中猪组织中的HSPs的转录和表达,研究HSPs的变化规律与猪应激性损伤之间的相关性；通过原代培养大鼠心肌细胞建立体外热应激模型,探讨HSPs的组织细胞保护机制。体内试验：选取皮特兰和二花脸杂交二代仔猪20头在相同的环境条件下饲养,待体重达到50±2kg时,随机分成4组,一组作为对照组饲养在正常饲养环境中,其余三组在常规路段分别进行1h、2h和4h的运输(车速为30-40千米/小时)。运输结束后,将所有运输组和对照组猪均先注射戊巴比妥钠(10 mg/kg)后再进行剖杀。分离血清,检测血清生化指标丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、肌酸激酶(CK)、乳酸脱氢酶(LDH)、肌酐(Cr)和尿素氮(BUN)。采取心脏、肝脏、胃和背最长肌,一部分于4%多聚甲醛溶液中固定,用于组织病理学检测,另一部分则速冻并于液氮中保存,用于ELISA和荧光定量RT-PCR研究。部分背最长肌用于肉质检测。体外试验：原代培养大鼠心肌细胞72h后,将细胞分成9组(对照组和热应激10min、20 min、40 min、60 min、120 min、240 min、360 min、480 min)。按照分组给予相应的热应激后,取各组培养液用于生化指标ALT、LDH、CK、CK的同工酶(CKMB)和α-羟基丁酸脱氢酶(Hydroxybutyrate dehydrogenase, HBDH)的检测；取各组心肌细胞分别进行免疫组织化学、Western blot、荧光定量RT-PCR、流式细胞术等检测。运输后血清生化指标ALT、AST、CK、LDH和Cr均极显著升高(P<0.01),肝脏和心脏实质细胞主要表现为以急性退行性变化为主要特征的急性损伤。Hsp27的表达水平在运输2h后的心脏内和运输4h的肝脏内显著升高(P<0.01),同时伴有hsp27mRNA在运输1h后的心脏和肝脏内的显著升高.01)。a B-crystallin在整个运输过程中的心脏和肝脏中的表达规律起伏不定(P>0.05),但是,αB-crystallin mRNA水平在运输1h后的心脏中显著升高,而在运输1h和2h的肝脏中却显著降低。结论：心脏和肝脏细胞的损伤在运输1h后最明显,Hsp27和αB-crystallin的表达在猪的运输过程中显示出了组织特异性。运输应激后猪胃脏黏膜皱褶上的主细胞有不同程度的脱落。表明经过4h的运输后,猪胃黏膜的完整性遭到破坏。运输2h后,胃组织中的Hsp90表达量显著下降(P<0.01),但经过1h、2h和4h运输后,各组猪胃脏中Hsp70水平均显著高于对照组(P<0.05)。Hsp27的表达水平在整个运输过程中相对稳定。胃中αB-crystallin的表达在运输4h后显著高于对照组(P<0.05)。HSPs的不同表达变化提示不同的Hsp在运输应激猪胃中发挥不同的功能。调节HSPs表达的HSF-1的水平在运输过程中基本保持稳定。提示在运输应激过程中,HSF-1不是调节HSPs表达的唯一因素。运输1h和2h后,猪背最长肌肉的初始和最终pH(pHi和pHu)均降低,汁液损失和L*值与对照组相比均升高,显示出PSE肉的特征。运输2h后的肉质低于运输1h和4h。运输应激后,四种被检热休克蛋白(αB-c、Hsp27、Hsp70和Hsp90)在猪背最长肌中的表达均呈降低趋势。导致运输应激后猪背最长肌肉品质下降的可能原因之一是HSPs的表达降低。体外实验结果显示：培养液中ALT、LDH、CK、CKMB在热应激后均有不同程度的升高。免疫组化结果显示,在热应激过程中,Hsp27在心肌细胞的细胞核和细胞浆中分布不同,HSF-1则相对稳定的分布于细胞核中。Wsstern Blot结果显示Hsp27的表达在热应激8h后显著升高,HSF-1的表达在热应激10 min、40 min、60 min和120 min时显著降低,而在240 min后极显著升高。荧光定量PCR检测结果显示hsp27mRNA和hsf-1 mRNA在热应激10 min后均显著或极显著升高。结果表明在持续热应激后,大鼠心肌细胞受到一定的损伤；Hsp27在热应激过程中发挥一定的作用；Hsp27和HSF-1的表达与其相应mRNA水平不同步,可能与其在热应激过程中被磷酸化有关。流式细胞仪检测结果表明,除热应激40 min和480 min时,细胞凋亡率分别极显著(P<0.01)和显著(P<0.05)高于相应的对照组外,热应激处理的其它各试验组离体培养的心肌细胞的凋亡率与相应对照组相比,差异均不显著。表明急性热应激可以诱导细胞启动某种自我保护机制以适应外界环境的改变。蛋白定量检测结果显示,在应激20 min后,Cleaved caspase-3极显著升高,并持续到热应激480 min后。Cyt c的水平与Cleaved caspase-3类似,提示两者变化在热应激过程中关系密切。更多还原

【Abstract】 The intensive management systems of the swine industry in China are resulting more and more long distance requirement of pig transportation, and increasing serious economic losses due to the transporting. Therefore, it is very important to study the characteristics of transport-induced injury for reduce the stress injury in pig industry. Heat shock proteins (HSPs) are a group of highly conserved proteins, which play important roles in the protection of cells and stabilization of internal envrionment. The main purpose of this study was to investigate the transcription and expression of HSPs and their corresponding mRNAs in the tissues of transport stressed pigs, and to correlate these HSPs levels with tissue damages at various stressing times, through the establishment of transport stress model in vivo; and to study on the mechanism of cytoprotective functions of HSPs, through establishment of heat stress model by primary culturing myocardial cells of rats in vitro.Twenty hybrid pigs from Erhualian and Pietrain strains were selected and raised in individual pens (2.5×3.0 m2). The pigs were randomly placed into 4 groups of 5 pigs each. The mean weight of the pigs was approximately 50±2 Kg (mean±SD). On the day of the transport trial, one group was maintained under normal housing conditions and served as control (not transported animals), while the other three groups were transported for 1,2 or 4 h, respectively. The route included an equivalent mix of local roads, including town traffic, state roads and highways. The average speed was 30 to 40 km per hour. Immediately after the end of the transport, all animals were euthanized by jugular injection with 10 mg/kg of 3% sodium pentobarbital while on the truck or in the animal house. Blood samples for serum enzyme activities were frozen at-20℃until further analysis. Tissue specimens were taken from heart, liver, stomach and M.longissimusdorsi (LD) and fixed in paraformaldehyde solution for histopathological examination. Additional samples were placed into 1.5 mL tubes and frozen in liquid nitrogen for later evaluation of HSPs expressions and their corresponding mRNA transcriptions. Primary cultured rat myocardial cells were divided into 9 groups (control and heat stress 10 min,20 min,40 min,60 min, 120 min,240 min,360 min and 480 min) after 72 h culture. After heat stress, the supernatants were obtained for enzyme detection. The myocardial cells were taken from each group for the experiments of immunohistochemistry, western blot, real-time quantitative PCR, and flow cytometry.A significant increase of ALT, AST, CK、LDH and Cr in the blood serum and acute parenchyma cell lesions characterized by acute degenerations in the heart and liver were observed. Hsp27 expression levels increased significantly in the heart after 2 h and in the liver after 4 h of transportation accompanying with the hsp27 mRNA increasing significantly in the heart and liver after 1 h of transportation. aB-crystallin expression levels were fluctuant (not significantly) in the heart and liver during transporting, however, aB-crystallin mRNA increase notably in the heart after 1 h and decrease significantly in the liver at 1 h and 2 h of transportation. In conclusion, the cellular damage to the heart and liver is highest after 1 h of transportation, Hsp27 and aB-crystallin play dissimilar roles and show tissue-specific response in different tissues during transportation.Pathological examination of all transported pigs showed that exfoliation of chief cells from the gastric surface occurred in pigs during transportation. These results imply that integrity of the gastric mucosa was compromised by damage occurring during the 4 h of transportation, despite the fact that gastric ulcers were not present. Levels of Hsp90 expression in stomach tissues were significantly decreased (P<0.01) after 2h of transportation, but Hsp70 levels increased significantly (P<0.05) after 1,2, and 4 h of transportation. Hsp27 levels remained relatively stable independent of the length of transport. Levels of aB-crystallin expression in the stomach were significantly increased (P <0.05) after 4 h of transportation. Variations in Hsp90, Hsp70, Hsp27, and aB-crystallin levels suggest that distinct protective functions are modulated by different HSPs in stomach tissues during transportation. Alterations in Hsp70 and aB-crystallin expression appear to be associated with protective functions, as no apparent gastric ulcers were present in pigs that underwent 4 h of transportation. Levels of HSF-1, which regulates the expression of HSPs, remained relatively stable independent of the transportation period. This observation indicates that the change of the HSF-1 expression levels is not the only factor which regluates the expressions of HSPs in pigs during transportation.The LD meat from 1 h and 2 h transported pigs had lower initial and ultimate pH values (pHi and pHu, respectively), higher drip loss and L* values compared to controls, indicating a higher likelihood of pale, soft and exudative (PSE) meat. Meat quality was lower after 2h compared to 1 h or 4 h of transport. All four HSPs tested (alpha-B-crystalline, Hsp27, Hsp70 and Hsp90) by ELISA in the LD tissue of pigs tended to decrease after transportation. One possible mechanism resulting in poor meat quality in the LD after transport seems to be a decline in Hsp expression.Levels of ALT, LDH, CK and CKMB in the medium were increased in different degrees. The results of immunofluorescence show that Hsp27 distributes in the cytoplasm and nucleus dynamically before and after heat stress. However, HSF-1 distributes in nucleus stably. Hsp27 protein level was increased after 8 h heat stress significantly, HSF-1 protein levels were decreased at 10 min,40 min,60 min, and 120 min after heat stress, and increased after 240 min stress significantly. The results of FQ-RT PCR show that hsp27 mRNA and hsf-1 mRNA levels were increased significantly after 10 min heat stress. The results indicating that the myocardial cells of rats were injuried during heat stressing, Hsp27 play a protective function during heat stressing. Hsp27 and HSF-1 protein levels were not coincides with their corresponding mRNA levels may be caused by phosphorylation.The result of flow cytometry shows that only at 40 min and 480 min are extremely significantly (p<0.01) and significantly (p<0.05) higher than that of the corresponding control groups. Suggesting that the myocardial cells start some kinds of self-protection mechanisms to adapt to the environment. Western blot results show that the levels of cleaved caspase-3 were increased significantly after 20 min, and continued to 480 min. The changes in levels of cytochrome C are similar to that of cleaved caspase-3, suggesting the two proteins are closely related during heat stress.更多还原