应激激素对团头鲂铁稳态和嗜水气单胞菌致病力的影响

发布时间：2019-06-21 14:31

【摘要】：应激是水生动物细菌病暴发的重要诱发因素。应激状态下机体的激素水平、代谢和免疫等方面发生显著改变。铁是鱼类和微生物中非常重要的微量元素,参与氧气运输、细胞增殖、电子传递等众多生命代谢过程;同时也是病原菌感染宿主后争夺的重要营养因子。目前关于应激抑制鱼类免疫机能的研究已有报道,但是关于应激时病原体侵袭宿主的具体机制仍不可知。本文从应激激素入手,研究了急性应激后鱼体内铁的变化规律和相关调控基因(铁调素和转铁蛋白)的表达变化及宿主应激激素对病原菌致病力的影响,以期为鱼类应激的致病机理和病理学研究提供基础资料。团头鲂是重要的大宗淡水鱼养殖品种,养殖过程中应激和嗜水气单胞菌感染导致的细菌性出血病非常严重。本研究通过注射皮质醇模拟急性应激,检测了团头鲂应激后不同时间点血浆和肝脏铁含量,并测定了铁调素和转铁蛋白基因在不同时间点的表达量,同时利用铁缺乏serum-SAPI培养基模拟生物体内环境,分析应激激素对病原菌的生长、毒力相关基因的表达、表型及毒力的变化。结果如下:1.定量注射皮质醇(10 mg/kg)后,团头鲂短时间内吸收了腹腔中的皮质醇,实验组0 h时血浆皮质醇浓度最高,此后皮质醇逐渐消退,但在2、4、8、10 h均维持着较高的皮质醇浓度(P0.05),12 h时与对照组皮质醇浓度基本持平,表明定量注射皮质醇适宜于建立团头鲂急性应激模型。团头鲂皮质醇胁迫后,实验组血浆铁含量在0-2 h无显著变化,4 h后血浆铁持续蓄积,8 h时血浆蓄积的铁有最大值,此后24 h时铁含量回落到对照组水平;实验组肝脏铁含量整体呈现“V”字形变化趋势,0-2 h肝脏铁含量保持稳定,4 h时肝脏铁急剧减少,此后肝脏铁不断流失,10 h时肝脏铁含量出现低谷,随后肝脏铁含量有所反弹,24 h后铁含量趋于稳定。皮质醇胁迫后,肝脏铁调素(LEAP1,LEAP2)和转铁蛋白(Tf)表达均呈单峰型变化趋势,分别在8,10,10 h时有最高的表达水平,在24,48,48h时恢复正常表达量;肠道中Tf表达量在0 h有略微的上升,8 h时Tf开始大量转录,并持续至24 h,最高表达量出现在10 h,48 h时Tf表达水平降为正常水平。由此说明,皮质醇胁迫造成团头鲂铁稳态失衡,诱导了铁稳态相关基因的表达,激活了机体的铁调控系统。2.嗜水气单胞菌AH196在serum-SAPI培养基中的生长曲线呈“S”形,较高的去甲肾上腺素(norepinephrine,NE)浓度(100μM和200μM)能够在对数期极大地提高嗜水气单胞菌AH196的增殖速度,而低浓度的NE(12.5,25和50μM)并不表现出此效应,同时嗜水气单胞菌NJ-35,AH33,AH189和AH301的生长亦可被100μM NE所诱导。荧光定量PCR结果显示,NE显著上调了嗜水气单胞菌热稳定肠毒素(ast),溶血素(hly),铁超氧化物歧化酶(sodB)和鞭毛蛋白B(flaB)基因mRNA表达水平(P0.05)。NE极显著增加了外膜蛋白W(omp W),外膜蛋白A(ompA),丝氨酸蛋白酶(ahp),弹性蛋白酶(ela),黏附素(aha),铁摄取调节子(fur)和ahyR的基因表达量。NE不能诱导气溶素(aerA),热不稳定肠毒素(alt)和脂肪酶(lip)基因mRNA表达水平的改变,却显著抑制了细胞毒性肠毒素(act)和鞭毛蛋白A(flaA)mRNA表达量。NE仅能增强嗜水气单胞菌AH196蛋白酶活性,对溶血性,运动性和脂肪酶活性无显著影响。活体攻毒试验显示,感染嗜水气单胞菌后,NE极大地提高了鲫的死亡率,从而说明NE增强了嗜水气单胞菌的毒性。
[Abstract]:Stress is an important cause of the outbreak of bacterial disease in aquatic animals. The hormone level, metabolism, and immunity of the body are changed significantly in the normal state. Iron is a very important trace element in fish and micro-organisms, and participates in many processes of life metabolism such as oxygen transportation, cell proliferation and electron transfer, and is also an important nutrient factor for pathogen infection and host. The current study on stress-inhibiting fish immune function has been reported, but the specific mechanism of pathogen invasion and host in stress is still unknown. The change of iron in fish after acute stress and the effect of host stress hormone on the pathogenic force of pathogenic bacteria were studied in this paper from the stress hormones. So as to provide basic data for the pathogenesis and the pathological study of fish stress. The cluster head is an important large freshwater fish culture variety, and the bacterial infection caused by stress and Aeromonas hydrophila infection in the culture process is very serious. In this study, the contents of plasma and liver iron at different time points were measured by injection of cortisol to simulate acute stress, and the expression of iron and transferrin genes at different time points was determined, and the internal environment was simulated by using iron deficiency serum-SAPI culture medium. The effects of stress hormones on the growth of pathogenic bacteria, expression, phenotype and virulence of virulence-related genes were analyzed. The results are as follows:1. After quantitative injection of cortisol (10 mg/ kg), the concentration of cortisol in the abdominal cavity was absorbed within a short period of time, and the concentration of the plasma cortisol was the highest at 0 h in the experimental group, and then the cortisol was gradually resolved. However, at 2,4,8 and 10 h, the concentration of cortisol (P0.05) was maintained, and the concentration of cortisol in the control group was substantially the same as that of the control group at 12 h, indicating that the quantitative injection of cortisol was suitable for the establishment of the acute stress model of the group head. In the experimental group, the iron content in the experimental group had no significant change at 0-2 h, and the iron content in the plasma after 4 h had a maximum value, and the iron content of the experimental group decreased to the control group at 24 h, and the iron content in the experimental group showed the change trend of the "V"-shape in the whole. The iron content of the liver was stable at 0-2 h, the iron content of the liver decreased sharply at 4 h, and the iron content in the liver was low at 10 h, and then the iron content of the liver rebounded, and the content of iron after 24 h was stable. After the stress of cortisol, the expression of liver iron (LEAP1, LEAP2) and transferrin (Tf) was unimodal. The highest expression level was found at 8,10 and 10 h. The expression of Tf in the intestinal tract was increased slightly at 24,48 and 48 h. The expression of Tf in the intestinal tract was slightly increased at 0 h, and Tf began to be transcribed in a large amount at 8 h. The expression level of Tf decreased to the normal level at the time of 10 h and 48 h for 24 h. As a result, the steady-state imbalance of the iron-iron homeostasis is induced by the stress of the cortisol, and the expression of the iron-state-related gene is induced, and the iron regulation system of the body is activated. The growth curve of Aeromonas hydrophila AH196 in the serum-SAPI medium was "S"-shaped, the higher noradrenaline (NE) concentration (100. mu.M and 200. mu.M) was able to greatly increase the proliferation rate of Aeromonas hydrophila AH196 in the logarithmic phase, The low concentrations of NE (12.5,25 and 50. mu.M) do not show this effect, while the growth of Aeromonas hydrophila NJ-35, AH33, AH189 and AH301 can also be induced by 100.mu. M NE. The results of quantitative PCR showed that NE significantly increased the expression level of heat-stable enterotoxin (ast), hemolysin (hly), iron-superoxide dismutase (sodB) and flagellin B (fla B) gene of Aeromonas hydrophila (P0.05). The NE of NE significantly increased the outer membrane protein W (omp W) and the outer membrane protein A (ompA). The amount of gene expression of the serine protease (ap), the elastase (ela), the adhesin (aa), the iron uptake regulator (fur) and the ayR. NE could not induce the changes of the level of expression of the expression of aerA, the heat labile enterotoxin (alt) and the lipase (lip), but significantly inhibited the expression of the cytotoxic and flagellin A (fla) mRNA. NE can only enhance the activity of Aeromonas hydrophila AH196, and has no significant effect on hemolytic, motility and lipase activity. The in-life challenge test shows that, after infection of Aeromonas hydrophila, the NE greatly increases the mortality of the crucian carp, thus indicating that the NE enhances the toxicity of Aeromonas hydrophila.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

【参考文献】