【摘要】：應(yīng)激是水生動物細菌病暴發(fā)的重要誘發(fā)因素。應(yīng)激狀態(tài)下機體的激素水平、代謝和免疫等方面發(fā)生顯著改變。鐵是魚類和微生物中非常重要的微量元素,參與氧氣運輸、細胞增殖、電子傳遞等眾多生命代謝過程;同時也是病原菌感染宿主后爭奪的重要營養(yǎng)因子。目前關(guān)于應(yīng)激抑制魚類免疫機能的研究已有報道,但是關(guān)于應(yīng)激時病原體侵襲宿主的具體機制仍不可知。本文從應(yīng)激激素入手,研究了急性應(yīng)激后魚體內(nèi)鐵的變化規(guī)律和相關(guān)調(diào)控基因(鐵調(diào)素和轉(zhuǎn)鐵蛋白)的表達變化及宿主應(yīng)激激素對病原菌致病力的影響,以期為魚類應(yīng)激的致病機理和病理學(xué)研究提供基礎(chǔ)資料。團頭魴是重要的大宗淡水魚養(yǎng)殖品種,養(yǎng)殖過程中應(yīng)激和嗜水氣單胞菌感染導(dǎo)致的細菌性出血病非常嚴(yán)重。本研究通過注射皮質(zhì)醇模擬急性應(yīng)激,檢測了團頭魴應(yīng)激后不同時間點血漿和肝臟鐵含量,并測定了鐵調(diào)素和轉(zhuǎn)鐵蛋白基因在不同時間點的表達量,同時利用鐵缺乏serum-SAPI培養(yǎng)基模擬生物體內(nèi)環(huán)境,分析應(yīng)激激素對病原菌的生長、毒力相關(guān)基因的表達、表型及毒力的變化。結(jié)果如下:1.定量注射皮質(zhì)醇(10 mg/kg)后,團頭魴短時間內(nèi)吸收了腹腔中的皮質(zhì)醇,實驗組0 h時血漿皮質(zhì)醇濃度最高,此后皮質(zhì)醇逐漸消退,但在2、4、8、10 h均維持著較高的皮質(zhì)醇濃度(P0.05),12 h時與對照組皮質(zhì)醇濃度基本持平,表明定量注射皮質(zhì)醇適宜于建立團頭魴急性應(yīng)激模型。團頭魴皮質(zhì)醇脅迫后,實驗組血漿鐵含量在0-2 h無顯著變化,4 h后血漿鐵持續(xù)蓄積,8 h時血漿蓄積的鐵有最大值,此后24 h時鐵含量回落到對照組水平;實驗組肝臟鐵含量整體呈現(xiàn)“V”字形變化趨勢,0-2 h肝臟鐵含量保持穩(wěn)定,4 h時肝臟鐵急劇減少,此后肝臟鐵不斷流失,10 h時肝臟鐵含量出現(xiàn)低谷,隨后肝臟鐵含量有所反彈,24 h后鐵含量趨于穩(wěn)定。皮質(zhì)醇脅迫后,肝臟鐵調(diào)素(LEAP1,LEAP2)和轉(zhuǎn)鐵蛋白(Tf)表達均呈單峰型變化趨勢,分別在8,10,10 h時有最高的表達水平,在24,48,48h時恢復(fù)正常表達量;腸道中Tf表達量在0 h有略微的上升,8 h時Tf開始大量轉(zhuǎn)錄,并持續(xù)至24 h,最高表達量出現(xiàn)在10 h,48 h時Tf表達水平降為正常水平。由此說明,皮質(zhì)醇脅迫造成團頭魴鐵穩(wěn)態(tài)失衡,誘導(dǎo)了鐵穩(wěn)態(tài)相關(guān)基因的表達,激活了機體的鐵調(diào)控系統(tǒng)。2.嗜水氣單胞菌AH196在serum-SAPI培養(yǎng)基中的生長曲線呈“S”形,較高的去甲腎上腺素(norepinephrine,NE)濃度(100μM和200μM)能夠在對數(shù)期極大地提高嗜水氣單胞菌AH196的增殖速度,而低濃度的NE(12.5,25和50μM)并不表現(xiàn)出此效應(yīng),同時嗜水氣單胞菌NJ-35,AH33,AH189和AH301的生長亦可被100μM NE所誘導(dǎo)。熒光定量PCR結(jié)果顯示,NE顯著上調(diào)了嗜水氣單胞菌熱穩(wěn)定腸毒素(ast),溶血素(hly),鐵超氧化物歧化酶(sodB)和鞭毛蛋白B(flaB)基因mRNA表達水平(P0.05)。NE極顯著增加了外膜蛋白W(omp W),外膜蛋白A(ompA),絲氨酸蛋白酶(ahp),彈性蛋白酶(ela),黏附素(aha),鐵攝取調(diào)節(jié)子(fur)和ahyR的基因表達量。NE不能誘導(dǎo)氣溶素(aerA),熱不穩(wěn)定腸毒素(alt)和脂肪酶(lip)基因mRNA表達水平的改變,卻顯著抑制了細胞毒性腸毒素(act)和鞭毛蛋白A(flaA)mRNA表達量。NE僅能增強嗜水氣單胞菌AH196蛋白酶活性,對溶血性,運動性和脂肪酶活性無顯著影響�；铙w攻毒試驗顯示,感染嗜水氣單胞菌后,NE極大地提高了鯽的死亡率,從而說明NE增強了嗜水氣單胞菌的毒性。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S943

【參考文獻】

相關(guān)期刊論文 前10條

1 宋凱;駱源;張春曉;王玲;游文煌;陳曉卉;陽達;;皮質(zhì)醇對斜帶石斑魚原代培養(yǎng)肝細胞糖代謝的影響[J];動物營養(yǎng)學(xué)報;2016年11期

2 陳凱;習(xí)丙文;滕濤;王亞冰;高金偉;梁利國;謝駿;;團頭魴促腎上腺皮質(zhì)激素釋放激素受體基因序列特征及表達分析[J];中國水產(chǎn)科學(xué);2016年04期

3 陳潔;趙明峰;曹小立;孟娟霞;邢藝;賀小圓;金鑫;徐萍;江嫣雨;;鐵過載對小鼠脾臟CD8~+ T淋巴細胞凋亡及分泌功能的影響[J];中國實驗血液學(xué)雜志;2016年03期

4 胡靜;葉樂;吳開暢;王雨;;急性鹽度脅迫對克氏雙鋸魚幼魚血清皮質(zhì)醇濃度和Na~+-K~+-ATP酶活性的影響[J];南方水產(chǎn)科學(xué);2016年02期

5 董純;潘磊;喬曄;;魚用麻醉劑概述[J];水產(chǎn)科技情報;2016年01期

6 張亞晨;溫海深;李蘭敏;馮啟超;;急性溫度脅迫對妊娠期許氏平湁血清皮質(zhì)醇和血液生理指標(biāo)的影響[J];水產(chǎn)學(xué)報;2015年12期

7 楊永波;扶曉琴;陳柏湘;王衛(wèi)民;劉紅;;嗜水氣單胞菌感染對團頭魴肝臟鐵代謝的影響[J];華中農(nóng)業(yè)大學(xué)學(xué)報;2015年04期

8 董晶晶;薛春雨;習(xí)丙文;梁利國;劉波;任鳴春;謝駿;;團頭魴黑素皮質(zhì)素受體2基因克隆、組織分布和應(yīng)激后的表達分析[J];江蘇農(nóng)業(yè)科學(xué);2014年06期

9 王宇;盧丹琪;李偉萍;徐跑;顧若波;姚謐;梁瑤思;張勇;林浩然;;急性操作脅迫對刀鱭應(yīng)激反應(yīng)相關(guān)神經(jīng)內(nèi)分泌因子的影響[J];水產(chǎn)學(xué)報;2014年06期

10 孫鵬;尹飛;王建建;高權(quán)新;鐘幼平;;操作脅迫對云紋石斑魚肝臟抗氧化和鰓Na~+-K~+ ATPase活力的影響[J];海洋漁業(yè);2014年03期

相關(guān)會議論文 前1條

1 邵楓;林文娟;王瑋雯;WELTON CRAIG WASHINGTON;鄭麗;;情緒應(yīng)激對大鼠原發(fā)性體液免疫功能的影響[A];第七屆北京青年科技論文評選獲獎?wù)撐募痆C];2003年

相關(guān)博士學(xué)位論文 前5條

1 劉波;高溫應(yīng)激與大黃蒽醌提取物對團頭魴生理反應(yīng)及相關(guān)應(yīng)激蛋白表達的影響[D];南京農(nóng)業(yè)大學(xué);2012年

2 倫立民;文昌魚谷丙轉(zhuǎn)氨酶組織定位及在LPS和溶藻弧菌處理后急性炎癥相關(guān)因子的變化[D];中國海洋大學(xué);2008年

3 趙敏;心理應(yīng)激對肝鐵代謝的影響及機制研究[D];第二軍醫(yī)大學(xué);2008年

4 陳慕雁;櫛孔扇貝Chlamys farreri生態(tài)免疫機制的基礎(chǔ)研究[D];中國科學(xué)院研究生院（海洋研究所）;2007年

5 李大鵬;環(huán)境因子對史氏鱘生長的影響及其調(diào)控機制的研究[D];華中農(nóng)業(yè)大學(xué);2003年

相關(guān)碩士學(xué)位論文 前10條

1 韋海明;黃芪和維生素C對大黃魚抗應(yīng)激的影響[D];中國海洋大學(xué);2014年

2 吳村;去甲腎上腺素對空腸彎曲菌的促生長作用鑒定及表達譜分析[D];山東農(nóng)業(yè)大學(xué);2014年

3 朱辰嫻;鯉魚轉(zhuǎn)鐵蛋白基因克隆、多態(tài)性、組織表達及建鯉與荷包紅鯉之間表達差異[D];南京農(nóng)業(yè)大學(xué);2014年

4 崔素麗;高溫下大黃素對團頭魴生長、血液指標(biāo)及抗氨氮應(yīng)激的影響[D];南京農(nóng)業(yè)大學(xué);2012年

5 黃云;維生素C、E對青魚氨氮脅迫的營養(yǎng)調(diào)控研究[D];湖南農(nóng)業(yè)大學(xué);2012年

6 張志偉;鰱低氧應(yīng)激相關(guān)基因的克隆與表達分析[D];華中農(nóng)業(yè)大學(xué);2011年

7 王晶晶;吡啶甲酸鉻對熱應(yīng)激建鯉生產(chǎn)性能、營養(yǎng)物質(zhì)代謝及抗氧化能力的影響[D];南京農(nóng)業(yè)大學(xué);2011年

8 李琴;草魚（Ctenopharyngodon idella）膠原凝集素（collectin）和轉(zhuǎn)鐵蛋白（transferrin）功能的初步分析[D];南昌大學(xué);2010年

9 吳學(xué)敏;嗜水氣單胞菌主要粘附素基因序列及其重組基因表達產(chǎn)物功能分析[D];福建農(nóng)林大學(xué);2010年

10 向建國;虹鱒對水溫應(yīng)激反應(yīng)的初步研究[D];湖南農(nóng)業(yè)大學(xué);2008年

，

本文編號：2504147