【作者】 邢明青；

【导师】 孙修勤；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 目前大规模高密度养殖造成的水环境污染,致使水产病害日趋严重,特别是病毒性疾病造成的损失更加惊人。淋巴囊肿病毒(Lymphocystis disease virus,LCDV),属于虹彩病毒科(Iridoviridae),是我国海水养殖鱼中大规模暴发的首例病毒。已知至少有42科、125种以上的鱼可被淋巴囊肿病毒感染,其中海水鱼占30科。国外报道蝶鲆类发病较多,这些鱼通常为经济鱼种,淋巴囊肿病的流行给该产业带来了巨大的损失。目前,对病毒性感染疾病尚无特效药物,在人类中通常是利用疫苗进行预防接种或干扰病毒复制的药物及抗生素对症治疗,而干扰病毒复制的药物常常给宿主带来危害。水产养殖者大多使用抗生素类药,易造成抗生素药物的积累、破坏生态平衡、诱变抗药菌株、污染水体,还导致了我国水产品的出口受阻。据观察,淋巴囊肿病毒感染有自限性,即疾病存在自愈现象。牙鲆出现自愈的主要表现在体表无肿瘤,病毒检测为阴性。牙鲆自愈现象与温度有密切的关系,一般在14℃或低于14℃时,肿瘤增长较慢;随水温的升高,18℃左右,肿瘤生长较陕,发病情况严重;但水温达到24以上,牙鲆身上的肿瘤脱落,病鱼出现自愈。通常认为,在低温下,鱼类的先天免疫成份的活性较大;在高温下,鱼类的适应性免疫如淋巴细胞活性和抗体活性变高。鱼类显示出季节影响的对病原体的敏感性。这无论是对于病原体的流行,还是宿主的敏感性增加是非常重要的。关于淋巴囊肿病的自愈机理从未见研究报道,但如果搞清这一问题、可能会给该病的防治提供思路。细胞因子与病毒的清除有密切关系,尤其是免疫相关因子,它们的表达可干扰病毒在体内的复制、组装和分泌,因此本论文以淋巴囊肿病的自愈机理为内容,从机体抗病因子和功能基因为切入点,利用real-time PCR技术探讨其免疫防御机理;同时,利用双向电泳技术,对牙鲆在自愈过程中血清蛋白质出现的变化,寻找差异表达的蛋白,为进一步探索鱼类淋巴囊肿病自愈的原因寻找依据。以期找到治愈疾病的关键因素,为水产养殖中的疾病防治提供有价值的参考。本论文的主要成果如下:1.利用PCR技术,对自愈牙鲆的各组织进行LCDV-cn的检测,结果显示自愈后的牙鲆在脾、肾、鳃、心脏、肠、肌肉、血液、表皮、体表粘夜、卵巢、脑和肝脏中都不含有病毒。说明自愈后牙鲆体内没有病毒。2.组织病理切片显示肝脏PAS染色结果,自愈组比患病组着色深,说明患病牙鲆肝脏由于LCDV-cn的感染而受损,糖原合成功能降低。肿瘤的AB-PAS染色结果,可见肿瘤细胞外层呈现蓝色、为酸性物质。初步分化的幼稚肿瘤细胞中,核仁非常发达、在某些细胞核中还出现了2-3个核仁,在某些细胞中核变成了哑铃状;肿瘤的AB-PAS染色结果目前还未见报道。3.利用real-time PCR技术,试验温度分别设置为14℃,18℃,22℃和26℃,对正常对照组和患病牙鲆组肝、脾、头肾中IL-1,TNF,TNFR,IL-8,IL-8R,Mx的表达研究结果表明:在肝脏中,实验组IL-1的表达随温度的升高而增加,22℃达到峰值,是标准对照组的3.8倍,(P＜0.05);对照组在22℃表达上升,增高1.9倍;在脾、头肾中,IL-1的表达随温度的升高而升高,在26℃达到峰值,分别为10.8和13.9倍(P＜0.05)。肝、脾中的Mx的表达均随温度的升高明显增加,26℃时与标准对照相比,增加到31.1和26.5倍(P＜0.05);只有头肾中在22℃达就达到峰值,为42.1倍:在对照组,随温度升高,Mx的表达虽有增加,但最高是肾脏在26℃的表达,增高仅为2.1倍。实验组肝脏TNF在22℃时表达最高,是标准对照组的8.51倍(P＜0.05);脾和头肾中的表达,无论是对照组还是实验组,均随温度的升高表达增加。实验组的肝、脾和头肾中,在22℃时,TNFR的表达都达到最高,分别为标准对照组的22.1、4.87和21.55倍(P值均＜0.05)。试验组肝脏和肾脏中IL-8在22℃表达量最高,分别为标准对照组的25.7和1.67倍(P＜0.05),而脾脏中IL-8在18℃即达到峰值,为标准对照组的3.31倍(P＜0.05).对照组的脾、肾组与其他温度相比,在18℃也达到最大表达量(P＜0.05)。试验组脾和头肾中IL-8R的表达在26℃最高,为标准对照组的4.5和5.6倍,(P＜0.05),肝脏中的IL-8R的表达量在22℃时最大,为9.73倍(P＜0.05):对照组肝脾中的表达在22℃最高为2.5和4.1倍(P＜0.05),头肾中在26℃最高,为2.6倍(P＜0.05)。这些数据显示随着水温的升高,细胞因子在实验组的不同组织内表达明显升高,在对照组表达升高不是很明显。由于这些细胞因子涉及到牙鲆的非特异性免疫,它们表达增强,说明高温下牙鲆的非特异性免疫力增强,也说明,随着水温的升高,牙鲆清除病毒的能力增强,使其自愈成为可能。4.本实验应用real-time PCR技术检测了MHCⅠα、MHCⅡα和TCR在疫苗接种后不同时间以及不同温度下表达水平的变化。在肝脏中,MHCⅠα、MHCⅡα和TCR峰值分别在22℃,26℃,22℃,增高倍数分别为14.03,16.33,10.6倍;在脾脏中MHCⅠα、MHCⅡα和TCR峰值分别在18℃,26℃,26℃,增高倍数分别为2.11,16.87,2.89倍;在头肾中MHCⅠα、MHCⅡα和TCR峰值分别在14℃,26℃,18℃,增高倍数分别为5.23,9.96,10.9倍。疫苗注射组,肝、脾、肾中MHCⅠα的表达第2天高于第8天:MHCⅡα的表达则相反,第8天高于第2天;TCR的表达无显著性差异。从结果我们可以看出,MHCⅡα和TCR的表达是温度依赖性的,随着水温的升高,表达量升高。MHCⅠ的表达峰值的温度相对较低。总体上来说,LCDV感染组MHCⅠα,MHCⅡα,TCR在不同组织的表达量要高于健康组。说明被感染的牙鲆处在-种较强的免疫应答状态。对于MHCⅠα和MHCⅡα在疫苗免疫后出现的应答不同,应该是它们不同的激活途径导致。目前普遍认为MHCⅠ类分子主要传递内源性抗原,而MHCⅡ分子主要传递外源性抗原。但部分内源性抗原也可以由MHCⅡ类分子传递,部分外源性抗原也可以由MHCⅠ类分子传递。可见MHCⅠ、Ⅱ类基因在表达上互相补充共同调节机体的免疫活动。所以,高温使MHC和TCR的高表达,有利于促进机体对病毒感染细胞的识别,增强细胞免疫和体液免疫,从而清除病毒。5.本实验利用双向电泳技术,对自愈牙鲆和患病牙鲆血清中表达的差异蛋白进行了鉴定。通过分析型上样,结果显示自愈组的蛋白点为328±30个点,患病组为315±21个点。自愈组有8个蛋白点表达上调,新增3个蛋白点,一个蛋白点下调;患病组新增两个蛋白点。制备型凝胶自愈组和病鱼组的血清蛋白图谱中分别分离198±33和301±41个点。对其中10个差异表达的蛋白质点,进行质谱分析后得到牙鲆补体C3和转铁蛋白。由于这两种物质是牙鲆主要的非特异性免疫因子。所以我们认为,非特异性免疫因子的高表达,是牙鲆自愈的主要因素之一。更多还原

【Abstract】 The requirement for additional or altemate sources of fish and shellfish has prompted the rapid growth of the aquaculture industry worldwide,which has resulted in the outbreak of aquaculture animal diseases,especially the prevalence of viral infectious diseases. Lymphocystis disease(LCD) is an infectious disease of several seawater fish species.LCD and its causative agent,fish lymphocystis disease virus(LCDV),exist worldwide,and the rate of incidence appears to be increasing.LCDV belongs to the Iridoviridae group of viruses. LCD is the first large scale prevalent disease in seawater fish species.Today,at least there are 42 osections,more than 125 fishses can drive LCDV infect,among them,the sea water fish have 30 sections.The prevalence of LCDV rsults to large loss in seawater cultured industry. Infection disease to the virus appeal to have no special effect medicine still currently,usually is the medicine and antibiotics that makes use of vaccine to carry on the vaccination or the interference virus replication to cure to the disease in the mankind.But the medicament that disturb virus copy usually brings damage to host.The marine products farming mostly uses chemistry to synthesize or antibiotic medicine,result in easily the backlog,the ecological devastation balance of the antibiotic medicine,guide to change anti- medicine germ stub and pollute the water body,also cause our country the exit of the marine products article get difficulty.According to the observation,LCD infection have from the limit,then the disease exist the phenomenon of self-cure.The primary phenomenon of self-cure exist in that the skin have no tumour and the check of virus is negative.The self-cure of the Japanese flounder(paralichthys olivaceus) have close relation to temperature,general at 14 or low in 14℃,the tumor growth compares slowly;But with go up of water temperature,18℃and or so,the tumor growth compares quickly,being taken bad circumstance severity;But water temperature attain 24℃above,the tumor section of body shed off in Japanese flounder,and the disease fish appear from self-cure.Asusual,under the low temperature,the activity of the fish inborn immunity composition compares greatly,under the heat,the fish adaptability immunity,such as the lymphoid cell activity and the antibody activity become effect.Fish displays the season influence of to the sensitivity of the cause body.It is very imporment for the prevalence of pathogeny and the increase of sensitivity to host.As for the researh on mechanism of LCDV self-cure have never be found,but if make pure this problem and may provide way of thinking for the prevention and cure that should get sick.The cytokine,especially the immunity related cytokine,have a ability in the clearance of the virus,by interfering the replication,construction and the secrete of virus in host,So this research,we discuss the immunity defense mechanism of Japanese flounder about disease self-cure by real-time PCR technique and 2D electrophoresis technique,and look for the difference expression serum protein appears in the process of variety.Find out to cure the paroxysm key factor with the period,provide a worthy reference for the disease prevention and cure in the marine products farming.The main results of this thesis are as follows:1 The Japanese flounder different organization were checked by the PCR technique,The result showed that there have no virus in spleen,liver,kidney,gill,heart,bowel,muscle,blood, epidermis of Japanese flounder,So the result indicated that the self-cure Japanese flounder have no virus.2 Organize pathology slice is about liver by PAS dye.It is thus clear that color is deep in self-cure group,showing liver damaged because of the infection of the LCDV-cn and then the synthesize of hepatin reduce.The AB-PAS dyeing of the tumor clear sthat the tumor layer outside the cell present a blue and is an acidity material.The nucleolus in the immature tumor cell that the primary divide flourishing,still appeared nucleolus,the pit became a dumbbell form in some cells.3 The expression of IL-1,TNF,TNFR,IL-8,IL-8R and Mx in different tissue:liver, spleen and kidney,under different temperature:14℃,18℃,22℃and 26℃,using the technique of real-lime PCR.The research indicated the expression of go up IL-1 with the temperature increase,and the expression reached tiptop at 22℃in liver in the expriment and the control groups,but the tiptop is 26℃in spleen and kidney tissue.The expression of Mx indicated that go up with the temperature increase,and the expression reached tiptop at 26℃in liver and spleen tissue,but 22℃in kidney in the expriment groups,and the increase degree isn’t obvious in different tissue in control group.The expression of TNF indicated that the expression go up with the temperature increase,and the expression reached tiptop at 22℃in the the liver tissue,but the expression reduced at 26℃in liver tissue in the control groups.The expression of TNFR indicated that the expression go up with the temperature increase,the expression reached tiptop at is 22℃in the liver,spleen and kidney in the expriment groups.The expression of IL-8 indicated that the expression reached tiptop at 22℃in the liver and kidney tissueses,but the expression reached tiptop at 18℃in spleen in the expriment and control groupses.The expression of the IL-8 R indicated that the reached tiptop at 26℃in liver,spleen and kidney,but the expression reached the tiptop at 22 t℃in liver in the expriment groups,the tiptop is 22 t℃in liver and spleen tissue,26℃in kidney tissue respectively,in the control groups.These data indicated that the expression of cell factors increase obviously with the go up of water temperature in the different organization of experiment group,but the increase is not very obvious in the control group,cytokine involve to the non-special immunity of Japanese flounder.The strengthened expression of these cytokineexplains that non-special immunity of Japanese flounder increase in the heat water, also explaining that the ability of clearance virus will strengtheded along with the water temperature,and then accelerate the possibility of self-cure in Japanese flounder..4 The expression level of TCR,MHCⅠand MHCⅡheavy chain of mRNA in different tissues from a group of vaccinated Japanese flounders(Paralichthys olivaceus) and different temperature was investigated using a real-time polymerase chain reaction(real-time PCR) approach.In the liver,the expression tiptop of MHCⅠ,MHCⅡand TCR is at 22℃,26℃,22℃respectively,and the expression multiple is 14.03,16.33,10.6 respectively.In the spleen,the expression tiptop of MHCⅠ,MHCⅡand TCR is at 18℃,26℃,26℃respectively,and the expression multiple is 2.11,16.87,2.89 respectively;In the kidney,the expression tiptop of MHCⅠ,MHCⅡand TCR is at 14℃,26℃,18℃respectively,and the expression multiple is 5.23,9.96,10.9 respectively;The level expression of MHCI was significantly increased in liver,spleen and kidney of the vaccinated group after two days and eight days,and the level of after two days is higher to after eight days,but the expression level of MHCⅡis contrary to MHCⅠ.The level of expression of TCR have no difference in the vaccinated group aider two and eight days.The expression of MHCⅡand TCR depended on temperature and the expression level increase with the temperature。As a whole,the expression level of from tissues in LCDV group The level of expression of MHCⅠ,MHCⅡ,TCR in defferent tissues was different in the LCDV group and control group.The level of expression was significantly increased in LCDV group.These results indicated that the infected Japanese flounders is placed in a kind of stronger immunity response and the activated route of MHCⅠand MHCⅡis different.It is clear that the mutual expression of MHCⅠ,Ⅱgene regulate the immunity activity of the body together.So,the the high expression of the MHC and TCR in the high temperature was advantageous to promote the body to identify to the virus infection cell,strengthen the cell immunity and the body fluid immunity,and thus clearance virus.5.The different protein of blood serum in selt-cure and ailing Japanese flounders was investigated using two-dimensional gel electrophoresis(2-DE).After silver staine,the results indcated that the protein dot is 328±30 in self-cure groups and 315±21 in infected groups. There are 8 protein spots that their expression were increased in selt-cure groups, and added 3 protein spots.After G-250 stain,there are 10 protein spots to be analysed.The two increased spots are identified to C3 and transferrin(TRF) in self-cure groups.Bacause C3 and transferrin are importment nonspecific immune response factors,the high expression of the non- particularity immunity factor is primary factors for the self-cure of Japanese flounders.更多还原