【作者】 曲悦；

【导师】 刘思当；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2012， 博士

【摘要】 ALV-J主要引起鸡的骨髓细胞瘤(Myelocytoma)或骨髓白血病(Myeloid Leukosis，ML)和多种细胞类型的恶性肿瘤。近年来我国蛋鸡群出现ALV‐J感染病例的报道不断增加，J亚群白血病已由最初肉用鸡感染传播向蛋鸡和中国的许多地方品种鸡发展，其宿主范围正在不断扩大。另外，ALV‐J的感染除了引起常见的髓样细胞瘤外，还出现血管瘤、成髓细胞性白血病、成红细胞性白血病、纤维肉瘤等多种组织的肿瘤，甚至同一鸡群或同一鸡体内出现多种肿瘤同时发生的现象。目前国内对J亚群白血病的研究，多偏向病原的分子病毒学和致病模型的病理形态学研究，未见从宿主靶细胞肿瘤相关基因的变化来探讨肿瘤的发病机制。我国当前流行的ALV-J的组织嗜性、发病过程及致瘤机制的系统病理学研究少见报道，这方面的研究探讨对揭示该病的病因学及发病学具有重要的理论意义。该研究旨在用从自然发病蛋鸡群分离鉴定的ALV‐J毒株人工接种5日龄SPF鸡胚，建立ALV‐J感染的动物病理模型，在不同时期剖杀试验鸡，通过定量检测实验鸡群的病毒血症、泄殖腔排毒及抗体阳性率，动态检测靶组织肿瘤相关基因的异常表达，观察靶组织的病理变化及肿瘤演变过程，研究ALV‐J的组织嗜性、致瘤机制及病理发生过程，丰富该病的病理学研究内容，进而为该病的有效防制提供理论依据。本研究选择山东、河南两个表现为血管瘤兼髓细胞瘤型的蛋鸡典型禽白血病病例，取病鸡的病料，接种DF-1细胞（C/E）系，采用ELISA抗原检测、PCR以及亚群特异性免疫荧光试验（IFA）等方法，分离鉴定了二株ALV-J病毒，分别命名为hn10py01和sd10xt03。根据GenBank中已发表的不同亚群序列设计合成3对连续、相互部分重叠的引物，完成了这两株病毒前病毒全基因组核苷酸序列测序，测序结果表明：hn10py01和sd10xt03全基因组长度分别为7652bp和7636bp，与已公开的ALV-J全基因组序列大小相比略有差异，但符合典型的复制完全型反转录病毒的基因组结构，基因序列中不含已知致癌基因。与国内外各参考毒株进行了同源性分析，两毒株具有如下序列特征：（1）5’LTR和5’UTR与参考序列相比变异较大，hn10py015’UTRleader区有一个19bp的插入，两株病毒在5’UTR区均有一个碱基的缺失；（2）3’UTR区rTM和DR区有一个连续的205bp的缺失，E元件相对保守，两毒株均有一个碱基的缺失，形成新基序元件；（3）gag和pol基因非常保守；env基因变异较大，呈现高度多样性，hn10py01有一个氨基酸缺失。为研究蛋鸡ALV-J分离株（hn10py01）的致病特性，接种5日龄SPF白莱航蛋鸡鸡胚，在感染后的第2、3、5、7、9、11、13、17、21、25、30、35周采集泄殖腔棉拭子、抗凝血、非抗凝血，进行泄殖腔排毒、病毒血症、ALV-J特异性抗体检测；每次采样时同时随机取部分鸡剖检，进行病理变化观察。结果表明：（1）感染组鸡只从第2周开始出现病毒血症，并随着日龄的增加，阳性率不断增加，11周后其阳性率达到100%，一直持续到实验结束；（2）感染组鸡只从第2周开始排毒率不断上升，在16周以后100%鸡只排毒；（3）感染组鸡只产生ALV-J抗体的阳性率很低，最高达50%，后期快速下降至0；（4）感染组鸡只产蛋后，蛋清内p27阳性率为100%，卵黄ALV-J抗体阳性率为0；（5）能诱导感染鸡发生骨髓瘤、血管瘤、骨髓瘤兼血管瘤和血管瘤兼纤维肉瘤多种类型肿瘤，肿瘤诱发率为25%。为研究癌基因、抑癌基因、凋亡基因及其它肿瘤相关基因在该病发病学中的作用，本研究成功建立了原癌基因（c-myc、c-myb）、抑癌基因（p53、p16、p27）、凋亡调节基因（Bcl-2）、血管内皮生长因子（VEGF）及其受体（VEGFR）基因荧光定量PCR检测方法，动态检测了抑癌基因、癌基因、凋亡基因和其它肿瘤相关基因在各脏器中的异常表达，对抑癌基因p53进行了突变检测，以探讨ALV-J人工感染SPF鸡的分子病理学发病机制。结果表明：感染组与对照组相比具有以下特征：（1）抑癌基因p53在各组织器官内（尤其是骨髓内）呈现高表达， p53的高表达与日龄关系不明显，各时间段之间没有明显差异。感染鸡高表达的p53基因主要为突变型，总突变率为60%以上，突变方式多种多样。7例标本同时存在两个以上位点的突变，DNA结合部位外显子的点突变率为51.6%，C-末端外显子的点突变率为17.3%；缺失突变为30.7%，N-末端外显子突变率为0.5%，p53基因突变和过量表达可能是使靶细胞癌变的重要原因；（2）感染组抑癌基因p16呈失表达，抑癌基因p27呈低表达；（3）凋亡基因bcl-2在攻毒早期（3月龄前），各组织呈现高表达，随着日龄的增加bcl-2的表达水平与对照组之间无明显差异；（4）原癌基因c-myc在骨髓、肝和脾内表达升高，但未检测到插入突变；（5）感染组鸡只组织与血液中的VEGF及其受体基因随着日龄的增加表达量也有一定程度的增加。根据九种肿瘤相关基因检测结果，J亚群禽白血病肿瘤的发生发展、转移、浸润，可能与抑癌基因p53变异及高表达、抑癌基因p16失活及p27的低表达、原癌基因c‐myc激活、凋亡基因Bcl‐2的过表达及VEGF基因和其受体基因（FLT1、Flk1/KDR）的过表达密切相关。本研究还据获得的突变型p53基因和野生型p53基因，构建了pEGFP-C1-p53和PCAGGS-p53真核表达载体，通过瞬时转染方法转染DF-1细胞，经间接免疫荧光、western blot、共聚焦激光扫描显微镜观察等检测方法，证实两种真核表达载体构建成功，为稳定转染细胞株的筛选和进一步研究p53基因在J亚群白血病发病过程中的作用奠定了基础。更多还原

【Abstract】 Avian leukosis virus subgroup J (ALV-J) can cause a variety of neoplasms, includingmainly myeloid leukosis (myelocytomatosis) and nephromas. In recent years reports of layerflocks infected by ALV-J continuously increased, host range of subgroup J avian leukosisvirus enlarge obviously, infecting from meat-type chickens at first to layer flocks and manylocal chickens in China. ALV-J can cause not only myelocytomatosis but also hemangioma,myeloblastosis, fibrosarcoma which could be found in one flock or a single.Nowadays, study of ALV-J mainly focus on epidemiological survey, virus isolation andidentification and virus molecular properties. However, there was no report on tissue tropism,oncogenic mechanism and the process of systematic pathology, which is of great theoreticalsignificance and practical application value in revealing etiology and pathogenesis of ALV-J.To establish animal pathological model infected by ALV-J，ALV-J was isolated fromclinical hemangioma and myelocytomatosis layer flock. Differentially expressed of tumor-related genes were tested and tumor progression and pathological change were observed intarget tissue as chicken killed at interval period.To study the tissue tropism, the oncogenicmechanism and the process of systematic pathology, expression of virus multiplication andtumor-related gene were quantified in vivo animal experiments. So it will enrich pathologystudy and support theoretical basis in preventing ALV-J.In this study, two ALV-J virus, named hn10py01and sd10xt03were isolated andidentificated by taking sick chicken disease material, inoculating with DF-1cells (C/E),detecting antigen by ELISA, using specific PCR and indirect immunofluorescence assay (IFA)method from chiken with typical cases of hemangioma and myeloid tumor type in Shandongand Henan. According to different subsets of sequences expressed in GenBank, three pairs ofconsecutive and mutually partially overlapping primers were designed and synthetized, andthen two virus proviral genome-wide nucleotide sequencing. Sequencing results showed that:the The complete genome nucleotide sequences of hn10py01and sd10xt03were7652bp and7636bp, respectively,slightly different with the published genome-wide sequence size, but fitwith the typical copy complete retroviral genome structure. Gene sequence does not containknown viral oncogenes. Compare with domestic and international reference strains, thehomology analysis of the two strains have the following sequence features： Comparing to the reference sequence,5’LTR and the5’UTR were large variational, hn10py015’UTR leaderregion with a19bp insertion and the5’UTR district with a base deletion in the two viruses； acontinuous205bp were absence in rTM and DR region of the3’UTR,but the E element isrelatively conservative, the two strains have a base deletion, which formated a new motifelement; gag and pol gene is very conservative; env gene was variation, showing a highdegree of diversity, an amino acid deletion in the hn10py01。To study the pathogenicity of isolated ALV-J popular in laying hens, a layers of ALV-J(hn10py01) inoculated5-day-old SPF white Leghorn laying hens chick embryo. Since2,3,5,7,9,11,13,17,21,25,30,35week after infection, samples of cloacal swabs, anticoagulantand non-anticoagulant blood were collected to study the virus-infected stutus such as virusshedding, viremia, and specific antibody to ALV-J and clinical symptoms and pathologicalchanges were also observed. The results show that：（1）Chickens of the infection beganviremia from the second week, and with the age increasing, the positive rate is increasing.thepositive rate reached100%after11weeks, continue to the end of the experiment；（2）Chicken of infection start the detoxificating from the second week,100%of chickensdetoxificating after16weeks；（3）ALV-J antibody-positive rate of infection chickens isvery low, up to50%, and rapid decline to0lately；（4）After chickens of infection egg, thep27-positive rate within egg white was100%, and ALV-J antibody positive rate of the yolkwas0%;（5）groups of chickens infected could induce myeloma tumor, hemangioma andfibrosarcoma with induction rate25%.Subgroup J avian leukosis is a neoplastic infectious diseases. Occurrence of the diseaserelate to oncogenes, tumor suppressor genes, apoptotic genes and other tumor-related genes.Therefore, this study has successfully established a proto-oncogenes (c-myc, c-myb), tumorsuppressor genes (p53, p16, p27), regulation of apoptosis gene (Bcl-2), vascular endothelialgrowth factor (VEGF) and its receptor (VEGFR) gene real-time RT-PCR detection method.After ALV-J artificially infected SPF chickens, tumor suppressor genes, abnormal expressionof oncogenes, apoptotic genes and other tumor-related genes in various organs were detecteddynamicly at interval times of experiment. Observed tumor evolution in target tissues anddetected tumor suppressor genes mutations p53. Wild-type p53and mutant p53expressionvector were built in order to explore the molecular pathology of pathogenesis and thepathogenesis of ALV-J artificially infected SPF chickens.Compared with the control group,infection group has the following characteristics (1)Tumor-suppressor gene p53in various organs (especially in bone marrow) shows high expression, no significant correlation was found between expression of p53and age. Ininfection of ALV-J group, p53gene mutation rate was beyond60%in total（19/30）,7casesspecimens in two above at the same time site mutations were found in this study. In p53genemutations, point mutation rate of specific DNA-binding domain is51.6%, point mutation rateof C-teminal domain is17.3%; deletion mutation rate is30.6%, point mutation rate of N-terminal domain is0.5%.(2) tumor-suppressor gene p16in affected group presents lostexpression, tumor-suppressor gene p27presents lower expression;(3) early (3months ago)afeter infection, apoptosis gene Bcl-2in the organization presents high expression, there is noobvious difference compare with control group in Bcl-2expression level with the increase ofday；(4) the oncogene c-myc expresses rise in bone marrow, liver and spleen, but notdetected insertional mutagenesis;(5)Expression of VEGF and its receptor gene in infectiontissue and blood is also a certain degree of increase with the increase of day age.According to test results of the nine tumor related gene, p53tumor-suppressor genevariations and p53high expression, p16tumor-suppressor gene inactivation and p27lowexpression, c-onc c-myc activation, apoptosis Bcl-2of gene high expression and VEGF geneand its receptor gene (FLT1, Flk1/KDR) high expression may closely related with tumorsdevelopment, metastasis, infiltration of ALV-J.Built a series of pEGFP-C1-of p53and PCAGGS-p53eukaryotic expression vector for themutant p53and wild-type p53gene, then transfected DF-1cells by transient transfectionmethod, indirect immunofluorescence, Western blot, a total offocusing the laser scanningmicroscope and other detection methods, and confirmed that two eukaryotic expression vectorwas successfully constructed.it laid the foundation stably for transfected cell line screeningand further research for the P53gene in leukemia pathogenesis of subgroup J.更多还原