禽病毒性肿瘤发生过程中血浆内Hsp90α蛋白的检测
发布时间：2018-09-19 19:51
【摘要】：病毒性肿瘤在鸡群中较为常见,导致鸡的生产性能下降及死亡率升高,给家禽业带来巨大的经济损失。肿瘤的发生和发展的分子机理、抗肿瘤疫苗的发现与研发、鸡场病毒的净化是目前亟待解决的科学问题,随着对肿瘤发生机理认识的深入,热休克蛋白在肿瘤发生中的重要作用被逐步揭示。热休克蛋白90α(Hsp90α)是伴侣蛋白,可以辅助蛋白折叠和维持细胞内多种信号传导,从而促进细胞存活和生长。2009年,清华大学罗永章团队在世界上首次宣布了人体肿瘤细胞特异分泌Hsp90α的调控机理,同时首次揭示了细胞外Hsp90α与细胞内Hsp90α的分子差异。该团队进一步证明了分泌型Hsp90α能促进肿瘤侵袭及转移,且其在血液中的含量与肿瘤恶性程度正相关。经过大量的临床试验,成功证明了Hsp90α是人肺癌相关肿瘤标志物,可用于患者病情监测和疗效评价。但是,Hsp90α与家禽肿瘤病的关系尚无明确的定论。本论文的工作主要研究家禽发生病毒性肿瘤病(禽白血病和马立克氏病)时,血液中Hsp90α的含量及其变化;以及急性转化型ALV对鹌鹑的致病性。本试验将鸡急性纤维肉瘤研磨液、辅助病毒(外源性非急性转化型禽白血病病毒)、PBS分别于腹腔和颈部皮下接种的两种方式对1日龄海兰褐鸡进行攻毒,连续三周采取抗凝血,2 000 r/min 4℃离心2min,取上层血浆,取其中100μL(剩余血浆-80℃保存)接种到已经贴壁的CEF 96孔细胞培养板,2h后换细胞培养维持液培养7天,取上清经ALV-p27抗原检测试剂盒检测。剩余血浆用于检测在病毒血症发展过程中血浆中Hsp90α的含量,通过对病理组织切片HE染色的方法对肿瘤组织进行病理组织学的观察与研究;同时本试验还将急性纤维肉瘤的研磨液,分别在鹌鹑的幼雏期和成年期,采取颈部皮下和腹腔接种的方式攻毒,以研究鹌鹑对急性纤维肉瘤的病毒易感性。GX0101株MDV是国内外从鸡体内分离到的整合有网状内皮组织增生症病毒长末端重复序列的第一株重组野毒株。本试验分别对7日龄SPF鸡人工腹腔接种MDV GX0101毒株、1日龄免疫7日龄腹腔接种MDV GX0101毒株、7日龄腹腔接种PBS,三种方式构建马立克病肿瘤模型,连续三周采取无菌抗凝血,2 000 r/min 4℃离心2min,取上层血浆接种CEF细胞,7天后检测细胞上清病毒感染情况,剩余的血浆用于检测病毒血症发展过程中Hsp90α的含量,并通过对病理组织切片HE染色的方法对肿瘤组织进行病理组织学的观察与研究。结果显示,禽白血病急性肿瘤组的Hsp90α平均含量高于辅助病毒组及空白对照组;禽马立克氏病攻毒组的Hsp90α平均含量高于疫苗组和空白对照组。说明受肿瘤病毒感染后的鸡血液中Hsp90α含量有一定程度的升高,病毒引起的应激促使Hsp90α表达量增加。该结论为后期建立家禽肿瘤病与Hsp90α含量之间的相关关系做铺垫,有助于研究在家禽上应用以Hsp90α为靶向确定家禽肿瘤病的血液学研究。而在急性纤维肉瘤对鹌鹑的易感性方面的研究中,无论是1日龄幼雏或是60日龄成年的鹌鹑进行腹腔或颈部皮下接毒,p27检测、斑点杂交试验,均为阴性,未检测出病毒的存在,剖检查看,无眼观可见的肿瘤结节等特征性的肿瘤病变。表明鹌鹑对致急性纤维肉瘤病毒不易感,体内不会有急性纤维肉瘤产生。。急性缺陷型病毒在鹌鹑体内的作用机制如何,需有关实验进一步研究。
[Abstract]:Viral tumors are common in chicken flocks, which lead to the decline of chicken production performance and the increase of mortality rate, and bring huge economic losses to poultry industry. Heat shock protein 90 alpha (Hsp90alpha) is a chaperone protein that assists protein folding and maintains multiple signal transductions in cells, thereby promoting cell survival and growth. In 2009, Luo Yongzhang's team at Tsinghua University announced for the first time in the world that human tumor cells are specifically differentiated. The team further demonstrated that secretory Hsp90a promotes tumor invasion and metastasis, and that its content in the blood is positively correlated with the malignancy of the tumor. A large number of clinical trials have successfully demonstrated that Hsp90a is associated with human lung cancer. Tumor markers can be used for patient monitoring and therapeutic evaluation. However, the relationship between Hsp90a and poultry oncology is still unclear. In this experiment, chicken acute fibrosarcoma grinding fluid, accessory virus (exogenous non-acute transformed avian leukemia virus) and PBS were inoculated subcutaneously in abdominal cavity and neck respectively to attack 1-day-old Hailan brown chickens. Anticoagulant was taken for three weeks, centrifuged at 2 000 r/min 4 C for 2 minutes, and 100 mu L (residual plasma - 80) was obtained. The supernatant was detected by ALV-p27 antigen detection kit. The remaining plasma was used to detect the content of Hsp90a in the plasma during the development of viremia. The tumor tissues were pathologically examined by HE staining of pathological tissue sections. In order to study the susceptibility of quails to acute fibrosarcoma virus, the GX01MDV strain was isolated from chickens at home and abroad, and was inoculated subcutaneously and intraperitoneally in the young and adult stages of quails. MDV GX0101 strain was inoculated intraperitoneally in 7-day-old SPF chickens, MDV GX0101 strain was inoculated intraperitoneally in 1-day-old SPF chickens, and PBS was inoculated intraperitoneally in 7-day-old SPF chickens. The malignant tumor model of Marek's disease was established by three methods: aseptic anticoagulation for three weeks, 2 000 r/min 4 C. The supernatant of CEF cells was detected after 7 days. The remaining plasma was used to detect the content of Hsp90a during the development of viremia. The tumor tissues were observed and studied by HE staining on pathological sections. The average content of Hsp90alpha was higher than that of adjuvant virus group and blank control group, and the average content of Hsp90alpha in marek's disease attack group was higher than that of vaccine group and blank control group. The correlation between Hsp90alpha content and tumorigenesis in poultry is helpful for the study of Hematology targeting Hsp90alpha in poultry. In the study of susceptibility to acute fibrosarcoma in quails, abdominal or cervical subcutaneous grafting was performed in either one-day-old or adult quails. Toxicity, p27 detection, dot blot hybridization test, are negative, not detected the presence of the virus, anatomical examination, no visible tumor nodules and other characteristics of tumor lesions. Further experiments were carried out.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S857.4
本文编号：2251156
[Abstract]:Viral tumors are common in chicken flocks, which lead to the decline of chicken production performance and the increase of mortality rate, and bring huge economic losses to poultry industry. Heat shock protein 90 alpha (Hsp90alpha) is a chaperone protein that assists protein folding and maintains multiple signal transductions in cells, thereby promoting cell survival and growth. In 2009, Luo Yongzhang's team at Tsinghua University announced for the first time in the world that human tumor cells are specifically differentiated. The team further demonstrated that secretory Hsp90a promotes tumor invasion and metastasis, and that its content in the blood is positively correlated with the malignancy of the tumor. A large number of clinical trials have successfully demonstrated that Hsp90a is associated with human lung cancer. Tumor markers can be used for patient monitoring and therapeutic evaluation. However, the relationship between Hsp90a and poultry oncology is still unclear. In this experiment, chicken acute fibrosarcoma grinding fluid, accessory virus (exogenous non-acute transformed avian leukemia virus) and PBS were inoculated subcutaneously in abdominal cavity and neck respectively to attack 1-day-old Hailan brown chickens. Anticoagulant was taken for three weeks, centrifuged at 2 000 r/min 4 C for 2 minutes, and 100 mu L (residual plasma - 80) was obtained. The supernatant was detected by ALV-p27 antigen detection kit. The remaining plasma was used to detect the content of Hsp90a in the plasma during the development of viremia. The tumor tissues were pathologically examined by HE staining of pathological tissue sections. In order to study the susceptibility of quails to acute fibrosarcoma virus, the GX01MDV strain was isolated from chickens at home and abroad, and was inoculated subcutaneously and intraperitoneally in the young and adult stages of quails. MDV GX0101 strain was inoculated intraperitoneally in 7-day-old SPF chickens, MDV GX0101 strain was inoculated intraperitoneally in 1-day-old SPF chickens, and PBS was inoculated intraperitoneally in 7-day-old SPF chickens. The malignant tumor model of Marek's disease was established by three methods: aseptic anticoagulation for three weeks, 2 000 r/min 4 C. The supernatant of CEF cells was detected after 7 days. The remaining plasma was used to detect the content of Hsp90a during the development of viremia. The tumor tissues were observed and studied by HE staining on pathological sections. The average content of Hsp90alpha was higher than that of adjuvant virus group and blank control group, and the average content of Hsp90alpha in marek's disease attack group was higher than that of vaccine group and blank control group. The correlation between Hsp90alpha content and tumorigenesis in poultry is helpful for the study of Hematology targeting Hsp90alpha in poultry. In the study of susceptibility to acute fibrosarcoma in quails, abdominal or cervical subcutaneous grafting was performed in either one-day-old or adult quails. Toxicity, p27 detection, dot blot hybridization test, are negative, not detected the presence of the virus, anatomical examination, no visible tumor nodules and other characteristics of tumor lesions. Further experiments were carried out.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S857.4
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