禽病毒性腫瘤發(fā)生過程中血漿內(nèi)Hsp90α蛋白的檢測
發(fā)布時間：2018-09-19 19:51
【摘要】：病毒性腫瘤在雞群中較為常見,導(dǎo)致雞的生產(chǎn)性能下降及死亡率升高,給家禽業(yè)帶來巨大的經(jīng)濟(jì)損失。腫瘤的發(fā)生和發(fā)展的分子機理、抗腫瘤疫苗的發(fā)現(xiàn)與研發(fā)、雞場病毒的凈化是目前亟待解決的科學(xué)問題,隨著對腫瘤發(fā)生機理認(rèn)識的深入,熱休克蛋白在腫瘤發(fā)生中的重要作用被逐步揭示。熱休克蛋白90α(Hsp90α)是伴侶蛋白,可以輔助蛋白折疊和維持細(xì)胞內(nèi)多種信號傳導(dǎo),從而促進(jìn)細(xì)胞存活和生長。2009年,清華大學(xué)羅永章團(tuán)隊在世界上首次宣布了人體腫瘤細(xì)胞特異分泌Hsp90α的調(diào)控機理,同時首次揭示了細(xì)胞外Hsp90α與細(xì)胞內(nèi)Hsp90α的分子差異。該團(tuán)隊進(jìn)一步證明了分泌型Hsp90α能促進(jìn)腫瘤侵襲及轉(zhuǎn)移,且其在血液中的含量與腫瘤惡性程度正相關(guān)。經(jīng)過大量的臨床試驗,成功證明了Hsp90α是人肺癌相關(guān)腫瘤標(biāo)志物,可用于患者病情監(jiān)測和療效評價。但是,Hsp90α與家禽腫瘤病的關(guān)系尚無明確的定論。本論文的工作主要研究家禽發(fā)生病毒性腫瘤病(禽白血病和馬立克氏病)時,血液中Hsp90α的含量及其變化;以及急性轉(zhuǎn)化型ALV對鵪鶉的致病性。本試驗將雞急性纖維肉瘤研磨液、輔助病毒(外源性非急性轉(zhuǎn)化型禽白血病病毒)、PBS分別于腹腔和頸部皮下接種的兩種方式對1日齡海蘭褐雞進(jìn)行攻毒,連續(xù)三周采取抗凝血,2 000 r/min 4℃離心2min,取上層血漿,取其中100μL(剩余血漿-80℃保存)接種到已經(jīng)貼壁的CEF 96孔細(xì)胞培養(yǎng)板,2h后換細(xì)胞培養(yǎng)維持液培養(yǎng)7天,取上清經(jīng)ALV-p27抗原檢測試劑盒檢測。剩余血漿用于檢測在病毒血癥發(fā)展過程中血漿中Hsp90α的含量,通過對病理組織切片HE染色的方法對腫瘤組織進(jìn)行病理組織學(xué)的觀察與研究;同時本試驗還將急性纖維肉瘤的研磨液,分別在鵪鶉的幼雛期和成年期,采取頸部皮下和腹腔接種的方式攻毒,以研究鵪鶉對急性纖維肉瘤的病毒易感性。GX0101株MDV是國內(nèi)外從雞體內(nèi)分離到的整合有網(wǎng)狀內(nèi)皮組織增生癥病毒長末端重復(fù)序列的第一株重組野毒株。本試驗分別對7日齡SPF雞人工腹腔接種MDV GX0101毒株、1日齡免疫7日齡腹腔接種MDV GX0101毒株、7日齡腹腔接種PBS,三種方式構(gòu)建馬立克病腫瘤模型,連續(xù)三周采取無菌抗凝血,2 000 r/min 4℃離心2min,取上層血漿接種CEF細(xì)胞,7天后檢測細(xì)胞上清病毒感染情況,剩余的血漿用于檢測病毒血癥發(fā)展過程中Hsp90α的含量,并通過對病理組織切片HE染色的方法對腫瘤組織進(jìn)行病理組織學(xué)的觀察與研究。結(jié)果顯示,禽白血病急性腫瘤組的Hsp90α平均含量高于輔助病毒組及空白對照組;禽馬立克氏病攻毒組的Hsp90α平均含量高于疫苗組和空白對照組。說明受腫瘤病毒感染后的雞血液中Hsp90α含量有一定程度的升高,病毒引起的應(yīng)激促使Hsp90α表達(dá)量增加。該結(jié)論為后期建立家禽腫瘤病與Hsp90α含量之間的相關(guān)關(guān)系做鋪墊,有助于研究在家禽上應(yīng)用以Hsp90α為靶向確定家禽腫瘤病的血液學(xué)研究。而在急性纖維肉瘤對鵪鶉的易感性方面的研究中,無論是1日齡幼雛或是60日齡成年的鵪鶉進(jìn)行腹腔或頸部皮下接毒,p27檢測、斑點雜交試驗,均為陰性,未檢測出病毒的存在,剖檢查看,無眼觀可見的腫瘤結(jié)節(jié)等特征性的腫瘤病變。表明鵪鶉對致急性纖維肉瘤病毒不易感,體內(nèi)不會有急性纖維肉瘤產(chǎn)生。。急性缺陷型病毒在鵪鶉體內(nèi)的作用機制如何,需有關(guān)實驗進(jìn)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S857.4
本文編號：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S857.4
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