鸡马立克病肝组织肿瘤病变与热休克蛋白转录、表达的相关性研究
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摘要
研究马立克病毒引发肿瘤的发生、发展过程中,热休克蛋白(heat shock proteins,HSPs)转录表达水平、组织细胞内定位与肝病理组织学损伤之间的相关性,为进一步阐述HSPs的生物学作用奠定基础。通过人工感染建立鸡MD肿瘤模型,定期剖杀试验鸡,采集肝组织,利用组织学、免疫组织化学、荧光定量RT-PCR及ELISA方法,检测肝病理组织学损伤、HSPs组织细胞内定位以及HSPs转录、表达水平的动态变化。结果:在MDV感染14d后,感染组鸡肝组织可见大小不等的淋巴样瘤细胞浸润和局灶性生长;HSP90、HSP60在淋巴瘤细胞的细胞质内强表达,而HSP27、HSP70主要分布于肿瘤细胞外的肝细胞的细胞质内;感染组HSP70和HSP90mRNA转录水平始终极显著高于空白对照组和疫苗免疫对照组;感染组HSP90的表达量始终极显著高于空白对照组,HSP70表达量在14和21日龄时,极显著高于空白对照组,在28和35日龄时显著高于空白对照组,42日龄后与空白对照组和疫苗免疫对照组相比差异不显著。在MDV引发鸡肿瘤发生、发展过程中,HSP60、HSP90与HSP70、HSP27之间在肝组织细胞内定位截然不同,且HSP70与HSP90在肝组织内表达量变化趋势差异明显,说明不同HSPs在MD肿瘤发生、发展过程中的生物学作用存在差异。
The experiment was conducted to discuss the biological function of HSPs during the progress of tumor induced by the Marek's disease,the relationship between pathological lesion of liver and distribution,expression level of HSPs were studied.The tumor animal model was established using MD virus.Chickens were timely killed post artificial infection and the livers were sampled.Pathological lesion of liver and distribution,transcription and expression level of HSPs were studied during the progress of tumor by the methods of histopathology,immunohistochemistry,FQ RT-PCR and ELISA.Results were as follows:At 14 days,obvious pathological damage appeared in the livers of chickens attracted by MDV,and unequal-sized lymphoid cells infiltration was found among liver cells.HSP60 and HSP90 were mainly located in the cytoplasm of tumor cell,while HSP27 and HSP70 were mainly located in the hepatocyte around the tumor tissues.Within the course of MD,the transcription level of HSP70 and HSP90 mRNA of challenge group were always significantly higher than those of blank control and vaccine control groups.The expression level of HSP90 of challenge group was always significantly higher than those of blank control group in the test process,while the expression level of HSP70 of challenge group was significantly higher than those of blank controrl group at 14 and 21 d,was significantly higher at 28 and 35 d,and no significant difference after 42 d.The distribution among HSP60,HSP90,HSP27,HSP70 and the trend of expression level between HSP90 and HSP70 are completely different during the progress of tumor induced by MDV,which indicates that different HSPs have different biological functions.
The experiment was conducted to discuss the biological function of HSPs during the progress of tumor induced by the Marek's disease,the relationship between pathological lesion of liver and distribution,expression level of HSPs were studied.The tumor animal model was established using MD virus.Chickens were timely killed post artificial infection and the livers were sampled.Pathological lesion of liver and distribution,transcription and expression level of HSPs were studied during the progress of tumor by the methods of histopathology,immunohistochemistry,FQ RT-PCR and ELISA.Results were as follows:At 14 days,obvious pathological damage appeared in the livers of chickens attracted by MDV,and unequal-sized lymphoid cells infiltration was found among liver cells.HSP60 and HSP90 were mainly located in the cytoplasm of tumor cell,while HSP27 and HSP70 were mainly located in the hepatocyte around the tumor tissues.Within the course of MD,the transcription level of HSP70 and HSP90 mRNA of challenge group were always significantly higher than those of blank control and vaccine control groups.The expression level of HSP90 of challenge group was always significantly higher than those of blank control group in the test process,while the expression level of HSP70 of challenge group was significantly higher than those of blank controrl group at 14 and 21 d,was significantly higher at 28 and 35 d,and no significant difference after 42 d.The distribution among HSP60,HSP90,HSP27,HSP70 and the trend of expression level between HSP90 and HSP70 are completely different during the progress of tumor induced by MDV,which indicates that different HSPs have different biological functions.
引文
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[16]ZHENG G P,ZHANG Z J,LIU H,et al.HSP27-mediated extracellular and intracellular signaling pathways synergistically confer chemoresistance in squamous cell carcinoma of tongue[J].Clin Cancer Res,2018,24(5):1163-1175.
[17]ZHAO Y G,KURIAN D,XU H T,et al.Interaction of Marek's disease virus oncoprotein Meq with heat-shock protein 70in lymphoid tumour cells[J].JGen Virol,2009,90:2201-2208.
[18]DIDELOT C,SCHMITT E,BRUNET M,et al.Heat shock proteins:endogenous modulators of apoptotic cell death[J].Handb Exp Pharmacol,2006(172):171-198.
[19]DJUZENOVA C S,BLASSL C,ROLOFF K,et al.Hsp90inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia[J].Cancer Biol Ther,2012,13(6):425-434.
[20]HASHIDA S,YAMAMOTO H,SHIEN K,et al.Hsp90inhibitor NVP-AUY922enhances the radiation sensitivity of lung cancer cell lines with acquired resistance to EGFR-tyrosine kinase inhibitors[J].Oncol Rep,2015,33(3):1499-1504.
[2]RATHORE A P S,HAYSTEAD T,DAS P K,et al.Chikungunya virus nsP3&nsP4interacts with HSP-90to promote virus replication:HSP-90inhibitors reduce CHIKV infection and inflammation in vivo[J].Antiviral Res,2014,103:7-16.
[3]LI Y B,BAO E D,WANG Z L,et al.Detection of HSP mRNA transcription in transport stressed pigs by fluorescence quantitative RT-PCR[J].Agric Sci China,2007,6(3):361-167.
[4]李玉保,裴兰英,鲍恩东,等.运输应激猪HSPs mRNA转录、分布及免疫器官病理性损伤相关性研究[J].中国农业科学,2008,41(6):1832-1837.LI Y B,PEI L Y,BAO E D,et al.Relationship between distribution,transcription level of HSPs mRNA and immunity tissue pathological lesion of transport stressed pigs[J].Scientia Agricultura Sinica,2008,41(6):1832-1837.(in Chinese)
[5]李玉保,鲍恩东,王志亮,等.荧光定量RT-PCR法检测运输应激猪热休克蛋白mRNA转录水平[J].中国农业科学,2006,39(1):187-192.LI Y B,BAO E D,WANG Z L,et al.Detection of HSP mRNA transcription in transport stressed pigs by fluorescence quantitative RT-PCR[J].Scientia Agricultura Sinica,2006,39(1):187-192.(in Chinese)
[6]SHEVTSOV M,MULTHOFF G.Heat shock protein-peptide and HSP-based immunotherapies for the treatment of cancer[J].Front Immunol,2016,7:171.
[7]LI Y B,LI J,WANG Z L,et al.Study on Hsp90expression in different tissues and its antibody in serum of chickens infected with Marek’s diseases[J].J Integr Agric,2014,13(6):1355-1362.
[8]李娟,李玉保,吴晓东,等.鸡MD肾组织肿瘤病变与HSP60表达的相关性研究[J].畜牧兽医学报,2013,44(4):610-616.LI J,LI Y B,WU X D,et al.Relationship between expression of HSP60and tumor lesion of Chickens kidney in progress of MD[J].Acta Veterinaria et Zootechnica Sinica,2013,44(4):610-616.(in Chinese)
[9]FELIP E,BARLESI F,BESSE B,et al.Phase 2study of the HSP-90inhibitor AUY922in previously treated and molecularly defined patients with advanced non-small cell lung cancer[J].J Thorac Oncol,2018,13(4):576-584.
[10]LORENZO G J,VINTRL X,DE MADRID M C P.HSP-90 expression as a predictor of response to radiotherapy in head and neck cancer patients[J].Acta Otorrinolaringol,2016,67(3):130-134.
[11]MEHTA P P,KUNG P P,YAMAZAKI S,et al.Retraction notice to“A novel class of specific Hsp90small molecule inhibitors demonstrate in vitro and in vivo anti-tumor activity in human melanoma cells”[J].Cancer Lett,2018,422:132.
[12]BIZARRO A,SOUSA D,LIMA R T,et al.Synthesis and evaluation of the tumor cell growth inhibitory potential of new putative HSP90inhibitors[J].Molecules,2018,23(2):407.
[13]裴兰英,冯秀娟,李娟,等.马立克病病毒感染鸡心组织肿瘤病变与热休克蛋白表达的相关性分析[J].畜牧兽医学报,2014,45(12):2013-2018.PEI L Y,FENG X J,LI J,et al.Relationship between expression level of HSPs and heart tissue pathological lesion of chickens in progress of Marek’s Disease[J].Acta Veterinaria et Zootechnica Sinica,2014,45(12):2013-2018.(in Chinese)
[14]雷蕾.肉鸡组织的热应激损伤与HSP90水平及其mRNA表达的变化[D].南京:南京农业大学,2008.LEI L.Tissue damages and expression of HSP90and its corresponding mRNA in the heat-stressed broilers[D].Nanjing:Nanjing Agricultural University,2008.(in Chinese)
[15]孙培明.肉鸡热应激损伤与热休克蛋白70表达的研究[D].南京:南京农业大学,2006.SUN P M.Study on heat stress damages and heat shock protein 70expression in broiler tissues[D].Nanjing:Nanjing Agricultural University,2006.(in Chinese)
[16]ZHENG G P,ZHANG Z J,LIU H,et al.HSP27-mediated extracellular and intracellular signaling pathways synergistically confer chemoresistance in squamous cell carcinoma of tongue[J].Clin Cancer Res,2018,24(5):1163-1175.
[17]ZHAO Y G,KURIAN D,XU H T,et al.Interaction of Marek's disease virus oncoprotein Meq with heat-shock protein 70in lymphoid tumour cells[J].JGen Virol,2009,90:2201-2208.
[18]DIDELOT C,SCHMITT E,BRUNET M,et al.Heat shock proteins:endogenous modulators of apoptotic cell death[J].Handb Exp Pharmacol,2006(172):171-198.
[19]DJUZENOVA C S,BLASSL C,ROLOFF K,et al.Hsp90inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia[J].Cancer Biol Ther,2012,13(6):425-434.
[20]HASHIDA S,YAMAMOTO H,SHIEN K,et al.Hsp90inhibitor NVP-AUY922enhances the radiation sensitivity of lung cancer cell lines with acquired resistance to EGFR-tyrosine kinase inhibitors[J].Oncol Rep,2015,33(3):1499-1504.