马立克病肿瘤组织内HSP60表达及生物学作用初步研究

doi:10.11843/j.issn.0366-6964.2019.12.017

摘要/Abstract

摘要： 本试验旨在研究HSP60与马立克病肿瘤发生、发展之间的相关性。通过人工感染，建立鸡马立克病肿瘤模型，定期剖杀，利用病理组织学和免疫组织化学方法，检测HSP60与肿瘤细胞定位之间的相关性；设计HSP60 RNA干扰序列，构建重组慢病毒，转染MSB-1细胞，利用流式细胞技术，探索降低HSP60转录表达对MSB-1细胞凋亡水平的影响。结果显示：HSP60在肿瘤细胞的细胞质内强表达；成功构建了HSP60 RNA干扰慢病毒，且5147序列干扰效果最佳；5147序列慢病毒转染48 h时，与对照序列组和空白对照组相比，HSP60转录、表达水平极显著降低（P<0.01），MSB-1细胞凋亡水平极显著升高（P<0.01）。在马立克病肿瘤发生、发展过程中，HSP60组织细胞定位与肿瘤细胞具有明显的相关性，降低HSP60表达水平能够导致MSB-1细胞凋亡升高，说明HSP60对肿瘤细胞的存活具有重要的生物学作用。

关键词: RNA干扰, 免疫组织化学, 热休克蛋白60, MSB-1细胞, 细胞凋亡

Abstract: This paper will focus on the relationship between HSP60 expression and the tumor induced by the Marek's disease(MD) virus. The animal tumor model was established using MD virus and chickens were timely killed post artificial infection. The relationship between pathological lesion and distribution of HSP60 were studied using histopathology and immunohistochemistry methods. Three small interfering RNA sequences of HSP60 were designed and recombinant lentivirus were constructed. Flow cytometry were used to study the effect of down-regulation of HSP60 to the apoptosis ratio of MSB-1 cells. Results showed that HSP60 was mainly located in the cytoplasm of tumor cells. Recombinant RNA interference lentiviruses targeting HSP60 were successfully constructed and the interference effect of 5147 sequence lentivirus was the best. At 48 h post transfection, the transcription, expression of HSP60 were significantly reduced (P<0.01), and the apoptosis ratio of MSB-1 cells was significantly induced (P<0.01) in lentivirus transfected group than those in control cell group and control sequence lentivirus transfected group. HSP60 is mainly expressed in the cytoplasm of tumor cells induced by the MDV and down-regulation of HSP60 is able to induce the apoptosis ratio of MSB-1 cells.

Key words: RNA interference, immunohistochemistry, heat shock protein 60, MSB-1 cells, apoptosis

中图分类号:

S852.3

王俊丽, 李玉保, 段世豪, 路建彪, 丁飞, 司振书, 李旭勇, 贾克金, 张云飞. 马立克病肿瘤组织内HSP60表达及生物学作用初步研究[J]. 畜牧兽医学报, 2019, 50(12): 2529-2536.

WANG Junli, LI Yubao, DUAN Shihao, LU Jianbiao, DING Fei, SI Zhenshu, LI Xuyong, JIA Kejin, ZHANG Yunfei. Study on the Distribution of HSP60 in the MD Tumor Tissues and the Biological Function[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12): 2529-2536.

参考文献 27

[1]	BERTHAULT C, LARCHER T, HARTLE S, et al. Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia[J]. Vet Res, 2018, 49:31.
[2]	蔡秀磊, 李玉保,李娟,等. 马立克病毒感染对鸡体液免疫的影响[J]. 黑龙江畜牧兽医, 2014(8):143-144.CAI X L, LI Y B, LI J, et al. Effect of Marek's disease virus infection on chicken humoral immunity[J]. Heilongjiang Animal Science and Veterinary Medicine, 2014(8):143-144. (in Chinese)
[3]	WU J M, LIU T E, RIOS Z, et al. Heat shock proteins and cancer[J]. Trends Pharmacol Sci, 2017, 38(3):226-256.
[4]	李玉保, 王志亮,鲍恩东. 热应激对SH-SY5Y细胞中PrP及HSP90 mRNA转录水平的影响[J]. 扬州大学学报:农业与生命科学版, 2006, 27(1):14-18.LI Y B, WANG Z L, BAO E D. Influence of heat stress on transcription of PrP and HSP90 mRNA in SH-SY5Y cells[J]. Journal of Yangzhou University:Agricultural and Life Science Edition, 2006, 27(1):14-18. (in Chinese)
[5]	BATRA J, TRIPATHI S, KUMAR A, et al. Human heat shock protein 40(Hsp40/DnaJB1) promotes influenza a virus replication by assisting nuclear import of viral ribonucleoproteins[J]. Sci Rep, 2016, 6:19063.
[6]	MANZOOR R, KURODA K, YOSHIDA R, et al. Heat shock protein 70 modulates influenza A virus polymerase activity[J]. J Biol Chem, 2014, 289(11):7599-7614.
[7]	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):361-367.
[8]	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)
[9]	HARTL F U, HAYER-HARTL M. Molecular chaperones in the cytosol:from nascent chain to folded protein[J]. Science, 2002, 295(5561):1852-1858.
[10]	ZHOU C, SUN H W, ZHENG C, et al. Oncogenic HSP60 regulates mitochondrial oxidative phosphorylation to support Erk1/2 activation during pancreatic cancer cell growth[J]. Cell Death Dis, 2018, 9(2):161.
[11]	CAPPELLO F, DE MACARIO E C, RAPPA E, et al. Immunohistochemistry of human Hsp60 in health and disease:from autoimmunity to cancer[M]//CALDERWOOD S K, PRINCE T L. Chaperones:Methods and Protocols. New York:Humana Press, 2018:293-305.
[12]	TANG H P, TENG R F, ZHAO X Y, et al. Isotope tracing assisted metabolic profiling:Application to understanding HSP60 silencing mediated tumor progression[J]. Anal Chim Acta, 2019, 1047:93-103.
[13]	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 HSP60 and tumor lesion of Chickens kidney in progress of MD[J]. Acta Veterinaria et Zootechnica Sinica, 2013, 44(4):610-616. (in Chinese)
[14]	段世豪, 路建彪,李旭勇,等. 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[J]. 畜牧兽医学报, 2019, 50(2):415-421.DUAN S H, LU J B, LI X Y, et al. Relationship between liver tissues pathological lesion and expression level of HSPs in progress of marek's disease[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(2):415-421. (in Chinese)
[15]	CHUN J N, CHOI B, LEE K W, et al. Cytosolic Hsp60 is involved in the NF-κB-dependent survival of cancer cells via IKK regulation[J]. PLoS One, 2010, 5(3):e9422.
[16]	GHOSH J C, DOHI T, KANG B H, et al. Hsp60 regulation of tumor cell apoptosis[J]. J Biol Chem, 2008, 283(8):5188-5194.
[17]	YIN B, TANG S, SUN J R, et al. Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress[J]. Cell Stress Chaperones, 2018, 23(4):735-748.
[18]	WENG D S, CALDERWOOD S K, GONG J L. A novel heat shock protein 70-based vaccine prepared from DC-tumor fusion cells[M]//CALDERWOOD S K, PRINCE T L. Chaperones:Methods and Protocols. New York:Humana Press, 2018:359-369.
[19]	SNIGIREVA A V, VRUBLEVSKAYA V V, SKARGA Y Y, et al. Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells[J]. Cell Stress Chaperones, 2019, 24(2):309-322.
[20]	NAGARAJU G P, WU C, MERCHANT N, et al. Epigenetic effects of inhibition of heat shock protein 90(HSP90) in human pancreatic and colon cancer[J]. Cancer Lett, 2017, 402:110-116.
[21]	DJUZENOVA C S, BLASSL C, ROLOFF K, et al. Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia[J]. Cancer Biol Ther, 2012, 13(6):425-434.
[22]	RICCI C, CARROTTA R, RAPPA G C, et al. Investigation on different chemical stability of mitochondrial Hsp60 and its precursor[J]. Biophys Chem, 2017, 229:31-38.
[23]	ZHANG L X, PELECH S, UITTO V J. Bacterial GroEL-like heat shock protein 60 protects epithelial cells from stress-induced death through activation of ERK and inhibition of caspase 3[J]. Exp Cell Res, 2004, 292(1):231-240.
[24]	CHANDRA D, CHOY G, TANG D G. Cytosolic accumulation of HSP60 during apoptosis with or without apparent mitochondrial release:evidence that its pro-apoptotic or pro-survival functions involve differential interactions with caspase-3[J]. J Biol Chem, 2007, 282(43):31289-31301.
[25]	SAMALI A, CAI J Y, ZHIVOTOVSKY B, et al. Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells[J]. EMBO J, 1999, 18(8):2040-2048.
[26]	FARIED A, SOHDA M, NAKAJIMA M, et al. Expression of heat-shock protein Hsp60 correlated with the apoptotic index and patient prognosis in human oesophageal squamous cell carcinoma[J]. Eur J Cancer, 2004, 40(18):2804-2811.
[27]	SCHNEIDER J, JIMÉNEZ E, MARENBACH K, et al. Immunohistochemical detection of HSP60-expression in human ovarian cancer. Correlation with survival in a series of 247 patients[J]. Anticancer Res, 1999, 19(3A):2141-2146.