HSPA2在牦牛不同组织器官中的表达差异

doi:10.3864/j.issn.0578-1752.2014.17.015

摘要/Abstract

摘要： 【目的】探索热休克蛋白70-2(heat shock 70kD protein-2, HSPA2)在牦牛不同组织器官中的表达差异。【方法】选取 3 头 1 岁龄的健康青海高原雄性牦牛为研究对象，在正常生理条件下，于 2013 年 9 月中旬采集不同组织器官（心脏、肝脏、脾脏、肺脏、肾脏、脑和睾丸）样本。（1）从牦牛不同组织器官中提取 RNA，将 RNA 反转录成第一链 cDNA，参照牦牛 HSPA2 和β-actin 基因序列（登录号为 KC790105.1和 DQ838049.1）设计特异性引物，首先采用 RT-PCR 来验证实时荧光定量 PCR（RT-qPCR）是否能应用于牦牛不同组织器官中 HSPA2表达差异的测定；然后采用 RT-qPCR 测定牦牛不同组织器官中 HSPA2相对表达量的差异。（2）将牦牛不同组织器官样本 4％多聚甲醛固定，制成石蜡切片，免疫组织化学法测定 HSPA2 在不同组织器官中的分布。采用 Image-Pro Plus 6.0 软件进行免疫组化图像分析，测定 HSPA2 阳性反应物的积分光密度，进行吸光度分析;通过 SPSS 19.0 统计软件，用单因素方差分析进行差异显著性测定。【结果】(1) RT-PCR 结果表明 RT-qPCR 法可用于牦牛不同组织器官中 HSPA2表达差异的测定。实时荧光定量 PCR 结果表明，HSPA2在睾丸中的相对表达量，分别是在脑、肾脏、心脏、脾脏、肺脏和肝脏中的 83.33、97.09、111.11、133.33、222.22和 285.71倍。(2)免疫组织化学结果显示，牦牛睾丸、肾脏、脑、心脏、肺脏、肝脏和脾脏中均有 HSPA2 的阳性表达。其中，在牦牛肾脏皮质肾小管、髓质肾小管，大脑皮质海马CA1区、小脑皮质，心肌细胞，肺泡上皮细胞，肝细胞，脾脏边缘区和红髓中有 HSPA2 阳性反应，着色深浅不等，大部分阳性反应位于细胞质，细胞核阳性反应极少；而在睾丸曲精小管中生精细胞细胞质和细胞核中均有 HSPA2 阳性反应，着色深浅不等；阴性对照组未观察到阳性反应。根据积分光密度值比较得出，HSPA2 蛋白的阳性表达量在睾丸中最高，脑、肾脏、心脏、肺脏和肝脏次之，脾脏最少。【结论】通过基因水平和蛋白水平两个层面的研究，发现 HSPA2 在牦牛各组织器官中存在表达差异；睾丸中 HSPA2 基因和蛋白表达量均高于脑、肾脏、心脏、脾脏、肺脏和肝脏，提示 HSPA2 可能与睾丸的生殖功能密切相关。

关键词: HSPA2 , 牦牛 , 不同组织器官 , 实时荧光定量 PCR , 免疫组化

Abstract: 【Objective】 This experiment was conducted to study the differences in expression of heat shock protein 70-2 (HSPA2) in different tissues and organs of yak. 【Method】 Three 1-year-old Qinghai Plateau male yaks were used in the present study. Under normal physiological conditions, the tissues and organs were obtained from healthy yak in mid-september 2013 (heart, liver, spleen, lung, kidney, brain and testis). The RNA were extracted from different tissues and organs of yak, and the RNA were reverse-transcribed into first-strand cDNA. The primers were designed specifically according to the Bos grunniens HSPA2 and β-actin gene sequences (HSPA2:KC790105.1, β-actin: DQ838049.1). Firstly, RT-PCR were used to verify the real-time quantitative PCR (RT-qPCR) could be applied to the determination of the differences in expression of HSPA2 gene in different tissues and organs of yak. Then, real time quantitative PCR was applied to analyze the expression of HSPA2 gene. Paraformaldehyde was used to fix different tissues and organs of yak, and then made into paraffin sections. The protein localization of HSPA2 was measured by immunohistochemistry. The Image-Pro Plus 6.0 software was used to analyze immunohistochemistry, the integrated optical density of HSPA2 positive reaction was measured, and absorbance was analyzed. The SPSS19.0 software was used for significance of difference analysis. 【Result】 RT-PCR results showed that RT-qPCR can be applied to the determination of differences in expression of HSPA2 gene in different tissues and organs of yak. The results of RT-qPCR showed that comparative expression quantity of gene HSPA2 in testis was 83.33 fold than that in brain, 97.09 fold in kidney, 111.11 fold in heart,133.33 fold in spleen, 222.22 fold in lung and 285.71 fold in liver, respectively. Immunohistochemistry showed that HSPA2 expressed in the testis, kidney, brain, heart, lung, liver and spleen of yak. HSPA2 positive reaction was observed in yak renal cortical tubular, medullary tubules, cortical hippocampal CA1 region, cerebellar cortex, cardiac cells, alveolar epithelial cells, liver cells, spleen marginal zone and red pulp. The most positive reaction was found in the cytoplasm, and positive reaction was rarely found in the nucleus. In the testis seminiferous tubules, HSPA2 positive reaction was showed in both the cytoplasm and nucleus of spermatogenic cells. No positive reaction was observed in the negative control group. Analysis of integrated optical density values showed that the HSPA2 protein expression in testis was the strongest, and followed by the brain, kidney, heart, lung and liver, and its expression in spleen was the weakest. 【Conclusion】 By studying the gene and protein levels, the findings of the study showed the differences in expression of HSPA2 in different tissues and organs of yak. The expression of HSPA2 gene and protein in testis were higher than that in brain, kidney, heart, spleen, lung and liver, which suggests that HSPA2 may be associated with testicular reproductive function.

Key words: HSPA2 , yak , different tissues and organs , RT-qPCR , immunohistochemistry

温泽星，余四九，翟羽佳，杨琨,刘鹏刚，何俊峰，崔燕. HSPA2在牦牛不同组织器官中的表达差异[J]. 中国农业科学, 2014, 47(17): 3475-3482.

WEN Ze-xing, YU Si-jiu, ZHAI Yu-jia, YANG Kun, LIU Peng-gang, HE Jun-feng, CUI Yan. Differences in Expression of HSPA2 in Different Tissues and Organs of Yak[J]. Scientia Agricultura Sinica, 2014, 47(17): 3475-3482.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.17.015

https://www.chinaagrisci.com/CN/Y2014/V47/I17/3475

参考文献

[1]Lindquist S. The heat-shock response. Annual Review of Biochemistry, 1986, 55: 1151-1191.
[2]Su C Y, Chong K Y, Owen O E, Dillmann W H, Chang C, Lai C C. Constitutive and inducible hsp70s are involved in oxidative resistance evoked by heat shock or ethanol. Journal of Molecular and Cellular Cardiology, 1998, 30: 587-598.
[3]李士泽, 任宝波, 杨焕民, 杨玉英, 计红, 倪江. 不同强度冷应激对大鼠肌肉、脾脏和肝脏中HSP70表达的影响. 应用与环境生物学报,2006,12(2): 235-238.
Li S Z , Ren B B, Yang H M, Yang Y Y, Ji H, Ni J. Effect of cold stress with different intensities on HSP70 expression in wistar rat muscle, spleen and liver. Chinese Journal of Applied and Environmental Biology, 2006, 12(2):235-238. (in Chinese)
[4]Fei G, Guo C, Sun H S, Feng Z P. HSP70 reduces chronic hypoxia-induced neural suppression via regulating expression of syntaxin. Advances in Experimental Medicine and Biology, 2008, 605:35-40.
[5]Meimaridou E, Gooljar S B, Chapple J P. From hatching to dispatching: the multiple cellular roles of the Hsp70 molecular chaperone machinery. Journal of Molecular Endocrinology, 2009, 42: 1-9.
[6]Kampinga H H, Hageman J, Vos M J, Kubota H, Tanguay R M, Bruford E A, Cheetham M E, Chen B, Hightower L E. Guidelines for the nomenclature of the human heat shock proteins. Cell Stress and Chaperones, 2009, 14:105-111.
[7]Obrenovitch T P. Molecular physiology of preconditioning-induced brain tolerance to ischemia. Physiological Reviews, 2008, 88:211-247.
[8]Feng H L, Sandlow J I, Sparks AE T. Decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility. Fertility and Sterility, 2001, 76(6):1136-1139.
[9]Pirkkala L, Nykanen P, Sistonen L. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB Journal, 2001, 15:1118-1131.
[10]Eddy E M. Role of heat shock protein HSP70-2 in spermatogenesis. Reviews of Reproduction, 1999, 4(1):23-30.
[11]Fourie A M, Peterson P A, Yang Y. Characterization and regulation of the major histocompatibility complex-encoded proteins Hsp70-Hom and Hsp70-1/2. Cell Stress and Chaperones, 2001, 6(3):282-295.
[12]David J C, Tanguay R M, Grongnet J F. Perinatal expression of heat shock proteins HSC 70 and HSP 70 in neural and non neural tissues of the piglet. Developmental Brain Research, 2001, 128:91-99.
[13]Vydra N, Winiarski B, Rak-Raszewska A, Piglowski W, Mazurek A, Scieglinska D, Widlak W. The expression pattern of the 70-kDa heat shock protein Hspa2 in mouse tissues. Histochemistry and Cell Biology, 2009, 132:319-330.
[14]Pei Y, Wu Y, Qin Y. Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis. Cell Stress and Chaperones, 2012, 17:81-87.
[15]Grosz MND, Womack J E, Skow L C. Syntenic conservation of HSP70 genes in cattle and humans. Genomics, 1992, 14:863-868.
[16]Redgrove K A, Nixon B, Baker M A, Hetherington L, Baker G, Liu D , Aitken R J. The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition. Plos One, 2012,7(11):e50851.
[17]王晓临, 徐存栓, 王修杰, 王冬杰, 王青山, 张宝琛. 热休克反应与哺乳动物高海拔(缺氧)适应. 中国科学C辑：生命科学, 2007, 37(1):96-106.
Wang X L, Xu C S, Wang X J, Wang D J, Wang Q S, Zhang B C. Heat shock response and mammal adaptation to high elevation (Hypoxia). Science China: Life Sciences, 2007, 37(1):96-106. (in Chinese)
[18]Hartl F U, Hayer-Hart M. Molecular chaperones in the cytosol：from nascent chain to folded protein. Science, 2002,295:852-1858.
[19]Turner C P, Scott-Panter S, Sharp F R. Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70，HO-1/HSP32，HSP47) following subarachnoid injections of lysed blood. Molecular Brain Research, 1999, 65:87-102.
[20]Sun Y, Ouyang Y B, Xu L, Chow A M, Anderson R, Hecker J G, Giffard R G. The carboxyl-terminal domain of inducible Hsp70 protects from ischemic injury in vivo and in vitro. Journal of Cerebral Blood Flow and Metabolism, 2006, 26: 937-950.
[21]Unno K, Asakura H, Shibuya Y, Kaiho M, Okada S, Oku N. Increase in basal level of Hsp70, consisting chiefly of constitutively expressed Hsp70 (Hsc70) in aged rat brain. Journal of Gerontology: Biological Sciences, 2000, 55(7): B329-B335.
[22]Van-Dullemen L F, Bos E M, Schuurs T A, Kampinga H H, Ploeg R J, van Goor H, Leuvenink H G. Brain death induces renal expression of heme oxygenase-1 and heat shock protein 70. Journal of Translational Medicine, 2013,11:22.
[23]Zhao Y, Wang W Y, Qian L J. Hsp70 may protect cardiomyocytes from stress-induced injury by inhibiting Fas-mediated apoptosis. Cell Stress and Chaperones, 2007,12 (1):83-95.
[24]Radford N B, Fina M, Benjamin I J, Moreadith R W, Graves K H, Zhao P, Gavva S, Wiethoff A, Sherry A D, Malloy C R, Williams R S. Cardioprotective effects of 70-kDa heat shock protein in transgenic mice. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93:2339-2342.
[25]Wu R, Zhao Y, Plopper C G, Chang M , Chmiel K, Cross J J, Weir A, Last J A, Tarkington B. Differential expression of stress proteins in nonhuman primate lung and conducting airway after ozone exposure. American Journal of Physiology, 1999, 277:L511-L522.
[26]Goralczyk A D, Obed A, Beilage A G, Sattler B, Füzesi L, Lorf T. Tissue damage with different surgical techniques in a porcine model of liver resection：implications for living-donor liver transplantation. Journal of Hepato-Biliary-Pancreatic Sciences, 2011, 18:436-442.
[27]甘乾福, 林峥, 梁学武, 刘庆华, 鲍若红. 热应激大白鼠脾脏和肝脏组织HSP70和NOS的表达变化. 福建农林大学学报: 自然科学版, 2010, 39(3):274-278.
Gan Q F, Lin Z，Liang X W, Liu Q H, Bao R H. The expression of HSP70 and NOS in the rat spleen and liver by heat stress. Journal of Fujian Agriculture and Forestry University: Natural Science Edition, 2010, 39(3): 274-278. (in Chinese)