干涉鸡热休克蛋白70基因表达诱导鸡胚成纤维细胞凋亡

畜牧兽医学报

干涉鸡热休克蛋白70基因表达诱导鸡胚成纤维细胞凋亡

杨帆^1,2，汤承¹ ，岳华^1*

（1.西南民族大学，成都 610041； 2. 成都康弘药业集团股份有限公司，成都 610036）

收稿日期:2012-09-20 出版日期:2013-03-23 发布日期:2013-03-23
通讯作者: 岳华，教授， E-mail：yhua900@163.com
作者简介:杨帆(1984-)，男，甘肃徽县人，硕士，主要从事病原分子生物学研究，E-mail：yfqh84@yahoo.com.cn
基金资助:
自然科学基金（31172307）

Interference of Chicken Heat Shock Protein 70 Gene Expression Induced ChickenEmbryo Fibroblast Apoptosis

YANG Fan^1,2, TANG Cheng¹, YUE Hua^1*

(1. Southwest University for Nationalities, Chengdu 610041, China; 2. Chengdu Kanghong Pharmaceutical Group Co., Ltd., Chengdu 610036, China)

Received:2012-09-20 Online:2013-03-23 Published:2013-03-23

摘要/Abstract

摘要：

为研究鸡热休克蛋白70（HSP70）与细胞凋亡的相关性及分子机制，通过构建表达靶向鸡hsp 70的短发夹样结构小干扰RNA (shRNA)质粒载体，转染鸡胚成纤维细胞（CEF），干涉后18、24、28 h用Annexin V-FITC方法检测干涉hsp70表达后细胞凋亡情况，同时用荧光定量RT-PCR测定凋亡相关基因Caspase3和Caspase8 的表达水平。结果显示，与未干涉组相比，干涉组hsp70被分别敲低了75%、83%和96%，凋亡细胞数随着干涉时间的延长而增加，Caspase3表达水平升高，其表达量分别上调至250%、140%和110%， Caspase-8表达水平下降，其表达水平分别下降至76%、80%和34%。由此可见，鸡的hsp 70可能主要通过线粒体途径保护CEF对抗凋亡。

Abstract:

The purpose of this paper is to study the relationship and molecular mechanisms between chicken heat shock protein 70 (HSP70) and apoptosis. Plasmid vectors expressing short hairpin-like structure small interfering RNA (shRNA) targeting chicken HSP70 were constructed and transfected into the chicken embryo fibroblast (CEF) in this study. At 18, 24, 28 h post interference, the CEF apoptosis was observed by Annexin V-FITC method following interference of hsp70 expression and caspase3 ＆ caspase8 were quantified by real-time RT-PCR. The results showed that, compared with the mock cell, 1) Apoptotic cells increased as the interference time extended. 2) The hsp70 in CEF cells decreased by 75%, 83% and 96% while caspase3 increased to 250%, 140% and 110%, and caspase8 decreased to 76%, 80% and 34% at 18, 24, 28 h post interference respectively. The results projected that chicken hsp70 protects the CEF cells against apoptosis mainly via the mitochondrial pathway.

中图分类号:

S852.23

杨帆，汤承，岳华. 干涉鸡热休克蛋白70基因表达诱导鸡胚成纤维细胞凋亡[J]. 畜牧兽医学报, doi: .

YANG Fan, TANG Cheng, YUE Hua. Interference of Chicken Heat Shock Protein 70 Gene Expression Induced ChickenEmbryo Fibroblast Apoptosis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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参考文献

［1］CASTELLI C, RIVOLTINI L, RINI F, et al. Heat shock proteins: biological functions and clinical application as personalized vaccines for human cancer［J］.Cancer Immunol Immunother, 2004, 53(3): 227-233.

［2］GUPTA R S, RAMACHANDRA N B, BOWES T, et al. Unusual cellular disposition of the mitochondrial molecular chaperones Hsp60, Hsp70 and Hsp10［J］. Novartis Found Symp, 2008, 291:59-68.

［3］ZHAO Y, WANG W, QIAN L. Hsp70 may protect cardiomyocytes from stress-induced injury by inhibiting Fas-mediated apoptosis［J］. Cell Stress Chaperones, 2007, 12(1): 83-95.

［4］YANG J, TOWER J. Expression of hsp22 and hsp70 transgenes is partially predictive of drosophila survival under normal and stress conditions ［J］. J Gerontol A Biol Sci Med Sci, 2009，64（8）: 828-838.

［5］朱青，张王刚，王立锋. Hsp701A的RNA干涉诱导K562细胞凋亡［J］. 细胞与分子免疫学杂志，2006，22(4):480-484.

［6］MOSSER D D, CARON A W, BOURGET L, et al. The chaperone function of hsp70 is required for protection against stress-induced apoptosis ［J］. Mol Cell Biol, 2000, 20(19): 7146-7159.

［7］PADMINI E, VIJAYA GEETHA B. Modulation of ASK1 expression during overexpression of Trx and HSP70 in stressed fish liver mitochondria［J］. Cell Stress Chaperones, 2009，14（5）:459-467.

［8］WILSON J A，JAYASENA S，KHVOROVA A，et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells［J］. Proe Natl Acad Sci USA, 2003, 100(5):2783-2788.

［9］BRENZ VERCA M S, WEBER P, MAYER C, et al. Development of a species-specific RNA polymerase I-based shRNA expression vector［J］. Nucleic Acids Res, 2007, 35(2): e10.

［10］MCINTYRE G J, FANNING G C. Design and cloning strategies for constructing shRNA expression vectors［J］. BMC Biotechnol, 2006，6:1 (doi:10.1186/1472-6750-6-1) .

［11］TAXMAN D J, LIVINGSTONE L R, ZHANG J, et al. Criteria for effective design, construction, and gene knockdown by shRNA vectors［J］. BMC Biotechnol, 2006, 6：7 (doi:10.1186/1472-6750-6-7) .

［12］张卓然. 培养细胞学与细胞培养技术［M］. 上海:上海科学技术出版社，2004: 654-673.

［13］GIULIETTI A，OVERBERGH L，VALCHX D，et a1．An overview of real-time quantitative PCR：applications to quantify cytokine gene expression［J］．Methods，2001，25(4)：386-401.

［14］马莉，谢秀兰，岳华．鸡β-actin基因实时荧光定量PCR方法的建立［J］．中国畜牧兽医，2007，34(2)：73-75.

［15］LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method ［J］．Methods, 2001, 25：402-408.

［16］REED J C, DOCTOR K, ROJAS A，et a1. Comparative analysis of apoptosis and inflammation genes of mice and humans ［J］. Genome Res, 2003, 13(6b): 1376-1388.

［17］HIRATA I, NAITO Y, HANDA O，et a1. Heat-shock protein 70-overexpressing gastric epithelial cells are resistant to indomethacin-induced apoptosis ［J］. Digestion, 2009，79(4):243-250.

［18］SREEDHAR A S, PARDHASARADHI B V, KHAR A, et al. A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma［J］. Free Radic Biol Med, 2002, 32(3): 221-227.

［19］XIANG T X, LI Y, JIANG Z, et al. RNA interference-mediated silencing of the Hsp70 gene inhibits human gastric cancer cell growth and induces apoptosis in vitro and in vivo［J］. Tumori, 2008，94(4):539-50.

［20］INOUE H, UYAMA T, SUZUKI T，et al. Phosphorylated hamartin-Hsp70 complex regulates apoptosis via mitochondrial localization ［J］. Biochem Biophys Res Commun, 2010, 391(1):1148-1153.

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