稳定转染HSP72奶牛乳腺上皮细胞系建立与表达鉴定
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摘要
为了构建HSP72慢病毒表达载体,建立稳定表达HSP72的牛乳腺上皮细胞系。以牛HSP72基因序列为模板,设计并合成引物,PCR扩增目的基因,连接到慢病毒表达质粒中,用包装获得的慢病毒感染牛乳腺上皮细胞,经一定浓度的嘌呤霉素筛选,Hochest33342法对细胞核定位,倒置荧光显微镜下观察转染效率,用免疫组化和Western Blot方法验证感染细胞是否稳定表达HSP72。结果显示,携带HSP72基因的慢病毒滴度约为1. 0×109TU/m L;确定嘌呤霉素的最佳筛选浓度为2μg/m L;牛HSP72慢病毒感染细胞表达HSP72,感染的奶牛乳腺上皮细胞在倒置荧光显微镜下可看到绿色荧光,转染效率可达90%以上;免疫组化结果表明,正常细胞和空载体感染细胞,HSP72呈现低表达,转染携带HSP72目的基因载体细胞,HSP72高表达,呈现颜色较深的棕色; Western Blot方法检测证实与正常转染细胞相比,慢病毒空载体转染的奶牛乳腺上皮细胞HSP72表达稍有降低,但无统计学差异,而携带HSP72基因的慢病毒载体转染的细胞HSP72呈现高表达,且差异极显著(P <0. 01),与慢病毒空载体转染的奶牛乳腺上皮细胞相比,携带HSP72基因的慢病毒载体转染的细胞HSP72呈现高表达,且差异极显著(P <0. 01)。成功建立稳定表达HSP72的牛乳腺上皮细胞系,可为研究奶牛乳腺炎分子调控机制以及抗性分子药物筛选提供新的理论依据和工作基础。
The objective of present study was to establish stably expressing HSP72 mammary epithelial cell( bMECs) line with expression vector of HSP72 slow virus constructed. The primers for PCR amplification purpose were designed and synthesized according to the template of bovine HSP72 gene sequence and was connected to plasmid virus. The bMECs were infected with the slow virus obtained after packaging. The cells with purpose gene or no-load virus were sorted out with puromycin and nuclear localization were determined by Hochest33342 method.The transfection efficiency was examined under an inverted fluorescence microscope. The positively expressed cell lines were verified by immunohistochemical technique and Western Blot method. The results showed that the titer of the lentivirus carrying HSP72 gene was about 1. 0 × 109 TU/m L,the optimum screening concentration of purinamycin was 2 μg/m L,the bovine HSP72 lentivirus infected cells expressed HSP72,and the infected cow mammary epithelial cells could see green fluorescence under the inverted fluorescence microscope,and the transfection efficiency could be over 90%. The results showed that normal cells and empty carriers infected cells,HSP72 showed low expression,transfected with HSP72 target gene carrier cells,HSP72 high expression and dark brown color,and Western Blot assay proved that the expression of HSP72 in milk gland epithelial cells transfected by lentivirus empty carrier was slightly lower than that of normal transfected cells. There was no statistical difference,but the expres-sion of HSP72 transfected by lentivirus carrier with HSP72 gene was highly expressed,and the difference was very significant( P < 0. 01). Compared with the mammary epithelial cells transfected with the lentivirus empty carrier,the HSP72 of the cells transfected with the lentivirus carrier with HSP72 gene showed high expression,and the difference was very significant( P < 0. 01). In conclusion the bovine mammary epithelial cell line,which stably expressing HSP72 protein,was successfully established. It laid solid foundation for further studying HSP72 function in inflammatory response of bovine mammary epithelial cells.
The objective of present study was to establish stably expressing HSP72 mammary epithelial cell( bMECs) line with expression vector of HSP72 slow virus constructed. The primers for PCR amplification purpose were designed and synthesized according to the template of bovine HSP72 gene sequence and was connected to plasmid virus. The bMECs were infected with the slow virus obtained after packaging. The cells with purpose gene or no-load virus were sorted out with puromycin and nuclear localization were determined by Hochest33342 method.The transfection efficiency was examined under an inverted fluorescence microscope. The positively expressed cell lines were verified by immunohistochemical technique and Western Blot method. The results showed that the titer of the lentivirus carrying HSP72 gene was about 1. 0 × 109 TU/m L,the optimum screening concentration of purinamycin was 2 μg/m L,the bovine HSP72 lentivirus infected cells expressed HSP72,and the infected cow mammary epithelial cells could see green fluorescence under the inverted fluorescence microscope,and the transfection efficiency could be over 90%. The results showed that normal cells and empty carriers infected cells,HSP72 showed low expression,transfected with HSP72 target gene carrier cells,HSP72 high expression and dark brown color,and Western Blot assay proved that the expression of HSP72 in milk gland epithelial cells transfected by lentivirus empty carrier was slightly lower than that of normal transfected cells. There was no statistical difference,but the expres-sion of HSP72 transfected by lentivirus carrier with HSP72 gene was highly expressed,and the difference was very significant( P < 0. 01). Compared with the mammary epithelial cells transfected with the lentivirus empty carrier,the HSP72 of the cells transfected with the lentivirus carrier with HSP72 gene showed high expression,and the difference was very significant( P < 0. 01). In conclusion the bovine mammary epithelial cell line,which stably expressing HSP72 protein,was successfully established. It laid solid foundation for further studying HSP72 function in inflammatory response of bovine mammary epithelial cells.
引文
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[13]李琪,李华涛,丛霞,等.稳定转染高表达HSP72的奶牛乳腺上皮细胞系的建立[J].中国兽医杂志,2016,52(7):3-5.
[14] Patel H,Shaw S G,Shi-Wen X,et al. Toll-like receptors in ischaemia and its potential role in the pathophysiology of muscle damage in critical limb ischaemia[J].Cardiol Res Pract,2012,2012:121237.
[15] Morteza A,Nakhjavani M,Larry M,et al. Heat shock protein 70 and albuminuria in patients with type diabetes a matched case control study[J]. Cell Stress&Chaperones,2013,18(6):815-819.
[16] Yang C X,Chen H Q,Chen C,et al. Microfilamentbinding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase[J].Cell Research,2006,16(4):367-376.
[17] Chen H,Wu Y,Zhang Y,et al. Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination[J].FEBS Letters,2006,580(13):3145-3152.
[18] Li H,Cuartas E,Cui W,et al. IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation[J]. The Journal of Experimental Medicine,2005,201(7):1169-1177.
[19] Chen H X,Wang S,Wang Z,et al. Overexpression of RUNX3 inhibits malignant behaviour of Ecal109 cells in vitro and vivo[J]. Asian Pacific Journal of Cancer Prevention,2014,15(4):1531-1537.
[20]赵轶君,董浩,潘红艳,等.绿色荧光蛋白原核表达载体的构建[J].华北农学报,2013,28(3):73-76.
[2] Yang Z T,Yin R L,Cong Y F,et al. Oxymatrine lightened the inflammatory response of LPS-Induced mastitis in mice through affecting NF-kappa B and MAPKs signaling pathways[J]. Inflammation,2014,37(6):2047-2055.
[3] Lindquist S,Craig E A. The heat-shock proteins[J].Annu Rev Genet,1988,22(1):631-677.
[4] Hsiang-Wen C,Hsu C,Tzong-Shi L,et al. Heat shock pretreatment prevents cardiac mitochondrial dysfunction during sepsis[J]. SHOCK,2003,20(3):274-279.
[5]郭辉,王少刚,余建华.慢性前列腺炎患者精浆中热休克蛋白的表达及临床意义[J].中华泌尿外科杂志,2004,25(6):411-413.
[6] Lagler H,Sharif O,Haslinger I,et al. TREM-1 activation alters the dynamics of pulmonary IRAK-M expression in vivo and improves host defense during pneumococcal pneumonia[J]. Journal of Immunology,2009,183(3):2027-2036.
[7] Guha M,Mackman N. LPS induction of gene expression in human monocytes[J]. Cellular Signalling,2001,13(2):85-94.
[8] Shi Y,Tu Z,Tang D,et al. The inhibition of LPS-induced production of inflammatory cytokines by HSP70 involves inactivation of the NF-kappaB pathway but not the MAPK pathways[J]. SHOCK,2006,26(3):277-284.
[9] Doeppner T R,Kaltwasser B,Fengyan J,et al. TATHsp70 induces neuroprotection against stroke via anti-inflammatory actions providing appropriate cellular microenvironment for transplantation of neural precursor cells[J]. Journal of Cerebral Blood Flow and Metabolism,2013,33(11):1778-1788.
[10] Collet G,Lamerant-Fayel N,Tertil M,et al. Hypoxiaregulated overexpression of soluble VEGFR2 controls angiogenesis and inhibits tumor growth[J]. Molecular Cancer Therapeutics,2014,13(1):165-178.
[11] Mohanan V,Grimes C L. The molecular chaperone HSP70 binds to and stabilizes NOD2,an important proteininvolved in crohn disease[J]. Journal of Biological Chemistry,2014,289(27):18987-18998.
[12]朱冬芳,晏春根,黄丹文.重组热休克蛋白72对体外培养大鼠肝细胞脂肪变性的影响[J].中华全科医学,2014,12(6):850-852.
[13]李琪,李华涛,丛霞,等.稳定转染高表达HSP72的奶牛乳腺上皮细胞系的建立[J].中国兽医杂志,2016,52(7):3-5.
[14] Patel H,Shaw S G,Shi-Wen X,et al. Toll-like receptors in ischaemia and its potential role in the pathophysiology of muscle damage in critical limb ischaemia[J].Cardiol Res Pract,2012,2012:121237.
[15] Morteza A,Nakhjavani M,Larry M,et al. Heat shock protein 70 and albuminuria in patients with type diabetes a matched case control study[J]. Cell Stress&Chaperones,2013,18(6):815-819.
[16] Yang C X,Chen H Q,Chen C,et al. Microfilamentbinding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase[J].Cell Research,2006,16(4):367-376.
[17] Chen H,Wu Y,Zhang Y,et al. Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination[J].FEBS Letters,2006,580(13):3145-3152.
[18] Li H,Cuartas E,Cui W,et al. IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation[J]. The Journal of Experimental Medicine,2005,201(7):1169-1177.
[19] Chen H X,Wang S,Wang Z,et al. Overexpression of RUNX3 inhibits malignant behaviour of Ecal109 cells in vitro and vivo[J]. Asian Pacific Journal of Cancer Prevention,2014,15(4):1531-1537.
[20]赵轶君,董浩,潘红艳,等.绿色荧光蛋白原核表达载体的构建[J].华北农学报,2013,28(3):73-76.