JSRV-Env重组质粒诱导转染TC-1、TC-1-Hyal2细胞后对TGF-α及VEGF因子表达的影响

doi:10.16431/j.cnki.1671-7236.2016.09.001

摘要/Abstract

摘要：

通过脂质体法将JSRV-Env重组质粒瞬时转染小鼠肺上皮细胞（TC-1）和表达绵羊Hyal-2的小鼠肺上皮细胞（TC-1-Hyal2），而后探讨两细胞中转化生长因子-α（TGF-α）及血管内皮生长因子（VEGF）mRNA和蛋白表达的变化，以此分析TGF-α、VEGF与绵羊肺腺瘤病（ovine pulmonary adenomatosis，OPA）发生的关联性及其在细胞癌变过程中的作用。体外培养TC-1和TC-1-Hyal2细胞，按照不同质粒可将TC-1和TC-1-Hyal2细胞分别设为pEGFP-C1-env转染组、pEGFP-C1转染组和未转染组。利用实时荧光定量PCR和ELISA方法对TGF-α、VEGF的表达水平进行检测。实时荧光定量PCR检测结果表明，相比对照组（pEGFP-C1转染组及未转染组），在转染pEGFP-C1-env的两细胞组中VEGF mRNA的表达量均显著升高（P<0.05）；而两细胞组中TGF-α mRNA的表达量均极显著升高（P<0.01）。ELISA检测结果表明，同两对照组相比，转染pEGFP-C1-env的TC-1-Hyal2细胞上清液中VEGF、TGF-α蛋白浓度均极显著升高（P<0.01）；且VEGF在相同转染处理的TC-1细胞组中其蛋白浓度也极显著升高（P<0.01），而TGF-α的蛋白浓度呈显著升高（P<0.05）。比较pEGFP-C1转染和未转染各细胞组中TGF-α、VEGF的mRNA和蛋白表达量，结果均无显著差异（P>0.05）。本研究发现，通过实时荧光定量PCR和ELISA方法检测经JSRV-Env诱导转染上述两细胞系后TGF-α、VEGF的mRNA和蛋白表达量均升高，结果提示JSRV-Env可能上调两细胞因子，进而可推测TGF-α、VEGF的表达变化与OPA发病存在一定的关联性，由此为衍生出的OPA针对二者进行基因靶向治疗方案提供重要的理论依据。

关键词: JSRV-Env重组质粒; TC-1细胞; TC-1-Hyal2细胞; TGF-α 因子; VEGF因子

Abstract:

JSRV-Env recombinant plasmid was transiently transfected with mouse lung epithelial cell (TC-1) and ovine Hyal-2 which could stablely express in TC-1 cell (TC-1-Hyal2) by the method of liposome,then we could explore the mRNA and protein expression of TGF-α and VEGF in order to analyze the correlation TGF-α and VEGF with ovine pulmonary adenomatosis (OPA) and what roles they played in the carcinogenesis process.Both cells were cultured in vitro,according to different plasmids,TC-1 and TC-1-Hyal2 cells were set pEGFP-C1-env transfected groups,pEGFP-C1 transfected and non-transfected groups.The expression of TGF-α and VEGF were detected through Real-time quantitative PCR and ELISA.The PCR results showed that compared with control groups (pEGFP-C1 and non-transfected cells groups),the mRNA expression of VEGF was significantly increased in two cell groups of both transfected with pEGFP-C1-env (P<0.05),while the mRNA expression of TGF-α was extremely significantly increased (P<0.01).The ELISA test results showed that compared with two control groups,the protein expression of VEGF and TGF-α were extremely significantly increased in TC-1-Hyal2 cell which transfected with pEGFP-C1-env (P<0.01),and in TC-1 cell which transfected with pEGFP-C1-env,the expression of VEGF protein was extremely significantly increased (P<0.01),the protein expression of TGF-α was significantly increased (P<0.05).There was no significant difference in mRNA and protein expression of two cytokines between pEGFP-C1-transfected and non-transfected cells groups (P>0.05).The results showed that the expression of TGF-α and VEGF both increased in two cell lines which transfected with JSRV-Env.The results suggested JSRV-Env might raise the expression of TGF-α and VEGF,futher we could assume that the TGF-α,VEGF expression changes had a correlation with OPA,and it could provide an important theoretical basis on OPA for gene targeting therapy.

Key words: JSRV-Env recombinant plasmid; TC-1 cells; TC-1-Hyal2 cells; TGF-α factor; VEGF factor

中图分类号:

Q786

骆爽, 孙丽红, 斯日古楞, 么宏强. JSRV-Env重组质粒诱导转染TC-1、TC-1-Hyal2细胞后对TGF-α及VEGF因子表达的影响[J]. 《中国畜牧兽医》, 2016, 43(9): 2215-2222.

LUO Shuang, SUN Li-hong, Siriguleng, YAO Hong-qiang. JSRV-Env Recombinant Plasmid Induced-transfection of TC-1 and TC-1-Hyal2 and Influence of Cytokine VEGF and TGF-α[J]. , 2016, 43(9): 2215-2222.

参考文献

[1] Maeda N,Palmarini M,Murgia C,et al.Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA[J].Proc Natl Acad Sci USA,2001,98(8):4449-4454.
[2] Rai S K,Duh F M,Vigdorovich V,et al.Candidate tumor suppressor Hyal2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus,the envelope protein of which mediates oncogenic transformation[J].Proc Natl Acad Sci USA,2001,98(8):4443-4448.
[3] Vogt P K.Historical Introduction to the General Properties of Retroviruses[M].Retroviruses.Cold Spring Harbor(NY):Cold Spring Harbor Laboratory Press,1997.
[4] 朱福余,马学恩,于立新,等.绵羊肺腺瘤病毒受体透明质酸酶-2的原核表达及纯化[J].中国畜牧兽医,2013,40(9):32-36.
[5] 张月梅,么宏强,斯日古楞,等.绵羊透明质酸酶-2的真核表达及鉴定[J].中国畜牧兽医,2012,39(11):35-39.
[6] 国际兽疫局(OIE)编.农业部畜牧兽医局译.国际动物卫生法典[M].北京:兵器工业出版社,2000.
[7] Aitken L.Desease of Sheep,Fourth edition[M].Australia,Blackwall Publishing,2008.
[8] 李明,裴晓宁,张成辉.大肠癌患者FOLFIRI方案化疗前后血清血管内皮生长因子和内皮抑素水平的变化[J].中国现代药物应用,2012,6(12):61-62.
[9] 杨桢华,陆晓华,伍巧源,等.脂联素对高糖培养的大鼠肾小球系膜细胞表达VEGF及PEDF的影响[J].临床和实验医学杂志,2012,11(16):1255-1257.
[10] 岳朝艳,马妍慧,沈立松,等.Treg、Ki67和VEGF在结直肠癌中的表达及意义[J].国际检验医学杂志,2012,33(11):1288-1290.
[11] Hainsworth J D,Spigel D R,Farley C,et al.Phase Ⅱ trial of bevacizumab and erlotinib in carcinomas of unknown primary site:The Minnie Pearl Cancer Research Network[J].J Clin Oncol,2007,25(13):1747-1752.
[12] 秦继宝.食管癌患者手术治疗前后血清TGF-α、TGF-β1和血浆VEGF检测的临床意义[J].放射免疫学杂志,2009,22(4):334-335.
[13] Ribas J,Ni X,Haffner M,et al.miR-21:An androgen receptor-regulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer growth[J].Cancer Res,2009,69(18):7165-7169.
[14] Amankwah E K,Anegbe E,Park H,et al.miR-21,miR-221 and miR-222 expression and prostate cancer recurrence among obese and non-obese cases[J].Asian J Androl,2013,15(2):226-230.
[15] Schmittgen T D,Zakrajsek B A.Effect of experimental treatment on housekeeping gene expression:Validation by Real-time,quantitative RT-PCR[J].J Bioehem Biophys Methods,2000,46(1-2):69-81.
[16] Wootton S K,Metzger M J,Hudkins K L,et al.Lung cancer induced in mice by the envelope protein of Jaagsiekte sheep retrovirus (JSRV) closely resembles lung cancer in sheep infected with JSRV[J]. Retrovirology,2006,3(1):94.
[17] Dunlap K A,Palmarini M,Adelson D L,et al.Sheep endogenous betaretroviruses (enJSRVs) and the hyaluronidase 2(Hyal2) receptor in the ovine uterus and conceptus[J].Biol Reprod,2005,73(2):271-279.
[18] 孙丽红.JSRV-Env真核表达产物与体外细胞作用后对细胞信号通路中关键酶的影响[D].呼和浩特:内蒙古农业大学,2015.
[19] 刘畅,敖威华,李磊,等.绵羊肺腺瘤病毒致瘤机制研究进展[J].动物医学进展,2015,1:79-82.
[20] 蒋晓东,戴鹏,宋大安,等.HIF-1α、VEGF、VEGFR2在非小细胞肺癌组织中的表达及临床意义[J].临床肺科杂志,2011,16(3):386-388.
[21] Goel H L,Mercurio A M.VEGF targets the tumour cell[J].Nat Rev Cancer,2013,13(12):871-882.
[22] Chen J C,Chang Y W,Hong C C,et al.The role of the VEGF-C/VEGFRs axis in tumor progression and therapy[J].Int J Mol Sci,2012,14(1):88-107.
[23] Siva A C,Nelson L J,Fleischer C L,et al.Molecular assays for the detection of microRNAs in prostate cancer[J].Mol Cancer,2009,8:17.
[24] Wang G,Wang Y,Feng W,et al.Transcription factor and microRNA regulation in androgen-dependent and independent prostate cancer cells[J].BMC Genomics,2008,Suppl 2:S22.
[25] Nassif P S,Simpson S Q.Epidermal growth factor and transforming growth factor-alpha in middle ear effusion[J]. Otolaryngol Head Neck Surg,1992,119(6):564-568.
[26] Hofacre A,Fan H.Jaagsiekte sheep retrovirus biology and oncogenesis[J].Viruses,2010,2(12):2618-2648.