王继东;罗爽;方政慧;

• 1:山东大学附属济南市中心医院妇科
• 2:遂宁市中心医院妇产科

摘要(Abstract)：

目的:构建inhba基因过表达的慢病毒载体(lenti-EGFP-inhba),观察体外转染大鼠卵巢颗粒细胞的有效性。方法:p LVX-EGFP-3FLAG质粒系统经EcoRI酶切使之线性化。inhba基因(NM_008380)PCR扩增后同源重组入线性化的表达质粒系统。Western blot法检测表达质粒目的基因表达。将构建成功的慢病毒载体(lenti-EGFP-inhba)转染大鼠卵巢颗粒细胞(MOI=20),检测目的基因表达。结果:PCR扩增产物与目的基因大小吻合,重组质粒系统经转化后获得阳性克隆,转染293T细胞可见EGFP-inhba融合蛋白表达。Western blot获得76k Da分子量大小阳性条带,与融合蛋白分子量大小一致。lentiEGFP-inhba体外转染大鼠卵巢颗粒细胞,倒置荧光显微镜下可见EGFP表达。结论:lenti-EGFP-inhba表达载体成功构建,并可在体外有效转染大鼠卵巢颗粒细胞。

关键词(KeyWords)： Activin A;慢病毒载体;转染;颗粒细胞

Abstract:

Keywords:

基金项目(Foundation): 山东省医药卫生科技发展计划(No:2016WS0127)

作者(Authors): 王继东;罗爽;方政慧;

DOI: 10.13283/j.cnki.xdfckjz.2018.06.005

参考文献(References)：

• [1]Marino G,Zanghi A.Activins and inhibins:expression and role in normal and pathological canine reproductive organs:a review[J].Anat Histol Embryol,2013,42(1):1-8
• [2]Lee J,Ko J,Yi JY.Histone deacetylase inhibitor(HDACi)upregulates activin A and activates the Smad signaling pathway in melanomas[J].J Dermatol Sci,2017,90(1):13-20
• [3]Spottiswoode N,Armitage AE,Williams AR,et al.Role of activins in hepcidin regulation during malaria[J].Infect Immun,2017,85(12).pii:e00191-17
• [4]Mc Neilly AS.Diagnostic applications for inhibin and activins[J].Mol Cell Endocrinol,2012,359(1-2):121-125
• [5]Kiagiadaki F,Kampa M,Voumvouraki A,et al.Activin-A causes Hepatic stellate cell activation via the induction of TNFαand TGFβin Kupffer cells[J].Biochim Biophys Acta,2017,1864(3):891-899
• [6]Pan C,Singh S,Sahasrabudhe DM,et al.TGFβsuperfamily members mediate androgen deprivation therapy-induced obese frailty in male mice[J].Endocrinol,2016,157(11):4461-4472
• [7]Refaat B,El-Shemi AG,Mohamed AM,et al.Activins and their related proteins in colon carcinogenesis:insights from early and advanced azoxymethane rat models of colon cancer[J].BMC Cancer,2016,16(1):879
• [8]王继东,罗爽,潘红娟,等.慢病毒载体介导绿色荧光蛋白基因转染大鼠卵巢的实验研究[J].生殖与避孕,2012,32(6):366-371
• [9]Hetzel M,Suzuki T,Hashtchin AR,et al.Function and Safety of Lentivirus-Mediated Gene Transfer for CSF2RADeficiency[J].Hum Gene Ther Meth,2017,28(6):318-329
• [10]Tokgun O,Fiorentino FP,Tokgun PE,et al.Design of a lentiviral vector for the inducible expression of myc:a new strategy for construction approach[J].Mol Biotechnol,2017,59(6):200-206
• [11]Chira S,Jackson CS,Oprea I,et al.Progresses towards safe and efficient gene therapy vectors[J].Oncotarget,2015,6(31):30675-30703
• [12]Garoon RB,Stout JT.Update on ocular gene therapy and advances in treatment of inherited retinal diseases and exudative macular degeneration[J].Curr Opin Ophthalmol,2016,27(3):268-273
• [13]Sharon D,Kamen A.Advancements in the design and scalable production of viral gene transfer vectors[J].Biotechnol Bioeng,2018,115(1):25-40
• [14]Borsotti C,Borroni E,Follenzi A.Lentiviral vector interactions with the host cell[J].Curr Opin Virol,2016,21:102-108
• [15]Oldham RA,Berinstein EM,Medin JA.Lentiviral vectors in cancer immunotherapy[J].Immunotherapy,2015,7(3):271-284
• [16]Rao MS,Malik N.Assessing i PSC reprogramming methods for their suitability in translational medicine[J].J Cell Biochem,2012,113(10):3061-3068
• [17]Levy C,Verhoeyen E,Cosset FL.Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells[J].Curr Opin Pharmacol,2015,24:79-85
• [18]Hirai H.Basic research on cerebellar gene therapy using lentiviral vectors[J].Cerebellum,2012,11(2):443-445