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Ad-sTNFRI-IgGFc腺病毒载体的构建及其转染人气道平滑肌细胞

Construction of Adenoviral Vector Encoding Soluble Human sTNFRI-IgGFc cDNA and Its Expression in Human Airway Smooth Muscle Cells (HASMCs)
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摘要 TNF-α是一种具有广泛生物学活性的前炎症细胞因子,参与哮喘整个病理生理过程。可溶性肿瘤坏死因子受体(sTNFR)可以拮抗肿瘤坏死因子活性,已被用来治疗与TNF相关的炎性疾病。将sTNFRI-IgGFc基因插入腺病毒穿梭质粒pDC316,与辅助质粒pBHGloxΔE1,3Cre共转染HEK293细胞,重组产生Ad-sTNFRI-IgGFc,PCR鉴定毒种正确后,进行扩增、纯化和滴度测定,转染人气道平滑肌细胞,利用RT-PCR、免疫组化方法,流式细胞仪,ELISA检测转染后细胞中sTNFRI-IgGFc的转录和表达。实验结果证明成功构建了Ad-sTNFRI-IgGFc腺病毒载体,感染性滴度达3×1010TCID50/mL,200moi转染气道平滑肌细胞阳性率达99.32%,转染后气道平滑肌细胞在mRNA和蛋白水平均有sTNFRI-IgGFc表达。转染上清稀释64倍后仍对TNF有拮抗活性。为将表达sTNFRI-IgGFc腺病毒基因治疗哮喘的实验研究提供了基础。 Tumour necrosis factor (TNF-α) is a pro-inflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma, and which has recently been highlighted as potentially important in refractory asthma. To study the feasibility of local treatment of asthma with recombinant adenovirus vector carrying soluble extra-cellular region of TNF receptor I-IgGFc (sTNFRI-IgGFc) fusion protein, The sTNFRI-IgGFc gene was subcloned into the adenovirus shuttle plasmid pDC316, the products were co-transfected into HEK293 cell line with helper plasmid pBHGloxAEI,3Cre. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was produced by homologous recombination of above 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI- IgGFc was amplified and purified, its titer was measured by TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in rransfected human airway smooth muscle cells (HASMCs) was detected by RT-PCR, ELISA and immunological histochemistry.The anti-TNF activity assay of transfected HASMCs culture supernatant was measured by MTT. Ad-sTNFRI-IgGFc was successfully constructed with the titer of 3×10^10TCID50/mL. Ad-sTNFRI-IgGFc can transfect HASMC with high efficacy. The transcription of sTNFRI-IgGFc mRNA and the expression of protein were confirmed in the transfected HASMCs. Moreover, the product in 100μL expression supernatant could completely antagonize the cytolytic effect of 2ng TNFa on L929 cells, even at 1/64 dilution. This study forms the basement of the experiment study on local treatment of asthma with adenovirus expressing sTNFRI-IgGFc.
出处 《生物工程学报》 CAS CSCD 北大核心 2008年第5期774-779,共6页 Chinese Journal of Biotechnology
基金 国家高技术研究发展计划(863)(No.2007AA02Z458)资助~~
关键词 可溶性肿瘤坏死因子受体 腺病毒载体 人气道平滑肌细胞 支气管哮喘 soluble tumor necrosis factor alpha receptor(sTNFR), adenovirus vector, human airway smooth muscle celIs(HASMCs), asthma
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参考文献14

  • 1Berry MA,Hargadon B,Shelley M,et al.Evidence of a role of tumor necrosis factor alpha in refractory asthma.N Engl J Med,2006,354(7):697-708.
  • 2Busse WW,Banks-Schlegel S,Wenzel SE.Pathophysiology of severe asthma,J Allergy Clin Immunol 2000,106(6),1033-1042.
  • 3Howarth PH,Babu KS,Arshad HS,et al.Tumour necrosis factor (TNFalpha) as a novel therapeutic target in symptomatic corticosteroid dependent asthma.Thorax,2005,60(12):1012-1018.
  • 4何婕,姚立红,陈爱君,喻宏,贾润清,程从升,郇丽芳,张智清.sTNFR-IgGFc融合基因在内皮细胞中的表达及其对小鼠关节炎模型的基因治疗研究[J].生物工程学报,2006,22(3):378-383. 被引量:4
  • 5Brightling C,Berry M,Amrani Y,et al.Targeting TNF-α:A novel therapeutic approach for asthma.J Allergy Clin Immunol,2008,121(1):5-10.
  • 6Erin EM,Leaker BR,Nicholson GC,et al.The effects of a monoclonal antibody directed against tumor necrosis factor-alpha in asthma.Am J Respir Crit Care Med,2006,174(7):753-762.
  • 7Rouhani FN,Meitin CA,Kaler M,et al.Effect of tumor necrosis factor antagonism on allergen-mediated asthmatic airway inflammation.Respir Med,2005,99(9):1175-1182.
  • 8Gutierrez MA,Lizarralde PE,Martinez OP,et al.Tuberculous peritonitis in a patient with rheumatoid arthritis treated with adalimumab.Clin Rheumatol,2007,26(3):452-453.
  • 9Bongartz T,Sutton AJ,Sweeting MJ,et al.Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies:systematic review and meta-analysis of rare harmful effects in randomized controlled trials.JAMA,2006,295(21):2482.
  • 10Moreland LW,Weinblatt ME,Keystone EC,et al.Etanercept treatment in adults with established rheumatoid arthritis:7 years of clinical experience.J Rheumatol,2006,33(5):854-861.

二级参考文献11

  • 1Bemelmans MH,van Tits LJ,Burman WA,Tumor necrosis factor:function,release and clearance.Crit Rev Immunol,1996,16(1):1-11.
  • 2Idriss HT,Naismith JH,TNF alpha and the TNF receptor superfamily:structure-fuction relationship(s).Microsc Res Tech,2005,50(3):184-195.
  • 3Pilz G,Fraunberger P,Apper R et al.Early prediction of outcome in score-identified,postcardiac surgical at high risk for sepsis,using soluble tumor necrosis factor receptor-P55 concentrations.Crit Care Med,1996,24(4):596-600.
  • 4Cope AP,Aderka D,Doherty M et al.Increased levels of soluble tumor necrosis factor receptors in the sera and synocial fluid of patients with rheumatic disease,Arthritis Rheum,1992,35(10):1160-1169.
  • 5Loetscher H,Steinmetz M,Lesslauer W.Tumor necrosis factor:receptors and inhibitors Cancer Cells,1991.3(6):221-226.
  • 6John E.Coligan,da M. Kruisbeek,David H.Margulies et al.Current protocols in immunology.John Wiley & Sons,Inc.1995.
  • 7Lee CG,Feang KT,Martin MA et al.effcient long-term coexpression of a hammerhead ribozyme targeted to the U5 refion of HIV-1 LTR by linkage to the multidrug-resistance gene.Antisense Nucleic acid Drug Dev,1997,7(5):511-522.
  • 8Sugimoto Y,Aksentijevich I,Mrray GJ et al.Retroviral coexpression of a multidrug resistance gene(MDR1)and human alpha-galactosidase A for gene therapy of fabry disease.Hum Gene Ther,1995,6(7):905-915.
  • 9Zhou Y,Aran J,Gottesman MM et al.Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector,Hum Gene Ther,1998,9(3):287-293.
  • 10Maruyama H,Higuchi N,Kameda S et al.Rat liver-targeted naked plasmid DNA transfer by tail vein injection.Mol Biotechnol,2004.26(2):165-172.

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