title: [Protective effect of cotransfection of A20 and HO-1 gene against the apoptosis induced by TNF-α in rat islets in vitro].
abstract: OBJECTIVE
  To establish the method for cotransferring human A20 gene and human heme oxygenase-1 (HO-1) gene into the isolated rat islets using lentiviral transfection system, and to study the protective effect of A20 and HO-1 protein against the apoptosis induced by cycloheximide (CHX) and TNF-α, and finally to explore the underlying mechanism.
  
  
  METHODS
  The A20 gene and HO-1 gene were cloned and inserted into the lentiviral transfection system. The efficacy of gene transfer was measured by the intensity of the enhanced green fluorescent protein (EGFP) fluorescence-positive islets. Western blot was applied to verify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, the isolated islets were treated with CHX+TNF-α, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the fluorescence-activated cell sorting (FACS) methods were used to evaluate the apoptosis of the islet cells and Western blot was used to detect caspase-3 activation.
  
  
  RESULTS
  (1) A20 and HO-1 genes were introduced into the isolated islets by lentiviral transfection, both of the genes were highly expressed in the islets after 96 hours culture detected by Western blot method. (2) The insulin levels in the cell culture medium from A20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls (P < 0.01). (3)After CHX + TNF-alpha treatment, the cell culture medium insulin concentration in the A20 gene transfected group [(93.58 ± 4.12)µg/ml], HO-1 gene transfected group [(88.98 ± 4.77) µg/ml ] and A20/HO-1 co-transfected group [(103.33 ± 3.16) µg/ml] were significantly higher than that in the EGFP group [(9.03 ± 0.65) µg/ml ] and the control group [(8.86 ± 0.38) µg/ml] (P < 0.001). Minimum expression level of the activated caspase-3 was found in the A20/HO-1 co-transfected group.
  
  
  CONCLUSIONS
  The lentiviral gene transfer system was an efficient and stable gene transfer vector, the over-expressed A20 and HO-1 protein delivered via lentivirus could preserve rats' islets function and act against the apoptosis induced by CHX and TNF-α.
authors.affiliation: Department of Endocrinology and Inborn Metabolic Diseases, Children's Hospital of Fudan University, Shanghai 201102, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Lu
authors.forename: Shui-xian
authors.initials: SX
authors.lastname: Shen
authors.forename: Di-jing
authors.initials: DJ
authors.lastname: Zhi
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.forename: Li-he
authors.initials: LH
authors.lastname: Guo
authors.forename: Fei-hong
authors.initials: FH
authors.lastname: Luo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D053148:Caspase 3