title: [Effect of adipose differentiation-related protein on the apoptosis of H9c2 cells by palmitic acid].
abstract: OBJECTIVE
  To construct a eukaryotic expression vector for expressing adipose differentiation-related protein(ADRP) protein and obtain a stable transfected H9c2 cell line.
  
  
  METHODS
  (1)The full length DNA fragment of ADRP gene was amplified by PCR from the myocardium of adult SD rats and was inserted into pEGFP-C1 with T4 ligase. After the identification of digestion and sequencing on the recombinant pEGFP-C1-ADRP, the recombinant was transformed into E.coli strain DH5α.and the positive recombinant plasmid was selected. (2)The selected positive recombinant clones were amplified and transfected into H9c2 cells by lipofectamine(TM);2000. Cells containing stable transformants were selected by the ability of resistance to G418. The stable transfected cell line expressing high level of ADRP were obtained. Expression of green fluorescent protein gene was observed under fluorescence microscope and the expression of ADRP was identified by RT-qPCR and Western blot analysis. (3)The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry.
  
  
  RESULTS
  (1)Restriction map analysis and DNA sequencing analysis revealed that our target gene fragment was inserted into the expressing vector pEGFP-C1 successfully. (2)Green fluorescent was observed by fluorescence microscope on the cells which were transfected with pEGFP-C1 and pEGFP-C1-ADRP and did not disappear even at the twentieth passage of H9c2 cells. RT-qPCR and Western blot analysis showed that recombinant cells exhibit higher levers of mRNA and protein. (3)After treatment with different levels of PA, the apoptosis percentage of recombinant cells was lower than those of the other two cells.
  
  
  CONCLUSIONS
  (1)The eukaryotic expression vector pEGFP-C1-ADRP and stable transfected H9c2 cells were established successfully. (2)The overexpression of ADRP gene could prevent apoptosis of cells caused by PA, which indicated that ADRP might play a protective role in H9c2 cells.
authors.affiliation: Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Fudan University, Shanghai 200040, China. kittycat1984@yahoo.cn
authors.forename: Yuan-yuan
authors.initials: YY
authors.lastname: Bao
authors.forename: Zhou
authors.initials: Z
authors.lastname: Fang
authors.forename: Li-nuo
authors.initials: LN
authors.lastname: Zhou
authors.forename: Ren-ming
authors.initials: RM
authors.lastname: Hu
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000502:physiology; D019308:Palmitic Acid / Q000494:pharmacology*; D000070780:Perilipin-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D000070780:Perilipin-2; C000602984:Plin2 protein, rat; D019308:Palmitic Acid