title: [Establishment of the Chang liver cell line stably overexpressing human UCP2 gene and its effect on mitochondrial membrane potential and reactive oxygen species].
abstract: To establish the Chang liver cell line stably overexpressing human uncoupling protein 2 (UCP2) and observe the effect of UCP2 on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). The Chang liver cell line was transfected with recombinant plasmid containing full-length human UCP2 cDNA (pcDNA3.1-hUCP2) or pcDNA3.1 empty vector. The stable cell line was established by antibiotic screening with Zeocin. UCP2 expression was detected by Western blotting and immunocytochemistry. The UCP2 overexpressing cells were pretreated with genipin at various doses (25, 50 and 100 munol/L). MMP and intracellular ROS were detected by fluorescence spectrophotometry. The total normalized protein content in UCP2 overexpressing cells was 1.6-fold higher than that in unmanipulated normal cells. The fluorescence intensities of Rhodamine123 and DCFH-DA in UCP2 overexpressing Chang liver cells (11.11+/-2.76 and 4.97+/-0.62, respectively) were significantly lower than those in unmanipulated normal cells (15.56+/-2.55, P less than 0.01 and 6.14+/-1.25, P less than 0.05, respectively) and in cells transfected with empty vector (16.11+/-2.93, P less than 0.01 and 6.23+/-1.13, P less than 0.05, respectively). Treatment of UCP2 overexpressing cells with 25, 50 and 100 munol/L genipin caused a dose-dependent increase in fluorescence intensities of Rhodamine123 (14.89+/-2.89, 17.89+/-2.93 and 24.00+/-2.55, respectively, all P less than 0.01) and DCFH-DA (9.16+/-0.78, 10.84+/-1.09 and 11.83+/-1.25, respectively, all P less than 0.01). The Chang liver cell line stably overexpressing UCP2 was established successfully. Using this cell system, UCP2 was found to play a role in mitochondrial function by regulating MMP and ROS.
authors.affiliation: Department of Physiology, Dalian Medical University, Dalian, China.
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Guan
authors.forename: Yao-Feng
authors.initials: YF
authors.lastname: Wang
authors.forename: De-Zheng
authors.initials: DZ
authors.lastname: Gong
authors.forename: Bo
authors.initials: B
authors.lastname: Yuan
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.forename: Liang
authors.initials: L
authors.lastname: Zhu
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Jia
authors.forename: Ming-Chuan
authors.initials: MC
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Zhao
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zou
mesh_terms: D002460:Cell Line; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis*; D053078:Membrane Potential, Mitochondrial; D024101:Mitochondrial Proteins / Q000096:biosynthesis*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000071246:Uncoupling Protein 2
chemical_list: D007473:Ion Channels; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; C000604389:UCP2 protein, human; D000071246:Uncoupling Protein 2