title: [Screening and cloning genes transactivated by TGF beta 1 in hepatic stellate cells using suppression subtractive hybridization technique].
abstract: OBJECTIVE
  To construct a cDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1.
  
  
  METHODS
  mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction twice it then was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search.
  
  
  RESULTS
  The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC was constructed successfully. The amplified library contained 146 positive clones, which contained 200-1000 bp of inserts. Randomly, thirty clones were analyzed by sequencing and bioinformatics, consisting of 28 known genes and 2 unknown genes.
  
  
  CONCLUSIONS
  The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC using SSH technique was constructed successfully. Some gene coding proteins are those involved in cell growth regulation, protein synthesis, signal transduction, extracellular matrix metabolism, and anti-lipid peroxidative, which gives us some new clues for the study of the mechanism of liver fibrosis.
authors.affiliation: Department of Infectious Diseases, First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Xiao
authors.forename: Jun
authors.initials: J
authors.lastname: Cheng
authors.forename: Jiang
authors.initials: J
authors.lastname: Guo
authors.forename: Li-ying
authors.initials: LY
authors.lastname: Zhang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Hong
authors.forename: Yong-zhi
authors.initials: YZ
authors.lastname: Lun
authors.forename: Xian-yong
authors.initials: XY
authors.lastname: Lan
authors.forename: Hui-juan
authors.initials: HJ
authors.lastname: Wu
authors.forename: Li-juan
authors.initials: LJ
authors.lastname: Zhang
authors.forename: Yue-xin
authors.initials: YX
authors.lastname: Zhang
authors.forename: Jian-long
authors.initials: JL
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015723:Gene Library; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017385:Sequence Homology; D053773:Transforming Growth Factor beta1 / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1