[Screening and cloning genes transactivated by TGF beta 1 in hepatic stellate cells using suppression subtractive hybridization technique].
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.
Xiao
Lin
L
Department of Infectious Diseases, First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, China.
Cheng
Jun
J
Guo
Jiang
J
Zhang
Li-ying
LY
Hong
Yuan
Y
Lun
Yong-zhi
YZ
Lan
Xian-yong
XY
Wu
Hui-juan
HJ
Zhang
Li-juan
LJ
Zhang
Yue-xin
YX
Zhang
Jian-long
JL
Li
Yan
Y
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*
D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1