[Screening up-regulated genes in hepatic stellate cells treated with PDGF-BB using suppression subtractive hybridization technique].
OBJECTIVE
To construct a subtractive cDNAs library of up-regulated genes in rat hepatic stellate cells (HSCs) stimulated with platelet-derived growth factor (PDGF)-BB by suppression subtractive hybridization (SSH) technique, to clone the up-regulated genes associated with its regulation effects, and to elucidate the mechanism of the molecular biology of hepatic fibrosis involved in PDGF-BB.


METHODS
The mRNA was isolated from HSCs stimulated with PDGF-BB and controlled with identical cells untreated with PDGF-BB, and then the cDNAs were synthesized. The cDNAs were designated as tester and driver. After being digested by restriction enzyme Rsa I, small-sized cDNAs were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, individually. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.


RESULTS
The subtractive cDNAs library of up-regulated genes in HSCs stimulated with PDGF-BB was constructed successfully. The amplified library contained 102 positive clones. Colony PCR showed that 93 clones contained 200-1000 bp inserts. Sequence analysis was performed in 31 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank; altogether 13 coding sequences were obtained, which were known ones. The genes mainly included voltage-dependent anion channel (VDAC), heat shock protein 47 and RAN-member RAS oncogene family genes.


CONCLUSIONS
Acting as one of the most effective mitogens, PDGF-BB up-regulated some gene expressions during stimulation of the HSCs, including some cell growth associated proteins, some proteins participating in intracellular metabolism and some molecular chaperone proteins. This work brings some new clues for studying the molecular biological mechanism involved in the up-regulated genes in PDGF-BB transactivated HSCs in hepatic fibrosis.
Zhu
Li-ying
LY
Department of Infectious Diseases, Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China.
Cheng
Jun
J
Ma
Ying-ji
YJ
Li
Yong-guo
YG
Zhong
Li-hua
LH
Hong
Yuan
Y
D000818:Animals; D000077214:Becaplermin; D015870:Gene Expression; D015723:Gene Library; D055166:Hepatic Stellate Cells; D009693:Nucleic Acid Hybridization; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D015854:Up-Regulation
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D000077214:Becaplermin