Screening and cloning genes transactivated by TGF beta 1 in hepatic stellate cells using suppression subtractive hybridization technique / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 854-857, 2008.
Artículo
en Chino
| WPRIM
| ID: wpr-250101
ABSTRACT
<p><b>OBJECTIVES</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
ARN Mensajero
/
Biblioteca de Genes
/
Línea Celular
/
Clonación Molecular
/
Homología de Secuencia
/
Factor de Crecimiento Transformador beta1
/
Células Estrelladas Hepáticas
/
Vectores Genéticos
/
Genética
/
Metabolismo
Tipo de estudio:
Estudio diagnóstico
/
Estudio de tamizaje
Límite:
Animales
Idioma:
Chino
Revista:
Chinese Journal of Hepatology
Año:
2008
Tipo del documento:
Artículo
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