RARs基因转染对肝星状细胞信号传导的影响

The activation of hepatic stellate cells(HSC) plays a key role in pathogenesis of liver fibrosis. Retinoic acid(RA) which is an important derivative of vitamin A regulates cellular proliferation, differentiation and apoptosis through its nuclear receptors (RARs). The most striking changes which occurs during activation of HSC is that the content of all trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) in cytoplasm decrease dramatically while the expression level of their nuclear receptors RAR-βand RXR-αdecline. The aim of this research is to see if HSC could transit from activation to relatively quiescent status (inhibition of proliferation and production of ECM and phenotype shift in HSC) through transfection of RAR-βand RXR-αgenes plus treatment with their corresponding ligands in vitro. And if these effects are associated with interference of signal transduction pathway elicited by cytokines. Firstly, the recombinant eukaryotic expression vectors pCMV-Script-RAR-βand pcDNA3.1-RXR-α were constructed. The HSC in culture which had been isolated and purified from rat livers was activated further with PDGF-BB for 48 h, after that, the vectors were trasfected into these cells using LipofectAMINE. Successful transfection which was demonstrated by high level expression of RAR-βand RXR-αwas confirmed by Western blot and semi-quantitative RT-PCR . Then,the proliferation of transfected HSC was assayed by cell number counting, BrdU incorporation and MTT method. And the expression levels in both protein and mRNA of Dm, α-SMA, type I,III,IV of procollagens and FN in transfected HSC treated with corresponding ligands ATRA and 9-cis-RA(dosage from 0→10-4 mol/L) were observed with immunocytological stains ,image-analysis, and in situ hybridization . The results showed that high expression of RAR-βor RXR-αwas sustained for at least as long as 144 hours and 168 hours respectively in transfected cells treated with pertinent ligand. Those are long enough for the experiment. Both protein and mRNA levels in HSC treated with PDGF-BB were reduced considerably. However, the proliferation, the expression of proteins DM, α-SMA, type I,III,IV of procollagens and FN as well as the mRNA levels of type I,III procollagens and FN in transfected HSC treated with relevant ligand ATRA and 9-cis-RA were inhibited or down-regulated more significant than in sham-transfected HSC except that there was no effect on protein and mRNA levels of type III and IV procollagens in HSC transfected with RXR-αgene. The effects mentioned above were ligand dose dependent . We used irrelevant ligand 13-cis-RA as a parallel control treatment in order to verify that the biological effects on HSC brought out by transfection of both nuclear retinoic acid receptors were specific. It was demonstrated that 13-cis-RA had no any effects on transfected HSC.A variety of cytokines could activates HSC, among them, PDGF and TGF-β are most important. To explain if the possible mechanism through which the biological effects (inhibition of proliferation, somewhat reversed activation phenotype, and reduction of ECM-producing) are produced by transfection of the genes is involved in interference with cellular signal transduction initiated by cytokines. We studied the main cellular signal transduction pathway of PDGF, as it is well understanded. Western blot, semi-quantitation RT-PCR, immunocytoxhemistry, in situ hybridization and electrophoretic mobility shift assay (EMSA) were utilized to observe the changes of major signal transduction pathway, ras→raf→MEK→ERK→c-fos,c-jun→AP-1 and an alterative pathway,ras→MEKK→MKK→JNK/SAPK→AP-1 with special reference to protein expression level and phosphoylation of some key signal molecules (MEK, ERK, JNK) and to mRNA level of MEK and ERK as well as activity of AP-1. It was revealed that the down-regulation of protein and mRNA levels of MEK-1,ERK and phosphorylation level of ERK,JNK was much more enhanced in HSC transfected with RAR-βor RXR-αand treated with relevant ligand ATRA or 9-cis-RA t

从人肝组织分离纯化人肝星状细胞(hHSC),并进行有限代培养.体外用PDGF再激活hHSC,然后梦姿岷四谑芴寤蜃靖胔HSC,观察维甲酸及核内受体对hHSC激活到"静息"的调节作用,⒔徊酱游姿崂嗉昂四谑芴宥訮DGF信号转导通路的影响去探讨这种调节作用的机制.为临床使用维生素A类及人工合成其高效衍生物来防治肝纤维化提供理论基础.