OBJECTIVE: To investigate the effects of PDGF on the proliferation of rat HSC, its gene expression of collagens and the autocrine excretion of PDGF in vitro. METHODS: (3)H-TdR incorporation was used to estimate the DNA synthesis elicited by PDGF, TGF-beta1 and EGF in HSC cultured in an absolutely serum-free medium. Northern blot was employed to detect the levels of mRNA expressions of type I and III procollagens and PDGF induced by PDGF added in the same medium for HSC. RESULTS: Both PDGF and EGF enabled to stimulate the proliferation of HSC in a dose-dependent manner, and the effect of the former was stronger. The proliferation was dose-dependently increased in PDGF and EGF with a dosage between 0.5 and 10 ng/ml, and the synthesis of DNA became saturated when the dosage was over 10 ng/ml. TGF-beta1 gave no effects on the proliferation of HSC under current experimental condition. Furthermore, PDGF was able to enhance the mRNA expression for type I and III procollagens and PDGF itself. The promoting effect for the expressions of type I and III procollagen mRNAs by PDGF was bi-phasic between 6 to 48 hours of the cultivation. For type I procollagen, mRNA expression began to increase 12 hours after PDGF stimulation, and the increase became prominent at 48 hours. For type III procollagen, an increase of mRNA expression was seen before the stimulation of PDGF, and the expression level was comparatively increased 48 hours later. The mRNA expression of PDGF was increased by 2-3 times 6 hours after PDGF stimulation, and back to the original level 24 hours later. CONCLUSIONS: PDGF enabled to promote the proliferation of HSC and expression of collagens in HSC. The bi-phasic effect on the expression of collagens may be interrelated with the autocrine excretion of PDGF.

OBJECTIVE: The expression of C/EBPalpha protein and mRNA during automatically 能 activation process in primary cultures of HSCs were observed in order to explore its possible association with the proliferation and activation of HSCs. METHODS: Immunocytochemistry, Western blot and RT-PCR were used to evaluated the expression of C/EBPalpha protein and mRNA; as well as the expression of alpha-SMA, Desmin, MMP2, type I procollagen (alpha1). The eukaryotic vector harboring the full length cDNA of C/EBPalpha was transfected into activated HSC, then immunocytochemistry was applied to confirm the transfection and evaluate the effect of transfection on the proliferation of HSC by calculating the PCNA-positive cells. The morphological changes of HSC were observed by use of phase-contrast microscope. RESULTS: Constitutive expression of mRNA and protein of C/EBPalpha were detected in primarily cultured HSCs, and the protein was seen in both nuclei and cytoplasm with the latter being dominant. Their expression levels reached highest at day 2 of the culture, then decreased gradually when continually cultured to the day 4, 7, 10, on the other hand, the expression of alpha-SMA, MMP2 and ColI (alpha1) increased steadily. Transient transfection was verified by the fact that much more and stronger C/EBPalpha stain was observed in transfected HSCs than in void-vector transfected cells. In C/EBPalpha gene transfected HSCs, the number of PCNA-positive cells dramatically decreased compared with the void-vector transfected cells 24h after transfection. In addition, the C/EBPalpha gene transfected HSCs died 36h after transfection, a few surviving cells became longer and thinner in morphology, however the void-vector transfected cells almost all remained alive. CONCLUSIONS: C/EBPalpha was likely involved in the HSCs activation, and over-expressed C/EBPalpha by transfection had inhibitory influence on the proliferation of cultured rat HSCs.

OBJECTIVE: To study the effects of retinoic acid on regulation of expressions of cyclin-dependent kinase inhibitors, i.e. p16, p21 and p27 in cultured rat hepatic stellate cells. METHODS: Stellate cells were isolated from healthy rat livers and cultured. After stimulated with 1 ng/ml transforming growth factor beta 1, subcultured hepatic stellate cells were treated with or without 1 nmol/ml retinoic acid. MTT assay, immunocytochemistry for p16, p21, p27 and alpha-smooth muscle actin protein, in situ hybridization for retinoic acid receptor beta 2 and p16, p21 and p27 mRNA and quantitative image analysis (partially) were performed. RESULTS: Retinoic acid markedly inhibited hepatic stellate cells proliferation (41.50%, P<0.05), decreased the protein level of alpha-smooth muscle actin (55.09%, P<0.05), and induced hepatic stellate cells to express retinoic acid receptor beta 2 mRNA. In addition, retinoic acid increased the protein level of p16 (218.75%, P <0.05) and induced p21 protein expression; meanwhile, p27 was undetectable by immunocytochemistry in both control and retinoic acid-treated hepatic stellate cells. However, retinoic acid had no influence on the mRNA level of p16, p21 or p27 as determined by in situ hybridization. CONCLUSIONS: Up-regulation of p16 and p21 on post-transcriptional level may contribute, in part, to retinoic acid inhibition of transforming growth factor beta 1-initiated rat hepatic stellate cells activation in vitro.

OBJECTIVE: To study the effects of heparin on the growth, extracellular matrix and matrix metalloproteinase (MMP) gene expression in rat hepatic stellate cells (HSC). METHODS: Activated HSC was treated by heparin or fetal calf serum without heparin. The cell growth was evaluated by actual cell count and BrdU-labelled immunocytochemical stain. The gene expressions of type I and IV procollagen, fibronectin, MMP-2 and membrane type matrix metalloproteinase (MT-MMP) were investigated by immunocytochemical stain and digoxigenin-labeled in situ hybridization technique, respectively. In addition, the gelatinase activity of MMP-2 was examined by zymography. RESULTS: Heparin could obviously reduce HSC growth, inhibit the synthesis of type I procollagen and fibronectin protein, and the gene expressions of type I procollagen, fibronectin and MT-MMP. The expressions of type IV procollagen, MMP-2 and MMP-2 activity were not affected by heparin. CONCLUSION: The results demonstrate that heparin can inhibit HSC proliferation, down-regulate interstitial collagen synthesis and inhibit MT-MMP gene expression.

In both the primary tumor and associated lymph-node metastases of 40 cases of Dukes'C colorectal adenocarcinomas, exons 5 to 9 of the p53 tumor-suppressor gene were examined by PCR amplification and single-strand-conformation-polymorphism (SSCP) analysis. Mobility shifts indicating p53 mutations, which were confirmed by direct sequencing, were identified in 14 primary cancers (35%) and in 19 of the 40 lymph-node metastases (48%). In 12 cases (30%), the p53-mutation status in the primary cancer and its lymph-node metastases was identical. This result is compatible with the hypothesis that when a p53 mutation occurs before the establishment of lymph-node metastasis, it subsequently persists in the metastatic nodes. In 7 cases (18%), p53 mutations were identified in lymph-node metastases that were not concordant with the p53 status in the primary tumor. This finding can be explained by assuming that (1) p53 heterogeneity existing in the primary tumor is not reflected in all metastases and/or (2) new p53 mutations may occur during the development of metastatic lesions.