Serial observations were performed on the sera α-fetoprotein (A F P) as well as on the immunohistochemical localization of A F P in liver tissues during hepatocarcinogenesis induced with 3'-Me-DAB in rats. Results showed that the dynamics of the sera A F P in rats bearing hepatocellular carcinomas (HCC) revealed saddle-like curve, whereas that of those rats without hearing HCC only increased at a sustained low level. The pathological bases which are responsible for precancerous increase of sera AFP are as follows: the small basophilic hepatocytes, "survival" hepatocytes, hyperplastic nodules, dysplastic hepatocytes, the hepacytic cords within the cholangiofibrosis foci. Solt-Farber model analysis indicated that the increase of sera AFP was related to the proliferation of the initiated cells. Immunoelectronic microscopy demonstrated the AFP was located on the membranes, which may be caused by the absorption of AFP from blood onto the cell membranes.

Hepatic stellate cells (HSCs) play key roles in hepatic fibrosis. One of the most striking alterations in activated HSCs is loss of cytoplasmic lipid droplets. However, the association of lipid storage with the activation of HSCs remains unclear. CCAAT/enhancer-binding proteins family (C/EBPs), especially C/EBP-alpha, controls differentiation of adipocytes. We suggested that C/EBP-alpha gene may be involved in HSCs activation. The present results showed that the expression levels of C/EBP-alpha and C/EBP-beta genes declined in activated HSCs. Over-expression of C/EBP-alpha gene in activated HSCs: (1) inhibited HSCs proliferation, extracellular matrix-producing, alpha-smooth muscle actin gene expression, and induced rebound of cytoplasmic lipid droplets; (2) reduced retinoic acid receptor-beta, C/EBP-delta and-beta gene expressions, but increased the active form C/EBP-beta PSer (105), and induced retinoid X receptor-alpha gene expression; and (3) did not affect the protein level of p16INK4a, p21Cip1/WAF1 or p27Kip1. In conclusions, C/EBP-alpha gene is involved in in vitro activation of rat HSCs.

BACKGROUND/AIM: In hepatic stellate cells isolated from rat fibrotic livers, the amount of retinoid X receptor-alpha (RXR-alpha) mRNA is greatly reduced. However, the effectiveness of retinoids in the treatment of liver fibrosis is controversial. We hypothesized that increasing the expression levels of RXR-alpha in livers will improve the response of liver fibrosis to retinoids treatment. METHODS: pTracer-CMV2 vector harboring both green fluorescent protein and RXR-alpha genes was given to mice with carbon tetrachloride (CCl(4))-induced liver fibrosis, by hydrodynamic-based in vivo transfection. Vitamin A was simultaneously administered to the mice. Sirius red staining and measurement of hydroxyproline content were performed to evaluate liver fibrosis. The incorporation of 5-bromo-2-deoxyribouridine (BrdU) was carried out to determine liver cell proliferation. RESULTS: Successful transfection and expression of exogenous RXR-alpha gene in the liver was determined by observance of green fluorescence under a confocal microscope, and detection of RXR-alpha protein by immunohistochemistry. Hepatic fibrosis, evaluated by both Sirius red staining with image analysis and quantity of hydroxyproline in livers of RXR-alpha-transfected group, tapered off remarkably. The hydroxyproline content and Sirius red-positive staining area on liver sections from RXR-alpha-transfected mice decreased by 34.3% and 54.63%, respectively, compared with the control group receiving empty vector. The labeling index of BrdU in non-parenchymal cells was much lower in livers from the RXR-alpha-transfected group than that of empty vector-transfected group. CONCLUSIONS: Hydrodynamic-based in vivo transfection of the RXR-alpha gene can enhance the vitamin A-induced attenuation of liver fibrosis in mice. One of the possible mechanisms of action for this gene treatment is inhibition of non-parenchymal cell proliferation mainly composed of hepatic stellate cells in fibrotic livers.

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.

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.