Effector Mechanisms Of TGF-β 1-mediated Regeneration And Differentiation And Apoptosis In Hepatocytes

Background Transforming growth factor-β 1 has been described as the prototypical multifunctional cytokine, participating in the regulation of vital cellular activities such as proliferation and differentiation as well as a number of basic physiological functions such as tissue development and immunity Another essential function of TGF-β1, which has recently come to light, is its role as a tumor suppressor in a variety of different human cell types. It is thought that loss of sensitivity to TGF-β1 may be an important contributing factor in the development of cirrhosis and hepatornas. To understand the molecular and cellular mechanisms involved in the growth control of hepatocytes and apoptosis may provide a new idea in the prevention and therapy of liver diseases.Objective The aim of the present study was to determine the relationships between the effect of TGF-β1 and apoptosis in normal and cirrhotic hepatocytes, as well as in human carcinoma cell lines. Whether normal hepatocytes are more susceptible to TGF-β1-induced apoptosis than cirrhotic hepatocytes? Whether TGF-β1 would induce apoptosis in human carcinoma cell lines? What's the role of p53 and Smads and caspases in the critical signal transdution pathways during the TGF-β1-induced apoptosis? Methods Cirrhosis was induced in BALB/c mice by means of injection with 50% carbon tetrachloride for 8W. Hepatocytes were isolated from cirrhotic and normal mice by means of an in situ perfusion method with collagenase solution with minor modifications. The induction of apoptosis by TGF-β1 was determined in these hepatocytes by electrophoresis on 1.5% agarose gel to observe DNA ladder. To detect apoptosis after treated with TGF-β1(5ng/ml) and caspases inhibitor, hepatocyte nuclei were stained with fluorescent DNA-binding dyes Hoechst 33342 and viewed under fluorescent microscope to investigate and compare the effects of caspase-3 and caspases-8 on TGF-β1-induced apoptosis. Three human hepatic carcinoma cell lines, involving different status of the p53 gene respectively, was used in this study. TGF-β1-induced apoptosis in hepatic carcinoma cell lines was quantitated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. For identification the mechanism of apoptosis induced by TGF-β1, these cell lines were quantitatively assessed by luciferase assay. Firstly cells were transfected with a TGF-β1-inducible luciferase reporterplasmid containing Smad binding elements (SBE) and luciferase gene using LF2000, then were treated with TGF-β 1. Relative luciferase activity was assayed respectively.Results In CCL4-treated mice, the liver surface was micronodular, and the liver showed the histologically characteristic features of cirrhosis. In present research a stable method for the culture of primary hepatocytes was established Internucleosomal fragmented DNA ladder, a characteristic feature seen during apoptosis, was observed using a 1.5% agarose gel in TGF-β1 treated normal hepatocytes. In comparison, no obvious DNA ladder from cirrhotic hepatocytes was observed. The percentage of apoptotic rate in TGF-β 1 treated normal hepatocytes stained with Hoechst 33342 were much higher than hepatocytes untreated with TGF-β1. There was significant difference between them And apoptosis could be blocked by treatment of caspase-3 and caspase-8 inhibitors. Among three cell lines studied with TUNEL assay, addition of TGF-β 1 induced apoptosis only in HepG2 cells (wild type p53). In contrast, Huh-7 (mutant p53) and Hep3B (deleted p53) cell lines lacked apoptosis. The detection of luciferase activity indicated that HepG2 cells dramatically increased the response to TGF-β 1 induction, but Huh-7 and Hep3B cell lines significantly lowered luciferase expression. It was indicated that there is positive correlation between the rate of apoptosis and Smads as well as status of p53 in hepatic carcinoma cell lines.Conclusion It was revealed that cirrhotic hepatocytes showed poor response to apoptosis induced by TGF-β 1, indicating t...