| Part I Expression of HIF-1 and VEGF in hepatocellular carcinomaObjective : Hypoxia-inducible factor 1 (HIF-1) is an important transcription factorin mammals, which can regulate all kinds of target genes such as vascular endothelial growth factor (VEGF) in the hypoxia condition. HIF-1 is a key factor in the hypoxia response of tumor. HIF-1 is made up of two subunits: a and 3. HIF-1 a is special regulative subunit : hypoxia can increase the level of HIF-1 a protein, then HIF-1 a subunits bind the hypoxia responsive elements (HREs) of target genes with the 3 subunits and co-active factors (such as CBP> P300), enhances the mRNA level of target genes . The amino acids 401-603 of fflF-1 a protein is the oxygen-dependent degradation domain (ODD). HIF-1 a protein are polyubiquitinated by composing the ubiquitin-ligase complex through binding the pVHL, then the HIF-1 a subunits are degraded rapid by protease (<5min). The low 02 concentration can inhibit the degradation of HIF-1 a , enhance the HIF-1 a protein. VEGF is one of the important target genes of HIF-1. Hypoxia can increase the level and stability of VEGF mRNA. HIF-1 can target the HRE (5'-TACGTGGC-3') to enhance the transcribe of VEGF gene. In addition, the RNA binding protein can bind the A, U richly sequence in 3' end of VEGF mRNA, to stop the degradation of mRNA. In thisstudy, the expression of HIF-1 a and VEGF in HCC were evaluated, and may be promote the therapeutic genie targets of HCC.Methods1. HepGa cells were cultivated in hypoxia (37 , 5%CO2, 1%O2) for 3h, 6h, 12h, 24h, 48h.2. RT-PCR was used to measure the mRNA levels of HIF-1 a and VEGF in HepG2 cells.3. Western-Blot was used to evaluate the protein levels of HIF-1 a and VEGF in HepG2 cells.Results1. The mRNA levels of HIF-1 a had no significant change when the HepG2 cells were hypoxia for different hours(p>0.05). But the mRNA levels of VEGF gene increased distinctly with the extension of hypoxia(p<0.05).2. The protein levels of HIF-1 a and VEGF both significantly increased, and the levels were more higher when the hypoxia extended. HIF-1 a protein almost had no expression in the cells which did not be cultivated in hypoxia.ConclusionsThe HIF-1 and VEGF play important roles in hypoxia response of HCC. Hypoxia can increase the HIF-1 a protein which form the active HIF-1 enhance the expression of VEGF.Part II An tip rolife native potential of EGCG in hepatocellular carcinomaObjective: The important component Epirigallocatechin-3-gallate (EGCG) ofgreen tea has the pharmic effect including antioxidation , clearing freedom units , antivirus, antitumorigenesis . EGCG can induce all kinds of malignant cells to start apoptosis such as lung cancer, stomach cancer , colon cancer; EGCG can stop the malignant cells proliferation through block the cellular growth cycle; it also can inhibit the activation of tyrokinase; Urokinase is the key enzyme in the tumorigenesis which take part in the invading course of tumor, EGCG can inhibitit it's active by binding the urokinase. On the other hand, EGCG can stop the tumorigenesis by inhibiting the expression of some antitumorigenesic genes by regulating the ODC, SOD; stopping the tumor-inducible ability of nitrite, aflatoxin ;building up the immuno-ability. In this study, we discuss EGCG can or not inhibit the proliferation of HCC, and the effect of EGCG in hypoxia response.Methods1. Cytomophology observation2. PI staining to gain the HepG2 flow cells/adhibit cells3. HepGa cells growth assay under EGCG congditon by MTT4. RT-PCR and Western-Blot evaluate the mRNA and protein levels of HIF-1 a and VEGF in HepGi cells, under hypoxia and/or EGCG.5. Observeing the planting HCC growth in null mouse, RT-PCR and Western-Blot evaluate the mRNA and protein levels of HIF-1 a and VEGF in HCC under therapy by EGCG.Results1. EGCG can inhibit the proliferation of HCC cells, the effect depend on the dosage and time of the thera... |
PDF Full Text Request