| Liver cancer takes many gene expression of the cells change to a series of elements of change. so from the liver cancer research on genetic level of oncogenes, the change of cancer suppresergenes, looking for differences of genes can not only for the pathological cause of the same time, they can to early diagnosis and treatment and is on offer.Ebpl for the epidermal growth factor receptor ErbB-3 intracellular juxtamembrane domain-binding protein, is a proliferation-related PA2G4 a member of the family, its important domains, including PKC phosphorylation sites. With ErbB-3/ErbB-4 ligand HRG treatment of breast cancer cells show Ebpl phosphorylation, and ErbB-3 from the translocation from the cytoplasm to the nucleus, this ectopic expression inhibited the ErbB-2/ErbB-3 positive expression of breast cancer, prostate cancer cell growth and induction of cell differentiation. In breast cancer cells in vivo and in vitro Ebpl binding Rb protein, inhibit cell cycle regulatory genes and endogenous genes such as E2F1, c-myc transcription,That Ebpl the ability to inhibit cell proliferation and its regulation of cell cycle-related genes. As the green fluorescent protein (GFP) can be expressed in eukaryotic cells, green fluorescence, can play a role in tracing. Therefore, we use of the northern blot methods to obserbation the genes of ebpl mRNA expression in liver cancer cell lines. a pEGFPCl quality of the carrier's pEGFPCl-Ebpl really express the quality of the dyed, and transfection to the HEK293 cells analyses the proliferation cells grow for ebpl over express.In this study, First, observation of ebpl genetic mRNA expression for northern blot analysis; the use of Ebpl gene was amplified by PCR and was constructed by gene recombination technology Ebpl gene and enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) gene fusion expression vector pEGFPCl-Ebpl. Further use in the lipofection method, the pEGFPCl-Ebpl gene was transfected into HEK293 cells, and observed under a fluorescence microscope, GFP expression of the transfected HEK293 cells were treated with G418 (expression vector with a neo gene fragment, the G418 has a resistance) Screening positive clones were prepared by stable expression of Ebpl in cell lines and analysis of clone colony formation,Pairs of positive expression clones by Western blot identification of Ebpl protein overexpression. The results are as follows:Northern blot show it with NIH3T3 and HEK293 cells, compared Hep3B,HepG2 and Huh-7 cells the Ebpl mRNA overexpressed; was successfully constructed Ebpl gene and enhanced green fluorescent protein gene fusion expression vector pEGFPCl-Ebpl, and can be seen under fluorescence microscope were transfected HEK293 cells emit green fluorescence; Western blot results of stable expression cell lines identified overexpression of Ebpl protein; clone colony-forming The experimental results, the target gene transfected cell clones than the number of cells transfected with empty vector.The above results suggest that:1. The gene Ebpl mRNA overexpression in hepatoma cells.2. This study constructed Ebpl gene and enhanced green fluorescent protein gene fusion expression vector pEGFPCl-Ebpl, the carrier can be used not only the fluorescent protein as a reporter gene can be used as a transient expression markers used in a qualitative, but also can serve as a stable expression of marker material, the Ebpl gene function studies laid the foundation.3. Ebpl over-expression increased colony-forming HEK293 cell clone. |
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