| Hepatocellular carcinoma (HCC) is one of the most frequent visceral neoplasms worldwide, with an estimated 564,000 new cases and almost as many deaths in 2000. Metastasis is the most life threatening event in cancer patients. For hepatocellular carcinoma (HCC), the 4th leading cause of cancer death worldwide and the second in China, surgery remains the treatment of choice, but the 5 year recurrence rate after section is as high as 40%-70% . Metastatic recurrence is the main obstacle to the improvement of treatment efficacy. Pathologic factors indicative of tumor invasiveness such as portal vein invasion, intrahepatic metastasis, presence of satellite nodules, large tumor size, and advanced TNM stage, are the best established risk factors for recurrence .However, the molecular events promoting invasiveness of HCC cells are still hardly known and routine biomarkers for HCC prognosis are not yet available.Proteomics is the characterization (structure and function) of the complete profile of proteins expressed in a given tissue, cell or biological system. Among the currently available techniques, proteomics by 2DE-MS permits the analysis of thousands of modified or unmodified proteins simultaneously, and becomes increasingly popular for identifying biomarkers for early detection, classification and prognosis of tumors, as well as pinpointing targets for improved treatment outcomes. However, a problem with the traditional 2-D gel approach has been the limited reproducibility, which makes it difficult to compare protein spots between large numbers of different complex cell samples. A multidimensional chromatographic 2-D liquid-phase separation method has been developed for differential display of proteins from cell lysates and applied to a comparison of tumors proteomics. The method involves fractionation according to pi using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pi fraction using nonporous reversed phase HPLC. Compared to traditional fractionation techniques, advantages of this chromatographic approach include: high loading capacity without band distortion; improved detection of low-abundance species, membrane or hydrophobic proteins, and low molecular weight proteins; enhanced reproducibility; a contamination-free liquid flow path; simple automation; and liquidfractions.For a better insight into the mechanisms of HCC metastasis, a high metastatic human HCC cell line MHCC97, its clonal cells, MHCC97H and MHCC97L,with high and low metastatic potentials, and progressively more metastatic cells from lung metastatic lesions were established via repeated in vivo selection.These cells provide appropriate model systems with similar genetic background for the comparative study on the molecular events involved in HCC metastasis.In 1975 mouse mAbs were isolated in Nobel Prize winning work by Dr Cesar Milstein and Dr Georges Kohler. mAbs' arrival revolutionised biomedical research and sparked an international billion-pound biotechnology industry. In 1995 Dr. Katherine Knight made plasmacytoma in rabbits, then Dr. Robert Pytela and Mr. Weimin Zhu developed this RabMAb Technology. The advantages of RabMAb technology over mouse mAb technology are: High affinity and specificity; Novel epitope recognition; High success rate generating desirable MAbs; Broad applications; Recognition of human antigens with high homology to mouse; Higher throughput; Stable hybridomas; Multiplex; High humanization probability; Higher homology to human germline sequence.In this study we use two-dimensional chromatographic liquid-phase separation method to compare the difference of whole cell lysates of SMMC7721, MHCC97-L and MHCC97-H and identified the different proteins by MOLDI-TOF/MS. In the meantime we immunized rabbits with liver cancer tissues and conduct hybridoma cells to screen the rabbit mAbs and identified their antigens.OBJECTIVES:1. Identifying metastatic specific proteins of HCC to evaluate the possibility of them as new diagnostic m... |
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