Change of AFP gene promotion control mechanism with the development of hepatocellular carcinoma : analysis through many clinical cases

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We have performed ultrasound-guided percutaneous fine needle biopsy on approximately 150 cases of small hepatocellular carcinoma and have reserved the specimens in liquid nitrogen. A part of each specimen has been reserved with isogen in order to abstract RNA.We have also obtained samples of non-cancerous liver tissue as the control from each cases.We expect that comparison of nucleic acid sequence between the cancerous tissue and the non-cancerous tissue of the same patient will make it clear whether the carcinogenesis has produced any change in the nucleic acid sequence. Thus, the reserved samples were handled with protenase K,DNA was abstracted by the phenol-chroloform method, and the obtained DNA was amplified by PCR using primers set in the promoter and enhancer of AFP gene. Then we tried to determine the nucleic acid sequence of each sample by a sequencer, but we have not had reproducible results yet.We also homogenated the reserved sample by Dounce homogenizer, and centrifuged the homogenate and precipitated the nucleic fragment. Then we homogenated the precipitate in a buffer again, dialyzed it in the buffer, removed unsolvable materials, and obtained the nucleic abstract. Then we tried to examine the presence and quantity difference of transcriptional factors, but we have not found a certain trend yet.We will continue the experiment until we obtain stable results.