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

我们在超声引导下对大约150例小肝细胞癌进行了细针活检，并将标本保存在液氮中。为了提取RNA，每个标本都保留了一部分同源基因。我们还从每个病例中获得了非癌肝组织作为对照。我们希望通过比较同一患者的癌组织和非癌组织的核酸序列，可以了解癌变过程中是否发生了核酸序列的变化。因此，保存的样品用蛋白酶K处理，用酚-铬仿法提取DNA，然后用在AFP基因启动子和增强子上设置的引物进行PCR扩增。然后我们尝试用测序仪测定每个样本的核酸序列，但还没有得到可重复性的结果。我们还用Doss匀浆机将保留的样本匀浆，并对匀浆进行离心，沉淀出核酸片段。然后，我们将沉淀在缓冲液中再次匀浆，在缓冲液中透析，去除不溶性物质，得到核酸摘要。然后我们试图检查转录因子的存在和数量差异，但我们还没有发现一定的趋势。我们将继续实验，直到得到稳定的结果。

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.