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应用DNA电泳转移、生物素探针Southern杂交及不同探针重复杂交等3项新技术研究肝炎肝癌的肝内HBV DNA分子状态。经过方法学比较以电泳印迹取代毛细渗吸法对65例肝炎肝癌病人的101份肝内组织DNA进行尼龙膜快速转移和HBV DNA杂交,结果检出HBV DNA分子状态里整合型15份、游离型31份、整合型合并游离型30份。其中12例乙型肝炎标本为经生物素探针杂交检测,结果全部检出游离型HBV DNA。还有3例乙型肝炎标本和2例其它肝炎标本为经重复杂交检测,即先用~(32)P探针杂交继去探针再用生物素探针杂交,结果乙型肝炎标本两次杂交HBV DNA都阳性,其它肝炎标本两次均阴性。阳性者表现为3.2kb和2.3kb电泳位置呈现杂交带,代表HBV之松弛环状和超螺旋状结构。由于两种探针各含不同序列,故结果可对待检基因进行初步序列分析。本实验表明,上述3项技术对临床医学研究有广泛的应用价值。
Three new technologies, DNA electrophoresis transfer, biotin probe Southern hybridization and repeated probe hybridization, were used to study intrahepatic HBV DNA status in hepatitis liver cancer. After methodological comparison, capillary electrophoresis was used instead of capillary aspiration method to detect the rapid DNA transfer and HBV DNA hybridization of 101 pieces of intrahepatic tissue DNA from 65 patients with hepatocellular carcinoma. The results showed that 15 molecules of HBV DNA were integrated, 31 copies, integrated merger free type 30 copies. Among them, 12 cases of hepatitis B were detected by biotin probe hybridization, and all the free HBV DNA was detected. There are 3 cases of hepatitis B and 2 cases of other hepatitis specimens were detected by repeated hybridization, that is, first with ~ (32) P probe hybridization probe followed by biotin probe hybridization, the results of hepatitis B specimens twice Hybrid HBV DNA were positive, and other hepatitis specimens were negative twice. The positive samples showed hybridization bands at 3.2kb and 2.3kb electrophoresis sites, representing the relaxed and supercoiled structures of HBV. Since both probes contain different sequences, the result can be a preliminary sequence analysis of the gene to be tested. The experiment shows that the above three technologies have a wide range of clinical application of value.