含CD44 cDNA片段细胞系的构建及在肝癌中的应用

• 作 者: 肖承志 戴益民 余宏宇 王建军 倪灿荣 徐冠南 许炳华
• 机 构: 第二军医大学,长征医院普通外科,江苏省启东市肝癌防治研究所病理室
• 出 处: 华人消化杂志
• 发表日期: 1998年02期
• 关键词: 细胞系 构建 肝癌

目的构建含ＣＤ４４ｃＤＮＡ质粒ＰＧＥＸ２Ｔ和ＰＡＺ的单克隆细胞系．并利用此细胞系合成特异的ＣＤ４４ｃＤＮＡ探针，原位检测肝癌中ＣＤ４４ｍＲＮＡ的表达．方法应用常规的质粒转化方法将质粒ＰＧＥＸ２Ｔ和ＰＡＺ转入ＤＨ５菌株中得到单克隆细胞系．并以从此细胞系中提取的质粒为模板，应用ＰＣＲ法合成ＣＤ４４ｃＤＮＡ探针．应用原位杂交法检测肝细胞癌中ＣＤ４４ｖ６ｍＲＮＡ的表达．结果从ＤＨ５１菌株中提取的质粒含ＣＤ４４（２ｖ－１０ｖ）ｃＤＮＡ（１１ｋｂ），从ＤＨ５２菌株中提取的质粒含全长的ＣＤ４４ｃＤＮＡ（１８ｋｂ），ＰＣＲ合成的ＣＤ４４ｖ６ｃＤＮＡ探针长１４０ｂｐ．ＣＤ４４ｖ６ｍＲＮＡ的阳性检出率：转移高危组为８００％（８／１０），转移低危组为２１７％（５／２３），两者相差非常显著（Ｐ＜００１）．结论从单克隆细胞系ＤＨ５１和ＤＨ５２中提取的质粒，经ＰＣＲ扩增可得到特异的ＣＤ４４ｃＤＮＡ探针．肝细胞癌中ＣＤ４４ｖ６ｍＲＮＡ的表达与肿瘤的转移倾向呈正相关．

Objective To construct a monoclonal cell line containing CD44cDNA PGEX-2T and PAZ. And use this cell line to synthesize specific CD44 cDNA probes to detect the expression of CD44 mRNA in liver cancer. Methods The plasmids PGEX 2T and PAZ were transformed into DH5 strain by conventional plasmid transformation method to obtain monoclonal cell lines. And using the plasmid extracted from this cell line as a template, the CD44 cDNA probe was synthesized by PCR. The expression of CD44v6 mRNA in hepatocellular carcinoma was detected by in situ hybridization. Results Plasmids extracted from DH51 strain contained CD44 (2v-10v) cDNA (11kb), plasmids extracted from DH52 strain contained full-length CD44 cDNA (18kb), and CD44v6 cDNA probes were synthesized by PCR. 140bp long. The positive detection rate of CD44v6 mRNA was 80.0% (8/10) in the high-risk group and 21.7% (5/23) in the low-risk group. The difference between the two groups was very significant (P<001). Conclusion The specific CD44 cDNA probe can be obtained by PCR amplification from the plasmids extracted from the monoclonal cell lines DH51 and DH52. The expression of CD44v6 mRNA in hepatocellular carcinoma was positively correlated with the metastasis tendency of the tumor.