摘要
建立优化的差示PCR体系,通过对转移潜能不同的人成骨肉瘤细胞系进行差示比较,获得人成骨肉瘤转移相关基因片段.方法:以低转移性人成骨肉瘤细胞系SOSP-96O7及由此筛选出的高转移亚系SOSP-M为研究对象,利用优化的差示PCR技术获得两细胞系mRNA的表达差异,选取差异明显、片段较长的条带回收并克隆后,经点杂交和Northern杂交排除假阳性,并进行测序和同源性分析.结果:获得满意的差示PCR反应参数,并以高敏感度荧光染料SYBR””GREENI替代同位素放射自显影,改良了显示系统.以此为基础,得到了两条高转移亚系所特有的差异DNA片段,命名为MGI和MGZ,测序后分析显示它们具有较长的开放读框,与Genebank和EMBL数据库已知基因比较无明显同源性.结论:优化了差示PCR反应体系;分离筛选出2个人成骨肉瘤转移基因片段,已向Genebank申请登录.本实验为骨肉瘤转移的机制研究奠定了实验基础.
AIM: To optimize conditions of differential displayPCR and isolate the partially metastatic gene by comparisonof two human osteosarcoma cell lines with different ability ofmetastasis. METHODS: A low metastatic human osteosarco-ma cell line SOSP-96O7 and its highly metastatic subpopula-tion SOSP-M were chosen as material, and differentia1 ex-pression of mRNA was analyzed by optimized DD-PCR. ThecDNA bands, which were long and different unambiguously,were recovered and cloned. They were sequenced and ana-lyzed following the elimination of false positive clone by dotblot and Northern blot. RESULTS: Appropriate condition ofDD-PCR was obtained and displaying system was optimizedby flurochrome dying instead of isotope. Two special frag-ments, MGl and MG2 from highly metastatic cell line, wereobtained, which had long ORF and low autoploidy withknown genes collected in Genebank and EMBL database.CONCLUSION: Reliable optimized conditions of differentialdisplay PCR has been established. Two fragments were iso-lated from highly metastatic SOSP-M cells.
出处
《第四军医大学学报》
1999年第12期1038-1041,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金!39330200