改良差示PCR技术分离人成骨肉瘤转移基因片段

Isolation of partial metastatic gene in human osteosarcoma by optimized differential display PCR

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.