良性与恶性葡萄胎基因表达差异的研究

Study of gene differential display of hydatidiform moles

目的:通过基因表达的差异分析,筛选良性与恶性葡萄胎间的分子标志,以探讨侵袭性葡萄胎早期诊断的方法和可能性。方法:采用mRNA差异显示、斑点杂交和DNA重组、测序等技术对良性与恶性葡萄胎差异表达的基因片段进行筛选和序列测定,通过和基因库数据对比,明确所获基因片段是否为已知序列。结果:筛选出 28条侵袭性葡萄胎高表达的基因片段,其中 20个可再扩增,斑点杂交分析确认有 13个片段在侵袭性葡萄胎中高表达,克隆其中 3个片段,经序列测定和NCBI数据库的BLAST检索比对,发现 16号片段序列(IHM F16)未见记载,为新发现的表达基因片段,并已被NCBI数据库所收录(dbEST_Id: 10875704;GenBank_Accn:BM403211),其他两个片段为已知的序列。结论:良性与恶性葡萄胎间基因表达存在明显差异,差异基因的检测分析对侵袭性葡萄胎的早期诊断具有潜在意义。

Objective: To screen the specific molecular maker of invasive hydatidiform moles (HM) by differential display analysis. Methods: For dot hybridization, about 1.0 μg of each cDNA sample of invasive and non-invasive HM were labeled as probes using the Dig DNA labeling and Detection Kit (Boehringer Mannheim). The specific expression fragments of invasive HM recovered from PAGE gel were re-amplified by PCR, and the PCR products were dotted onto nylon membrane and hybridized by two probes of invasive and non-invasive HM cDNA respectively. Some fragments with a strong positive hybridization signal were cloned into the polylinker of lasmid PUC19 and were sequenced. The fragments’ sequences were searched for homology in the NCBI data using the BLAST (Database: GenBank Human EST entries;Posted date:Aug 31, 2004;Number of letters in database: 1 697 659 032;Number of sequences in database: 3 677 722). Results: The 20 fragments in 28 bands with specific expression in invasive HM were re-amplified, of which 13 showed positive hybridization signals, and 3 were cloned into the polylinker of lasmid PUC19. A fragment in the 3 was a new expressed sequence tag (EST) and the sequence was submitted to NCBI data (dbEST_Id:10875704;GenBank_Accn:BM403211). Conclusion: There are more differences in gene expression between invasive and non-invasive HMs, and differential display analyses are of a potential significance to early diagnosis of invasive HM.