【摘要】 目的探索人胚胎肺组织中是否存在一种新的血管内皮生长因子(VEGF)基因可变剪切形式。方法对1例4月龄引产胎儿肺组织的总RNA进行逆转录聚合酶链反应、克隆并测定扩增片段的序列。结果用一对引物(引物5’端-21~7 bp,3’端554~576 bp)可扩增出3条片段,一条为长度正常的VEGF16(5619 bp)全长序列,另一条为长度正常的VEGF12(1487 bp)全长序列,第三条为新的VEGF mRNA异常剪切片段。测序检测显示,此扩增片段长度为639 bp,同样为VEGF165全长核苷酸序列,但在VEGF165的第3和第4外显子之间插入了长度为20 bp的碱基片段。序列分析显示此插入序列为滞留的第Ⅲ内含子终末序列,含内含子剪切信号。上述移码突变导致VEGF基因阅读框架发生改变,在第4外显子中游出现终止密码,故仅含VEGF165外显子1~3及4部分序列,在蛋白水平上可能编码含97个氨基酸的新肽链。结论在一引产胎儿的肺组织中发现了新的VEGF mRNA可变剪切形式,其生物学意义有待进一步研究。更多还原

【Abstract】 Objective To characterize a new alternative splicing isoform of human vascular endothelial growth factor(VEGF) gene.Methods The total RNA was extracted from the lung tissue of a legally aborted 4-month-old fetus and amplified by RT-PCR.The amplified product was cloned into the plasmid pMD18-T and plasmid pcDNA3.1-for sequence analysis.Results Electrophoresis of the RT-PCR products displayed one short band for VEGF121(487 bp) and a long band.The latter was characterized to contain two fragments: one was normal VEGF165(619 bp),and the other(639 bp) had an identical nucleotide sequence to VEGF165 with a 20 bp fragment inserted between exons 3 and 4.Sequence analysis showed that this 20-bp nucleotide was inserted from the 3’ end of the third intron containing a splicing signal,thus causing shift mutation in the reading frame of VEGF gene and early appearance of the stop codon UAG in the middle of exon 4.Conclusion A new alternative splicing isoform of VEGF probably exists in the lung tissue of a legally aborted human fetus,and its biological significance remains to be further investigated.更多还原