妊娠高血压综合征胎盘组织差异表达基因的筛选与确定

Screening and confirmation of differentially expressed genes in placentas from PIH patients

目的 了解妊娠高血压综合征 (妊高征 )胎盘组织基因表达的改变 ,确定与妊高征发病相关的基因 ,为研究妊高征的发病机理提供线索。 方法 收集 2 0 0 1年 3月至 2 0 0 2年 9月西安地区以剖宫产终止妊娠的 4 2例妊高征患者及 2 2例正常产妇的产前、产后临床资料 ;收集胎盘组织 ,制作胎盘组织芯片 ;建立妊高征患者数据库。采用抑制消减杂交技术 (suppressionsubtractivehybridi za tion ,SSH)建立妊高征胎盘组织cDNA消减文库 ;利用差异筛选技术筛选出妊高征胎盘高表达基因 ,用RT PCR和免疫组织化学技术验证部分高表达基因。 结果 建立了由 4 2例中、重度妊高征患者和 2 2例正常产妇组成的数据库 ;用 6 6例胎盘组织制成 15 0点的胎盘组织芯片。利用SSH和差异筛选共分离出 10 3个阳性克隆 ,测序成功 90个。测序结果显示 90个cDNA片断与GenBank/EMBL中36条已知功能基因有 95 %以上的同源性。 结论 妊高征患者胎盘组织基因表达发生较大变化 ;有必要研究血管内皮生长因子受体、卵巢抑制素、丝氨酸蛋白酶抑制素、细胞角蛋白 7以了解妊高征的发病机理。

Objective To unmask clues for the investigation of PIH pathogenesis by detecting the altered gene expression profile of placentas from PIH patients. Methods Clinical data and placentas were collected from 42 PIH patients and 22 normotensive pregnancies in seclective cesarean section to construct PIH database from Mar. 2001 to Sep. 2002 in Xi’an district. Suppression subtractive hybridization (SSH) was performed to set the subtractive cDNA library and differential screening was used to identify the positive clones;insertion of positive clones were sequenced by T7 primer method. RT-PCR and immunohistochemistry were employed to confirm the putative gene inhibin A expression. Results A database was set up that consists of pre- and postpartum clinical data and placenta samples of 42 preeclamptic pregnancies and 22 normotensive ones From which,150 features of placental tissue microarray were made. One hundred and three positive clones were isolated by SSH and differential screening. Sequencing and BLAST analysis showed that 90 insertion shared more than 95% homology with sequences in the GenBank/EMBL database. We identified 36 putative genes including pregnancy-specific glycoproteins gene (BC005924),serine protease inhibitor gene (BC012868),VEGFR-1 gene (AF063657),cytokeratine 7 gene(CK7) (AF509887),etc. Inhibin beta A gene was highly expressed in placentas in PIH patients. Conclusion The gene expression profile in PIH placenta was greatly changed;it might be necessary to investigate the role of VEGFR1,serine protease inhibitor,inhibin and CK7 in the pathogenesis of PIH.