胚胎干细胞来源的微囊体分离提取及促造血作用的观察  

Microvesicle isolation from embryonic stem cell and its role in hematopoietic recovery

作　　者：何志旭[1] 李色 舒丽萍[1] 

机构地区：[1]贵阳医学院附属医院儿科,贵州贵阳550001 [2]贵阳市第一人民医院检验科,贵州贵阳550002

出　　处：《临床儿科杂志》2013年第5期465-469,共5页Journal of Clinical Pediatrics

基　　金：国家自然科学基金资助项目(No.30960412)

摘　　要：目的探讨分离提取及鉴定胚胎干细胞(ESC)产生的微囊体(MV)的实验方法,初步研究其促造血的生物学作用。方法利用超滤离心的方法从小鼠ESC培养上清液中分离提纯MV(ES-MV),以小鼠胚胎成纤维细胞(MEF)培养上清来源的MV(MEF-MV)为对照;采用透射电镜形态学观察,RT-PCR检测特异性基因表达的方法对ES-MV进行鉴定;将ES-MV用于环磷酰胺所致骨髓抑制小鼠,通过骨髓涂片初步观察其对造血的恢复作用。结果透射电镜下观察,ES-MV呈明显异质性的圆形或椭圆形小囊泡,直径约30 nm-1μm不等,有完整的包膜,内为低电子密度物质。RT-PCR方法从ES-MV中可检测到Oct-4、Wnt-3、Hoxb4基因的表达,而MEF-MV内不能检测到这些基因的表达;将ES-MV用于骨髓抑制小鼠后,骨髓涂片显示成熟红细胞和有核细胞比例明显升高。结论 ESC可以产生MV,并且是MV的丰富来源;超滤离心法是一种方便实用的提取ES-MV的方法;ES-MV对促进骨髓造血有一定的作用。Objectives To investigate the isolation and identification of microvesicle （MV） from embryonic stem cell （ESC）, and study its biological role in hematopoietic recovery. Methods Microvesicles were extracted from embryonic stern cell culture fluid by ultra-filtered centrifugation and identified by transmission electron microscopy （TEM） and RT-PCR with mouse embryonic fibroblast derived microvesicles （MEF-MV） as the control. The effect of hematopoietic recovery of embr- yonic stem cells derived microvesicles （ES-MV） was observed in bone marrow smear of myelosuppression mice. Results Under TEM, microvesicles were significantly heterogeneous round or oval vesicles with diameters of about 30nm - llim in size. The intact capsule of microvesicle contained materials with low electron density. The expression ofgene Oct-4, Wnt-3 and Hoxb4 was detected in ES-MV using RT-PCR, but was undetectable in MEF-MV. After treated with ES-MV, the bone marrow smear showed that the proportions of mature red blood cells and nucleated cells were significantly higher in myelosuppressed mice than those in the control. Conclusions ES-MV, which can be isolated from ESC using ultra-filtered centrifugation conve- niently, can promote hematopoiesis.

关 键 词：胚胎干细胞 微囊体 超滤离心 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]