胚胎干细胞来源的微囊体分离提取及促造血作用的观察

发布时间：2018-06-17 13:12

本文选题：胚胎干细胞 + 微囊体　；参考：《临床儿科杂志》2013年05期

【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。
[Abstract]:Objective to investigate the method of isolation and identification of microthylated MV produced by embryonic stem cell ESCs and to study the biological effect of MV on hematopoiesis. Methods MVGE-MVV was isolated from the supernatant of mouse ESC culture by ultrafiltration centrifugation, and the MVN MEF-MV derived from the supernatant of mouse embryonic fibroblast cells (MEF) was used as control. The specific gene expression of ES-MV was identified by transmission electron microscopy (TEM) and RT-PCR. ES-MV was used in cyclophosphamide induced bone marrow suppression mice and its hematopoietic recovery was preliminarily observed by bone marrow smear. Results under transmission electron microscope, the oval or circular vesicles were observed to be heterogeneous, with a diameter of about 30 nm-1 渭 m and a complete capsule. The expression of Oct-4 Wnt-3Hoxb4 gene was detected from ES-MV by RT-PCR with a low electron density substance. The expression of these genes was not detected in MEF-MV, and the ratio of mature erythrocytes and nucleated cells was significantly increased in bone marrow smears of mice treated with ES-MV. Conclusion ESC can produce MV and is a rich source of MV, and ultrafiltration centrifugation is a convenient and practical method to extract ES-MV in bone marrow hematopoiesis.
【作者单位】：贵阳医学院附属医院儿科;贵阳市第一人民医院检验科;
【基金】：国家自然科学基金资助项目(No.30960412)
【分类号】：R329

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