摘要
目的妊娠期间,胎盘可释放外泌体到母体循环,在正常妊娠或胎盘相关妊娠并发症中发挥重要作用。文中旨在建立简便、高效、低耗的母血清胎盘来源外泌体的分离纯化方法,为妊娠疾病研究奠定基础。方法蔗糖梯度离心联合2次8%PEG6000沉淀,分离纯化正常孕妇血清中胎盘来源外泌体,Western blot检测胎盘来源外泌体标记蛋白分子（CD63、CD81、PLAP）,银染法分析各密度层中所有蛋白质条带,透射电镜鉴定胎盘来源外泌体形态,动态光散射测定外泌体粒径及分布。结果通过蔗糖密度梯度离心后得到的各分层液经蛋白质SDS-PAGE胶电泳,银染后可见各蛋白分子条带清晰可见,Western blot结果显示CD63、CD81和PLAP同时表达于21%～34%蔗糖密度层,证明血清胎盘外泌体主要存在于1.09～1.16g/m L蔗糖密度层。透射电镜鉴定获得的胎盘来源外泌体形态符合一般外泌体特征,且特异表达PLAP。动态光散射测定获得的胎盘外泌体直径分布于28～91 nm,且大小均一。结论成功建立了一种简便、快捷、高效分离血清中胎盘来源外泌体的改良方法,为研究胎盘外泌体在正常妊娠及胎盘相关并发症中的作用奠定实验基础。
Objective During pregnancy,exosomes can be released from the placenta into maternal circulation and play important roles in normal pregnancy or placenta-related diseases. We aimed to establish a simple and efficient method for isolating and identifying placental exosomes from maternal serum and lay a foundation for the studies of pregnancy-related diseases. Methods Using sucrose gradient centrifugation with 8% PEG6000 precipitation twice,we isolated and purified placenta-derived exosomes from normal maternal serum and detected their molecular markers CD63,CD81 and PLAP by Western blot,followed by silver staining analysis of the protein profile of the exosome pellet. We identified the morphology of the placenta-derived exosomes by transmission electron microscopy（ TEM） and measured the size and distribution of the particles by dynamic light scattering（ DLS）. Results Silver staining of the protein profiles of the exosomes after sucrose gradient centrifugation clearly revealed the bands of the protein molecules.Western blot showed the expressions of CD63,CD81,and PLAP in the 21- 34% density layer,which demonstrated the presence of serum placental exosomes mainly in the 1. 09- 1. 16 g / m L density layer. TEM exhibited that the placenta-derived exosomes were round or oval cup-shaped,specifically expressing PLAP,and the particles were uniform in size,with a mean diameter of（ 41. 79 ± 11. 94）nm. Conclusion A simple,fast,and efficient method was successfully established for isolating placenta-derived exosomes from maternal serum,which provides a basis for studying the roles of placental exosomes in normal pregnancy and placenta-related diseases.
出处
《医学研究生学报》
CAS
北大核心
2015年第6期632-636,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金(81370724)
江苏省科技发展计划项目(BL2012015)
关键词
外泌体
胎盘
分离
胎盘碱性磷酸酶
Exosome
Placenta
Isolation
Placental alkaline phosphatase
