血清中胎盘来源外泌体的分离与鉴定

发布时间：2018-03-29 01:14

本文选题：外泌体　切入点：胎盘　出处：《医学研究生学报》2015年06期

【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。
[Abstract]:Objective during pregnancy, placenta can release exocrine to maternal circulation and play an important role in normal pregnancy or placental associated pregnancy complications. Methods sucrose gradient centrifugation combined with 8%PEG6000 precipitation was used for the study of pregnancy diseases. Western blot was used to detect the placental exocrine marker protein molecule (CD63nCD81) in normal maternal serum. All protein bands in each density layer were analyzed by silver staining, and the morphology of exocrine from placenta was identified by transmission electron microscopy (TEM). The particle size and distribution of exocrine were measured by dynamic light scattering. Results the layers obtained by sucrose density gradient centrifugation were electrophoretic with protein SDS-PAGE gel. After silver staining, the protein molecular bands were clearly observed. Western blot showed that CD63, CD81 and PLAP were simultaneously expressed in the density layer of 34% sucrose. The results showed that the exocrine of the placenta mainly existed in the sucrose density layer of 1.09~1.16g/m L. the morphology of the exocrine from the placenta identified by transmission electron microscope was consistent with the general exocrine characteristics. The diameter of placental exocrine was 2891 nm, and the size was uniform. Conclusion A simple, fast and efficient method for isolation of exocrine from placenta was established. To study the role of placental exocrine in normal pregnancy and placental related complications.
【作者单位】：东南大学医学院;南京大学医学院附属鼓楼医院妇产科;南京医科大学鼓楼临床医学院妇产科;
【基金】：国家自然科学基金(81370724) 江苏省科技发展计划项目(BL2012015)
【分类号】：R714.2

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