CAJ | 학술논문

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1．09～1．16 g／mL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。
목적：임신기간，태반가석방외비체도모체순배，재정상임신혹태반상관임신병발증중발휘중요작용。문중지재건립간편、고효、저모적모혈청태반래원외비체적분리순화방법，위임신질병연구전정기출。방법자당제도리심연합2차8％PEG6000침정，분리순화정상잉부혈청중태반래원외비체，Western blot검측태반래원외비체표기단백분자（ CD63、CD81、PLAP），은염법분석각밀도층중소유단백질조대，투사전경감정태반래원외비체형태，동태광산사측정외비체립경급분포。결과통과자당밀도제도리심후득도적각분층액경단백질SDS-PAGE효전영，은염후가견각단백분자조대청석가견， Western blot결과현시CD63、CD81화PLAP동시표체우21％～34％자당밀도층，증명혈청태반외비체주요존재우1．09～1．16 g／mL자당밀도층。투사전경감정획득적태반래원외비체형태부합일반외비체특정，차특이표체PLAP。동태광산사측정획득적태반외비체직경분포우28～91 nm，차대소균일。결론성공건립료일충간편、쾌첩、고효분리혈청중태반래원외비체적개량방법，위연구태반외비체재정상임신급태반상관병발증중적작용전정실험기출。
Objective During pregnancy , exosomes can be released from the placenta into maternal circulation and play im-portant roles in normal pregnancy or placenta-related diseases .We aimed to establish a simple and efficient method for isolating and i-dentifying 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 CD 63 , CD81 and PLAP by Western blot , followed by silver staining anal-ysis 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 stai-ning 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 CD 63, 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/mL 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 ma-ternal serum, which provides a basis for studying the roles of placental exosomes in normal pregnancy and placenta -related diseases.