小鼠囊胚胞外囊泡对子宫内膜上皮细胞E-cadherin和vimentin表达的影响

Mouse blastocyst-derived extracellular vesicles regulate expression of Ecadherin and vimentin in endometrial epithelial cells

目的:探讨小鼠囊胚细胞外囊泡(EVs)对小鼠子宫内膜上皮细胞(mEECs)上皮钙黏素(E-cadherin)和波形蛋白(vimentin)表达的影响。方法:以孕4.5 d的BABL/c小鼠囊胚为材料,利用超高速离心分离纯化小鼠囊胚EVs,粒度仪分析粒径,透射电子显微镜观察形态,Western blot分析Tsg101和CD63的表达;酶消化结合差速贴壁筛选法制备原代mEECs,流式细胞术分析细胞角蛋白8(CK8)的阳性率鉴定mEECs纯度。建立囊胚EVs与mEECs共培养模型,共聚焦显微镜观察PKH26标记的囊胚EVs进入mEECs的形态过程,Western blot和细胞免疫化学法分析共培养中mEECs中E-cadherin和vimentin表达的变化。结果:(1)小鼠囊胚EVs平均粒径(226.6±3.8) nm,表现囊泡超微形态,表达Tsg101和CD63蛋白标志物。(2)原代mEECs形态均匀,CK8阳性细胞率80%以上。(3)与0 h相比,mEECs与囊胚EVs共培养48和96 h时E-cadherin表达水平显著降低(P<0.01),vimentin表达水平显著升高(P<0.01)。结论:小鼠囊胚EVs可下调E-cadherin表达,上调vimentin表达,诱导mEECs发生上皮-间充质转化。

AIM: To find out whether the extracellular vesicles(EVs) secreted by mouse blastocysts affect the expression of E-cadherin and vimentin in mouse endometrial epithelial cells(mEECs). METHODS: A female mouse uterus was utilized to gather mouse blastocysts on day 4. 5 of pregnancy. Transmission electron microscopy, Western blot, and nanoparticle tracking were applied to describe the EVs. Enzyme digestion and differential adhesion screening were applied to obtain primary mEECs. Flow cytometry was used to quantify the proportion of mEECs that were positive for cytokeratin 8(CK8). The morphological processes of EVs labeled by PKH26 into mEECs were observed by fluorescence confocal microscopy. Western blot and cellular immunofluorescence were utilized to evaluate the expression of E-cadherin and vimentin of mEECs in co-culture system. RESULTS: The mean particle size of EVs was(226. 6±3. 8) nm and characterized with Tsg101 and CD63 proteins. The primary mEECs labeled with CK8 were enriched, with a positive rate of more than 80%. After 48 and 96 h of co-culture with EVs, mEECs expressed significantly lower levels of E-cadherin and significantly higher levels of vimentin(P<0. 01). CONCLUSION: Mouse blastocyst EVs may induce epithelial-mesenchymal transition of mEECs by decreasing E-cadherin expression and increasing vimentin expression.