子宫内膜异位症患者与健康女性的经血源间充质干细胞自噬功能比较研究

Comparative study of autophagy in menstrual blood-derived mesenchymal stem cells from patients with endometriosis and healthy women

目的比较子宫内膜异位症(EMT)患者与健康女性的经血源间充质干细胞(MenSCs)的自噬功能。方法分别从EMT患者及健康女性的月经血中提取EMT MenSCs(E-MenSCs)和正常MenSCs(H-MenSCs),并借助成脂、成骨诱导分化鉴定其干细胞属性。通过CCK-8法比较E-MenSCs和H-MenSCs在12、24、48、72、96、120 h时刻于波长450 nm的吸光度(A)值,并绘制细胞增殖曲线,比较2组细胞的细胞活力;运用透射电镜观察E-MenSCs与H-MenSCs的细胞形态,检测比较其中自噬体和自噬溶酶体的数量;采用Western blotting法检测E-MenSCs和H-MenSCs中自噬标志物微管相关蛋白轻链3-II(LC3-II)和Beclin1的蛋白表达。结果E-MenSCs和H-MenSCs均为长梭形,呈辐射状扩散,且经成脂、成骨诱导分化后均有脂滴、钙结节形成。与H-MenSCs比较,E-MenSCs在72、96、120 h的细胞活力均显著升高(P<0.01、0.001),自噬体和自噬溶酶体数量显著减少(P<0.05、0.01),且LC3-Ⅱ、Beclin1蛋白表达量也显著降低(P<0.05)。结论与H-MenSCs比较,E-MenSCs的自噬功能减弱,这可能是EMT发病进展的潜在机制。

Objective To compare the autophagy of menstrual blood-derived mesenchymal stem cells(MenSCs)in patients with endometriosis(EMT)and healthy women.Methods EMT MenSCs(E-MenSCs)and healthy MenSCs(H-MenSCs)were isolated from the menstrual blood of patients with endometriosis and healthy volunteers,respectively.Then their stem cell properties were identified using adipogenic and osteogenic differentiation.The absorbance(A)value at 450 nm of E-MenSCs and H-MenSCs at 12,24,48,72,96,and 120 h time points were obtained using CCK-8 assay,the cell proliferation curves were plotted,and the cell viability of E-MenSCs and H-MenSCs were compared.The cell morphology of E-MenSCs and H-MenSCs was observed by transmission electron microscope,and the number of autophagosome and autophagic lysosome was detected and compared.The expression of autophagy marker proteins(LC3-II and Beclin1)in E-MenSCs and H-MenSCs were detected by Western blotting.Results E-MenSCs and H-MenSCs,long spindle-shaped,diffused radially.After adipogenic and osteogenic differentiation,the lipid droplets and calcium nodules in E-MenSCs and H-MenSCs were observed.In contrast to H-MenSCs,E-MenSCs showed increased cell viability at 72,96,and 120 h(P<0.01 and 0.001),decreased expressions of autophagosomes and autolysosomes(P<0.05 and 0.01),and reduced LC3-II and Beclin1 proteins(P<0.05).Conclusion E-MenSCs autophagy is weakened compared with H-MenSCs,which might be the underlying mechanism of EMT pathogenesis and progression.