小鼠脂肪间质干细胞源胞外囊泡激活蛋白激酶B促进卵巢癌细胞增殖迁移和上皮间质转化

ADSC-EVs activate protein kinase B to promote the proliferation and migration and epithelial-mesenchymal transition of ovarian cancer cells

目的:探讨小鼠脂肪间质干细胞(adipose-derived mesenchymal stem cells,ADSCs)源胞外囊泡(extracellular vesicles,EVs)对卵巢癌ID8细胞株生物功能的影响及其可能机制。方法:采用酶消化法分离获得ADSCs,利用其形态特征、成骨成脂多向分化能力和流式细胞术表面标志检测对ADSCs进行鉴定;采用超速离心法从ADSCs上清液中分离和纯化EVs(ADSC-EVs),并通过透射电子显微镜、纳米粒径分析和蛋白质印迹实验进行鉴定;以PBS作为对照组,实验组采用不同浓度ADSC-EVs(20μg/mL和40μg/mL)分别预处理ID8细胞,通过平板克隆实验检测ADSC-EVs对ID8细胞增殖能力的影响;划痕实验和Transwell实验检测ADSC-EVs对ID8细胞迁移能力的影响;蛋白质印迹实验检测ID8细胞上皮间质转化(epithelial-mesenchymal transition,EMT)和蛋白激酶B(即AKT)相关蛋白的表达。结果:分离的ADSCs具有典型的干细胞形态,能够向成骨成脂分化,并符合干细胞表面一般特征;ADSC-EVs表达CD9和CD81蛋白;与PBS组相比,ADSC-EVs组ID8细胞增殖和迁移能力明显增强(P均<0.05),且40μg/mL ADSC-EVs组细胞增殖和迁移能力较20μg/mL ADSC-EVs组明显增强(P均<0.05);随着ADSC-EVs浓度增加,ID8细胞E-钙黏蛋白表达明显降低,N-钙黏蛋白、波形蛋白和p-AKT表达明显增加(P均<0.05)。结论:ADSC-EVs可能通过激活AKT信号的磷酸化促进ID8细胞EMT,进而增强其增殖和迁移能力。

Objective:To explore the effect of extracellular vesicles(EVs)derived from mouse adipose-derived mesenchymal stem cells(ADSCs)on the biological behaviors of ovarian cancer ID8 cell line and its possible mechanism.Methods:ADSCs were isolated by enzyme digestion method,and identified by morphological characteristics,osteogenic and adipogenic multidirectional differentiation ability and flow cytometry surface marker detection;EVs were isolated and purified from supernatant of ADSCs by ultracentrifugation,and ADSC-EVs were identified by transmission electron microscopy,nanoparticle size analysis and Western blotting;ID8 cells were treated with ADSC-EVs at different concentrations(20μg/mL and 40μg/mL)and PBS as the control group.The effect of ADSC-EVs on the proliferation of ID8 cells was detected by plate cloning assay.Wound healing and Transwell test were used to detect the effect of ADSC-EVs on the migration ability of ID8 cells.The expression of epithelial-mesenchymal transition(EMT)and protein kinase B(AKT)related proteins in ID8 cells were detected by Western blotting.Results:The isolated ADSCs had typical stem cell morphology,could differentiate into osteogenesis and adipogenesis,and consistent with the general characteristics of the stem cell surface.ADSC-EVs expressed CD9 and CD81 proteins.Compared with the PBS group,the proliferation and migration of ID8 cells in ADSC-EVs group were significantly enhanced(P<0.05),and the proliferation and migration of ID8 cells in 40μg/mL ADSC-EVS group was stronger than that in 20μg/mL ADSC-EVS group(P<0.05).With the increase of ADSC-EVS concentration,the expression of E-cadherin in ID8 cells was significantly decreased,while the expression of N-cadherin,vimentin and p-Akt was greatly increased(P<0.05).Conclusion:ADSC-EVs may promote the EMT of ID8 cells by activating the phosphorylation of AKT signal,thereby enhancing their proliferation and migration ability.