脂肪间充质干细胞经超声刺激后分泌的细胞外囊泡对脂肪间充质干细胞生物学特性的影响

The effect of extracellular vesicles secreted by adipose-derived mesenchymal stem cells after being stimulated by ultrasound on the biological properties of adipose-derived mesenchymal stem cells

目的通过超声刺激脂肪间充质干细胞后,收集其分泌的细胞外囊泡,与正常脂肪间充质干细胞分泌的细胞外囊泡(Extracellular vesicles,EVs)进行比较,探究经超声刺激后分泌的细胞外囊泡对脂肪间充质干细胞生物学特性(包括细胞增殖、细胞迁移和成脂分化能力)的影响。方法首先,通过低强度超声处理部分脂肪间充质干细胞,收集细胞培养上清并采用超滤法提取细胞外囊泡(U-EVs),部分间充质干细胞正常培养,并收集其细胞外囊泡(N-EVs)。在不同浓度细胞外囊泡处理下对脂肪间充质干细胞进行CCK-8实验检测细胞活力,选取提升细胞活力的最佳浓度,在这一浓度下采用划痕实验检测细胞迁移,EdU法检测细胞增殖,并使用成脂诱导培养基诱导并比较脂肪间充质干细胞成脂分化能力。结果超声刺激后产生的细胞外囊泡符合国际外囊泡协会的鉴定标准,对脂肪间充质干细胞作用的最适浓度约为150μg/mL。在这一浓度下,U-EVs的细胞增殖率(19.2%±1.8%)显著高于N-EVs(11.9%±3.4%),并且U-EVs组的油红O染色面积是对照组的(2.78±0.23)倍,显著高于N-EVs组的(2.11±0.18)倍,但N-EVs组(16.3%±2.0%)和U-EVs组(21.5%±2.3%)的细胞迁移率没有显著差异。结论经超声刺激的脂肪间充质干细胞分泌的细胞外囊泡显著促进脂肪间充质干细胞的活力,提高细胞增殖、迁移和成脂分化的能力。

Objective To collect extracellular vesicles(EVs)secreted by adipose mesenchymal stem cells(ADSCs)after ultrasound stimulation,compare with EVs secreted by normal ADSCs,and to explore the effects of EVs secreted after ultrasound stimulation on the biological characteristics of ADSCs,including cell proliferation,migration,and adipogenic differentiation abilities.Methods Firstly,some ADSCs were treated with low-intensity ultrasound,cell culture supernatant was collected and extracellular vesicles(U-EVs)were extracted by ultrafiltration method.Some ADSCs were cultured normally and their extracellular vesicles(N-EVs)were also collected.The experiments were performed in the following three groups:PBS(blank control group),EVs secreted by untreated cells(N-EVs group),and EVs secreted by ultrasoundtreated cells(U-EVs group).The CCK-8 assay was used to assess the viability of ADSCs under treatment with EVs at different concentrations.The concentration that most strongly enhanced cell viability was selected.At this concentration,cell migration was detected by scratch test,cell proliferation was detected by EdU method,and adipogenic induction medium was used to induce and compare adipogenic differentiation ability of ADSCs.Results EVs secreted after ultrasound stimulation met the identification standards,and the optimal concentration for affecting ADSCs was about 150μg/mL.At this concentration,the cell proliferation rate of the U-EVs group(19.2%±1.8%)was significantly higher than that of the N-EVs group(11.9%±3.4%),and the Oil Red O staining area of the U-EVs group was(2.78±0.23)times that of the control group,significantly higher than(2.11±0.18)times of the N-EVs group.However,there was no significant difference in cell migration rates between the N-EVs(16.3%±2.0%)and U-EVs(21.5%±2.3%)groups.Conclusion EVs secreted by ultrasound-stimulated ADSCs significantly enhance the vitality of ADSCs and improve their abilities in cell proliferation,migration,and adipogenic differentiation.