Alpha-ketoglutarate engineered small extracellular vesicles delay skin aging

doi:10.12307/2026.538

Abstract

Abstract: BACKGROUND: Cell-free therapy is a research hotspot in the field of medical cosmetic anti-aging. It is still unknown for paracellular secretion of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles loaded with the antiaging drug α-ketoglutaric acid to delay skin aging.
OBJECTIVE: To investigate the effect of the anti-aging agent α-ketoglutarate engineered human umbilical cord mesenchymal stem cell-derived small extracellular vesicles in a D-galactose-induced model of dermal fibroblast senescence.
METHODS: (1) Biological characteristics of primary human umbilical cord mesenchymal stem cells were identified by osteogenic-lipogenic differentiation staining and flow cytometry. (2) The small extracellular vesicles derived from human umbilical cord mesenchymal stem cell were obtained by using differential-ultracentrifugation. α-Ketoglutarate-engineered human umbilical cord mesenchymal stem cell-small extracellular vesicles were constructed by electroporation, and biologically characterized by transmission electron microscopy and nanoparticle tracking analyzer, while the encapsulation rate was assessed using high-performance liquid chromatography. (3) The effect of α-ketoglutarate on the proliferative capacity of dermal fibroblasts was assessed by CCK-8 and Edu cell proliferation assay kits. (4) The effect of α-ketoglutarate-engineered human umbilical cord mesenchymal stem cell-small extracellular vesicles on delaying the senescence of dermal fibroblasts was evaluated by reactive oxygen species detection kit, western blot assay, and cellular immunofluorescence.
RESULTS AND CONCLUSION: (1) The obtained human umbilical cord mesenchymal stem cell and human umbilical cord mesenchymal stem cell-small extracellular vesicles were biologically compatible. (2) There was no toxic effect on dermal fibroblasts when α-ketoglutarate was used in the concentration range of 0.5-8 mmol/L. (3) D-gal induced senescence in dermal fibroblasts, while α-ketoglutarate-engineered human umbilical cord mesenchymal stem cell-small extracellular vesicles treatment reduced the level of oxidative stress, DNA damage, and collagen loss, which was further verified that α-ketoglutarate-engineered human umbilical cord mesenchymal stem cell-small extracellular vesicles could effectively slow down the skin aging process.

Key words: ">small extracellular vesicle, human umbilical cord mesenchymal stem cell, α-ketoglutarate, senescence, dermal fibroblast, electroporation, oxidative stress, DNA damage, engineered extracellular vesicle

CLC Number:

Wu Zhijing, Li Jiali, Zhang Jiaxin, Wang Tangrong, Zheng Yuzhou, Sun Zixuan. Alpha-ketoglutarate engineered small extracellular vesicles delay skin aging[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(1): 120-129.

Figures/Tables 3

2.1 人脐带间充质干细胞及hucMSC-sEV的生物学特征分离培养的原代人脐带间充质干细胞从组织块周围爬出，细胞形态呈梭状，细胞状态良好呈漩涡状生长，见图1A；经过成骨和成脂培养基诱导分化后进行茜素红或油红O染色，可见明显的钙结节和脂滴，表明人脐带间充质干细胞成骨、成脂分化能力良好，见图1B；流式细胞术结果表明，人脐带间充质干细胞高表达阳性标志物CD105、CD73、CD166，阴性标志物CD11b、 CD34、CD45和ISO几乎不表达，见图1C，符合间充质干细胞的生物学特征。收集人脐带间充质干细胞上清液，进行差速-超速离心法得到hucMSC-sEV，在透射电镜下观察到hucMSC-sEV有典型的囊泡膜结构并且呈茶托状，见图2A；纳米粒子跟踪分析仪检测粒径分布在180.8 nm处，Zeta电位为(-29.32±1.16) mV，见图2B；Western blot结果显示，hucMSC-sEV表达小细胞外囊泡特征性标志物CD63、CD9、Alix、TSG101，不表达阴性标志物Calnexin，见图2C。 2.2 AKG对真皮成纤维细胞增殖能力的影响分离培养的真皮成纤维细胞从大鼠乳鼠的皮肤组织块中爬出，细胞形态呈短梭状，细胞状态良好呈漩涡状生长，见图3A；细胞免疫荧光结果显示，真皮成纤维细胞表达特征性标志物波形蛋白和α-平滑肌肌动蛋白，不表达阴性标志物即表皮标志物角蛋白10，见图3B，符合真皮成纤维细胞的生物学特征。分别将0.5，1，2，4，8 mmol/L的AKG与真皮成纤维细胞共孵育24 h和48 h，CCK-8结果显示AKG没有抑制真皮成纤维细胞的增殖，表明AKG对真皮成纤维细胞无毒性，见图3C，由于在传统的电穿孔过程中投入的药物量会有一定的损失，同时通过参考相关文献[35]，最终选择2 mmol/L浓度进行后续实验。 "

2.4 AKG工程化hucMSC-sEV延缓D-半乳糖诱导的真皮成纤维细胞衰老进程建立真皮成纤维细胞老化模型，见图5A，在D-半乳糖处理24 h后，向细胞中分别加入AKG或hucMSC-sEV或AKG工程化hucMSC-sEV处理12 h进行后续实验。在共聚焦显微镜下观察到hucMSC-sEV与真皮成纤维细胞共孵育12 h后能被有效摄取，而AKG工程化hucMSC-sEV组的摄取效率要略低于hucMSC-sEV组，可能与电穿孔造成小细胞外囊泡数量上的损失有关，见图5B。 2.4.1 AKG工程化hucMSC-sEV促进衰老真皮成纤维细胞的增殖通过CCK-8法来评估不同浓度AKG对D-半乳糖诱导的衰老真皮成纤维细胞增殖的影响。使用0.5，1，2，4，8 mmol/L AKG与真皮成纤维细胞共孵育，CCK-8结果显示AKG浓度在2 mmol/L以内时，对衰老的真皮成纤维细胞有促增殖作用，且当浓度为1 mmol/L和2 mmol/L时效果最为显著，Edu细胞增殖实验也具有相同的结果，见图5C-E，结合AKG对真皮成纤维细胞的毒性实验，以及药物在电穿孔过程中存在损失，最终选取2 mmol/L进行后续实验。 2.4.2 AKG工程化hucMSC-sEV降低衰老真皮成纤维细胞氧化应激水平使用活性氧评估不同处理组衰老真皮成纤维细胞内氧化应激水平，结果显示，与D-半乳糖组相比，各处理组显著抑制了细胞内活性氧的产生，降低了氧化应激水平，其中AKG工程化hucMSC-sEV组效果更优，见图5F-G。 2.4.3 AKG工程化hucMSC-sEV减少真皮成纤维细胞的胶原蛋白流失并降低DNA损伤水平 Western blot结果显示，与D-半乳糖组相比，AKG工程化hucMSC-sEV组能有效增加Ⅰ型胶原蛋白表达，并下调基质金属蛋白酶9表达，在一定程度上抑制了胶原蛋白的降解，同时下调Bax蛋白表达，有效抑制了细胞凋亡，此外衰老相关标志物P21表达水平也显著下调。细胞免疫荧光结果也证实，AKG组、hucMSC-sEV组和AKG工程化hucMSC-sEV组均能提升Ⅰ型胶原蛋白表达水平，下调DNA双链断裂标志物γ-H2AX表达水平，表明经处理后，衰老真皮成纤维细胞的胶原蛋白含量有所恢复，DNA断裂引起的损伤减轻，其中AKG工程化hucMSC-sEV组的效果最好，见图5H-M。"

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