关键词: 秃发, 间质干细胞, 纳米囊泡, 毛发再生, 毛乳头细胞, 毛囊周期

Abstract: 【Abstract】 Objective To investigate the role of umbilical cord mesenchymal stem cell （MSC）-derived nanovesicles in hair regeneration. Methods （1） Nanovesicles were prepared by continuously extruding umbilical cord MSCs through polycarbonate membranes, and were identified using transmission electron microscopy and nanoparticle tracking analysis. （2） Six C57BL/6 female mice with full-thickness skin wounds were randomly divided into a nanovesicle group （subcutaneously injected with nanovesicles once at the wound margin） and a control group （subcutaneously injected with an equal volume of phosphate-buffered saline ［PBS］ at the wound margin）; skin samples were collected on day 16 for hematoxylin-eosin （HE） staining to assess wound healing and hair follicle regeneration. （3） Human hair follicle dermal papilla cells （DPCs） were isolated using a two-step enzyme method; the uptake of PKH26-pre-labeled nanovesicles by DPCs was observed by fluorescence microscopy; the proliferative activity of DPCs co-cultured with nanovesicles was evaluated using cell counting kit-8 （CCK8） and 5-ethynyl-2'-deoxyuridine （EdU） assays. （4） Six healthy C57BL/6 female mice were randomly divided into two groups after anesthesia, and subcutaneously injected with either fluorescent dye DIR-pre-labeled nanovesicles or PBS; an in vivo imaging system was used to observe the uptake and metabolism of nanovesicles in the mouse skin. （5） Twenty-four C57BL/6 female mice with depilated backs were randomly divided into a nanovesicle group （subcutaneously injected with nanovesicles on days 0, 8, and 15） and a control group （subcutaneously injected with an equal volume of PBS at the same time points）; skin samples were collected on days 4, 18, and 21 for HE staining to analyze differences in hair follicle cycling; transcriptome sequencing was performed on skin samples collected on day 4. Statistical analyses were conducted using the t test. Results （1） Transmission electron microscopy showed that nanovesicles exhibited a spherical membranous structure with diameters of 141.3 ± 60.0 nm. （2） In 6 C57BL/6 female mice with full-thickness skin wounds, the wound area on day 12 was significantly smaller in the nanovesicle group （1.27 ± 0.50 mm2） than in the control group （4.13 ± 1.03 mm2, t = 4.34, P = 0.012）. （3） Fluorescence microscopy revealed that nanovesicles were taken up by DPCs within 20 hours; the absorbance of DPCs was significantly higher in the nanovesicle group than in the control group （t = 20.23, P < 0.001）, and the percentage of EdU-positive cells was also significantly higher in the nanovesicle group （49.62% ± 6.45%） than in the control group （37.58% ± 3.42%, t = 3.69, P = 0.006）. （4） In vivo imaging of the 6 C57BL/6 female mice showed strong fluorescence in the back of mice in the nanovesicle group on day 0, which markedly decreased by day 8, while no fluorescence was observed in the control group throughout the experiment. （5） Hair follicle cycle experiments on the 24 C57BL/6 female mice with depilated backs showed that the hair follicle length on day 4 after depilation was significantly longer in the nanovesicle group （368.00 ± 63.17 μm） than in the control group （266.90 ± 34.41 μm, t = 9.87, P < 0.001）, and the hair bulb diameter was also significantly longer in the nanovesicle group （54.83 ± 10.32 μm） than in the control group （39.12 ± 7.54 μm, t = 16.02, P < 0.001）; on day 18, the nanovesicle group showed a significantly higher hair follicle density （19.12 ± 0.90） compared with the control group （11.07 ± 1.51, t = 7.92, P = 0.001）; on day 21, 46.13% ± 8.64% of hair follicles in the nanovesicle group remained in the anagen phase Ⅵ to the catagen phase Ⅱ, and 46.24% ± 3.29% were in the catagen phases Ⅲ to Ⅳ, while 78.89% ± 18.36% of hair follicles in the control group were in the telogen phases Ⅶ to Ⅷ. Transcriptome sequencing showed that differentially expressed genes in the nanovesicle group were significantly positively enriched in the keratinization process （NES = 2.23, P < 0.001）. Conclusion Umbilical cord MSC-derived nanovesicles could promote the proliferation of DPCs, advance the entry of hair follicles into the anagen phase, delay their entry into the catagen phase, and induce hair regeneration.

Key words: Alopecia, Mesenchymal stem cells, Nanovesicles, Hair regeneration, Dermal papilla cells, Hair follicle cycle