经LED黄光照射的人间充质干细胞旁分泌效应对成纤维细胞光老化的作用研究

Study on the paracrine effects of LED yellow light irradiated human mesenchymal stem cells on fibroblast photoaging

目的研究经LED黄光照射的人脂肪间充质干细胞(hADSCs)旁分泌效应对人真皮成纤维细胞(HDFs)光老化的影响。方法利用中波紫外线(UVB)照射HDFs构建光老化模型,并进行细胞增殖、细胞衰老、细胞迁移实验。使用不同能量梯度(2.5、5.0、7.5、10.0 J/cm^(2))LED黄光照射hADSCs进行细胞增殖实验,确定最佳照射能量(10 J/cm^(2))。实验组用LED黄光10 J/cm^(2)照射的hADSCs上清液培养光老化HDFs,对照组用未经照射的hADSCs上清液培养光老化HDFs,用CCK-8法检测2组细胞增殖能力,用β-半乳糖苷酶染色检测2组细胞衰老水平,用微丝绿色荧光探针标记细胞骨架,用流式细胞仪检测细胞活性氧(ROS)含量,并进行转录组测序,对测序发现的显著差异基因进行实时荧光定量聚合酶链反应(qRTPCR)验证、京都基因与基因组百科全书(KEGG)通路富集分析。结果与未照射组比较,UVB照射组HDFs增殖能力下降,衰老细胞(蓝染细胞)明显增多,HDFs迁移受到抑制,差异均有统计学意义(P<0.05)。与对照组比较,经LED黄光照射的hADSCs上清液干预的实验组光老化HDFs增殖能力较强、蓝染细胞较少、细胞形态较为细长、ROS含量较低,差异均有统计学意义(P<0.05)。转录组测序发现6个有意义的差异表达基因:驱动蛋白家族成员20B(KIF20B)、异常纺锤体样小头畸形相关蛋白(ASPM)、A型激酶锚定蛋白9(AKAP9)、着丝粒蛋白F(CENPF)、着丝粒蛋白E(CENPE)、RB1的诱导卷曲蛋白1(RB1CC1)。qRTPCR结果显示,实验组KIF20B、ASPM、AKAP9、CENPF、CENPE、RB1CC1 mRNA表达高于对照组,差异均有统计学意义(P<0.05),与转录组测序结果一致。KEGG通路富集分析显示,显著差异基因富集在长寿调节通路(longevity regulating pathway)。结论经LED黄光照射的hADSCs旁分泌效应对HDFs的光老化具有抑制作用,可能与激活长寿调节通路有关。

Objective To investigate the impact of paracrine effects of LED yellow light irradiated human adipose derived mesenchymal stem cells(hADSCs) on human dermal fibroblasts(HDFs) photoaging. Methods A photoaging model was established by irradiating HDFs with medium-wavelength ultraviolet(UVB) light, followed by assays to evaluate cell proliferation, senescence, and migration. Cell proliferation assays were conducted on hADSCs exposed to LED yellow light with varying energy gradients(2.5, 5, 7.5, 10 J/cm^(2)) to determine the optimal irradiation energy. In the experimental group, HDFs subjected to photoaging were cultured with the supernatant from hADSCs irradiated with LED yellow light at 10 J/cm^(2). In the control group, HDFs were cultured with the supernatant from non-irradiated hADSCs. Cell proliferation was assessed using the CCK-8 assay, cellular senescence levels were evaluated by β-galactosidase staining, the cytoskeleton was labeled with green fluorescent probes targeting actin filaments, while reactive oxygen species(ROS) content was measured by flow cytometry. Additionally, transcriptome sequencing was performed, and significant differentially expressed genes identified through sequencing were validated by quantitative real-time PCR(qRT-PCR). The KEGG pathway enrichment analysis was also conducted for the differentially expressed genes. Results Compared to the non-irradiated group, the UVB-irradiated group showed a significant decrease in HDFs proliferation, a marked increase in senescent cells(blue-stained cells), and inhibited HDFs migration. All differences were statistically significant(P < 0.05). Compared to the control group, HDFs in the experimental group treated with the supernatant from LED yellow light irradiated hADSCs showed enhanced proliferation, fewer blue-stained senescent cells, a more elongated cell morphology, and lower ROS levels. All differences were statistically significant(P < 0.05). Transcriptome sequencing identified six significantly differentially expressed genes, including Kinesin family member 20B(KIF20B), Abnormal spindlelike microcephaly associated protein(ASPM), A-kinase anchor protein 9(AKAP9), Centromere protein F(CENPF), Centromere protein E(CENPE), and RB1-induced coiled-coil protein 1(RB1CC1). qRT-PCR results showed that the mRNA expression levels of KIF20B, ASPM, AKAP9, CENPF, CENPE, and RB1CC1 were higher in the experimental group compared to the control group, with all differences being statistically significant(P < 0.05). These results were consistent with the transcriptome sequencing data. KEGG pathway enrichment analysis revealed that the significantly differentially expressed genes were primarily enriched in the longevity regulating pathway. Conclusion The paracrine effects of LED yellow light irradiated hADSCs exhibited an inhibitory effect on HDFs photoaging,which may be related to the activation of the longevity regulating pathway.