目的:本研究的目的是研究正常口腔黏膜间充质干细胞(OM-MSC)转录组基因表达的差异。口腔白斑来源的MSC(OLK-MSC)和口腔鳞状细胞癌来源的MSC(OSCC-MSC)。此外,该研究旨在比较体外增殖,迁移,入侵能力,以及这三种MSC对光动力疗法(PDT)的反应,HOK,DOK,白细胞1和Cal27细胞系。
方法:HOK,DOK,白细胞1、Cal27细胞体外培养。从OM获得3个MSC细胞,OLK,OSCC组织(n=3)并通过流式细胞术鉴定。它们还在体外培养用于成骨和成脂诱导的分化。基于IlluminaHiSeq高通量测序平台,OM-MSC,OLK-MSC,OSCC-MSC(n=3)进行转录组测序,功能注释,并对差异表达基因和相关基因进行富集分析。CCK8测定,伤口愈合试验,和transwell分析进行比较增殖,迁移,和入侵七种类型的细胞。将7个细胞与0,0.125mM,0.25mM,0.5mM,1mM,和2mM的光敏剂(5-氨基乙酰丙酸,5-ALA)体外。随后,它们用150mM辐照,635nm激光1min,24h后用CCK8法检测细胞活性。用JC-10探针检测PDT处理前后7个细胞的线粒体变化,并测定PDT治疗前后ATP含量的变化。
结果:OM-MSC,OLK-MSC,和OSCC-MSC表达阳性MSC表面标志物。成骨和成脂诱导分化培养后,可见染色的钙结节和脂滴,符合MSC的识别标准。通路富集分析显示,与OLK-MSC相比,OSCC-MSC的差异表达基因(DEGs)主要与PI3K-Akt信号通路和肿瘤相关通路相关。与Cal27相比,OSCC-MSC表现出更强的迁移和侵袭能力。OM所需的IC50值,OLK,OSCC来源的MSC低于PDT处理的上皮细胞所需的MSC,1.396mM,0.9063mM,和2.924mM,分别。PDT处理24小时后,在七种类型的细胞中观察到细胞膜和线粒体破坏。然而,HOK,DOK,白细胞1和Cal27细胞的ATP含量增加。
结论:OLK,OSCC上皮细胞比相同组织来源的MSC需要更高浓度的5-ALA用于PDT治疗。所需的5-ALA浓度随着细胞恶性程度的增加而增加。上皮细胞和MSC对PDT治疗的反应差异可能对OLK复发和恶性肿瘤有不同的影响。
OBJECTIVE: The objective of this study is to investigate the variances in transcriptome gene expression of normal oral mucosa-derived mesenchymal stem cell (OM-MSC), oral leukoplakia-derived MSC (OLK-MSC) and oral squamous cell carcinoma-derived MSC(OSCC-MSC). as Additionally, the study aims to compare the in vitro proliferation, migration, invasion ability, and response to photodynamic therapy (PDT) of these three MSC, HOK, DOK, leuk1, and Cal27 cell lines.
METHODS: HOK, DOK, leuk1, Cal27 cells were cultured in vitro. 3 MSC cells were obtained from OM, OLK, OSCC tissue (n = 3) and identified through flow cytometry. They were also cultured in vitro for osteogenic and lipogenic-induced differentiation. Based on the Illumina HiSeq high-throughput sequencing platform, OM-MSC, OLK-MSC, OSCC-MSC (n = 3) were subjected to transcriptome sequencing, functional annotation, and enrichment analysis of differentially expressed genes and related genes. CCK8 assay, wound healing assay, and transwell assay were performed to compare the proliferation, migration, and invasion of the seven types of cells. The 7 cells were incubated with 0, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM of the photosensitizer (5-aminolevulinic acid, 5-ALA) in vitro. Subsequently, they were irradiated with a 150 mM, 635 nm laser for 1 min, and the cell activity was detected using the CCK8 assay after 24 h. The mitochondrial changes in the 7 cells before and after the treatment of PDT were detected using the JC-10 probe, and the changes in ATP content were measured before and after the PDT treatment.
RESULTS: OM-MSC, OLK-MSC, and OSCC-MSC expressed positive MSC surface markers. After osteogenic and lipogenic-induced differentiation culture, stained calcium nodules and lipid droplets were visible, meeting the identification criteria of MSC. Pathway enrichment analysis revealed that the differentially expressed genes (DEGs) of OSCC-MSC compared to OLK-MSC were primarily associated with the PI3K-Akt signaling pathway and tumor-related pathways. OSCC-MSC exhibited stronger migratory and invasive abilities compared to Cal27. The IC50 values required for OM, OLK, and OSCC-derived MSC were lower than those required for epithelial cells treated with PDT, which were 1.396 mM, 0.9063 mM, and 2.924 mM, respectively. Cell membrane and mitochondrial disruption were observed in seven types of cells after 24 h of PDT treatment. However, HOK, DOK, leuk1, and Cal27 cells had an ATP content increased.
CONCLUSIONS: OLK, OSCC epithelial cells require higher concentrations of 5-ALA for PDT treatment than MSC of the same tissue origin. The concentration of 5-ALA required increases with increasing cell malignancy. Differences in the response of epithelial cells and MSC to PDT treatment may have varying impacts on OLK recurrence and malignancy.