背景:寻常痤疮是一种物种特异性人类疾病。迄今为止,尚未建立亚洲起源的人类皮脂腺细胞系。我们先前的研究已经证明了5-氨基乙酰丙酸光动力疗法(ALA-PDT)治疗寻常痤疮的疗效,主要归因于其细胞毒性;然而,其调控机制在很大程度上仍然未知。
目的:建立源自中国人群的永生化人类皮脂腺细胞系,并研究ALA-PDT的潜在机制。
方法:用人tert基因(h-tert)转染人原代皮脂腺细胞。生物学特性,包括细胞增殖,细胞标记,和皮脂分泌功能,在原代皮脂腺细胞和永生化皮脂腺细胞(XL-i-20)之间进行比较。ALA-PDT等刺激,分别应用于原代皮脂腺细胞和XL-i-20细胞以评估其细胞功能的变化。使用RNA-seq分析研究了原代皮脂腺细胞和XL-i-20皮脂腺细胞之间的转录组差异。XL-i-20细胞系用于建立皮脂腺(SG)类器官培养,作为SG研究ALA-PDT的代表模型。
结果:h-tert永生化皮脂细胞细胞系表现出连续培养超过50代的能力。原代和永生化细胞均表达皮脂腺标记,上皮膜抗原(EMA,或MUC-1),细胞角蛋白7(CK7)和脂肪分化相关蛋白相关抗原(ADRP),维持皮脂分泌功能。发现XL-i-20的增殖能力明显高于原代皮脂腺细胞。XL-i-20对ALA-PDT的反应与原代皮脂腺细胞引起的反应没有区别。ALA-PDT后两种细胞系的细胞活力和皮脂分泌均降低,和脂质相关蛋白(SREBP-1/PPARγ)下调。转录组数据一致证明了PDT后两种细胞类型中与炎症反应相关的基因的上调和与脂质代谢相关的基因的下调。ALA-PDT后原代皮脂腺细胞和XL-i-20皮脂腺细胞的常见差异基因分析表明,MAPK信号通路和JAK-STAT信号通路被激活。SG类器官呈球形,表达FANS和PLET1的标记。ALA-PDT后Ki-67下调。
结论:我们首先从亚洲人开发了一种h-tert永生化的皮脂细胞细胞系,保持其亲本原代皮脂腺细胞的基本特征。此外,XL-i-20皮脂细胞表现出对ALA-PDT的显著反应,显示与原代皮脂腺细胞具有可比性的表型和分子变化。因此,XL-i-20及其衍生的SG类器官用作研究ALA-PDT在SG相关疾病中的功效和机制的合适的体外模型。
BACKGROUND: Acne vulgaris is a species-specific human disease. To date, there has been no established human sebocyte cell line of Asian origin. Our previous study has demonstrated the efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in the treatment of acne vulgaris, primarily attributed to its cytotoxic properties; however, its regulatory mechanism remains largely unknown.
OBJECTIVE: To establish an immortalized human sebocyte cell line derived from Chinese population and investigate the underlying mechanism of ALA-PDT.
METHODS: Human primary sebocytes were transfected with the human tert gene (h‑tert). The biological characteristics, including cell proliferation, cell markers, and sebum secretion function, were compared between primary sebocytes and the immortalized sebocytes (XL-i-20). Stimulations such as ALA-PDT, were applied respectively to both primary sebocytes and XL-i-20 cells to assess changes in their cellular functions. The transcriptome differences between primary sebocytes and XL-i-20 sebocytes were investigated using RNA-seq analysis. The XL-i-20 cell line was used to establish a sebaceous gland (SG) organoid culture, serving as a representative model of SG for the investigation of ALA-PDT.
RESULTS: The h‑tert immortalized sebocyte cell line exhibited the ability to be consecutively cultured for more than fifty passages. Both primary and immortalized cells expressed sebocyte markers such as epithelial membrane antigens (EMA, or MUC-1), Cytokeratin 7 (CK7) and adipose differentiation-related protein associated antigens (ADRP), and maintained sebum secretion function. The proliferative capacity of XL-i-20 was found to be significantly higher than that of primary sebocytes. The responses of XL-i-20 to ALA-PDT were indistinguishable from those elicited by primary sebocytes. Cell viability and sebum secretion were decreased after ALA-PDT in both two cell lines, and lipid-related proteins (SREBP-1/PPARγ) were down-regulated. The transcriptome data consistently demonstrated upregulation of genes related to inflammatory responses and downregulation of genes involved in lipid metabolism in both cell types following PDT. The analysis of common differential genes of primary sebocytes and XL-i-20 sebocytes post ALA-PDT showed that TNF signaling pathways, MAPK signaling pathways and JAK-STAT signaling pathways were activated. The SG organoids were spherical, which expressed markers of FANS and PLET1. Ki-67 was down-regulated after ALA-PDT.
CONCLUSIONS: We have developed an h‑tert immortalized sebocyte cell line from an Asian population. The cell line, XL-i-20, maintains the essential characteristics of its parent primary sebocytes. Moreover, XL-i-20 sebocyte exhibited a significant respond to ALA-PDT, demonstrating comparable phenotypic and molecular changes to primary sebocytes. Therefore, XL-i-20 and its derived SG organoid serve as appropriate in vitro models for investigating the efficacy and mechanisms of ALA-PDT in SG-related diseases.