Mei Xingyu, Wu Zhouwei, Huang Jie, Sun Yue, Shi Weimin
Department of Dermatology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine Shanghai 200080, China.
Am J Transl Res. 2019 May 15;11(5):2657-2667. eCollection 2019.
This study aims to screen the key genes and possible signaling pathways involved in the differentiation and proliferation of human melanocytes (MCs) by in vitro culture of mixed skin cells. This will be helpful to further study the mechanisms and treatment strategies of pigment-related diseases such as vitiligo.
Mixed skin cells were obtained by digesting and separating normal human foreskin tissues. Ribonucleic acid (RNA) was extracted from sorting cells and high-throughput transcriptome sequencing was performed at different culture time points. Differentially expressed genes (DEGs) were obtained by comparing the expression abundance of genes at different culture time points. Then the key genes and signaling pathways involved in the differentiation and proliferation of MCs were screened and verified by real-time quantitative polymerase chain reaction (qPCR) test.
Twenty one DEGs were finally screened for further qPCR validation, mainly involved in 4 signaling pathways. The expressions of Wnt5A, Wnt5B, FZD2 and FZD3 in Wnt pathway were continuously up-regulated, and that of Wnt4 gene was continuously down-regulated, however, all the above hadn't been verified by qPCR. The expressions of COL5A2, COL6A3, ITGB1, ITGA4, ITGAV, AKT3, PIK3CD, PIK3R1 and PIK3R2 in phosphoinositide 3-kinase (PI3K) pathway were continuously up-regulated, of which PIK3CD, PIK3R2, COL5A2, ITGA4, ITGAV and AKT3 were verified by qPCR. PDGFB and GRB2 gene expressions were down-regulated in platelet-derived growth factor (PDGF) pathway, while PDGFRB was continuously up-regulated, of which PDGFB and PDGFRB were verified. The DHRS3, DHRS9, RDH10 and SDR16C5 genes in retinol metabolic pathway were continuously down-regulated and verified by qPCR.
We suggested that Wnt5A gene in Wnt/β-catenin classical pathway, integrin combining with extracellular matrix through PI3K signaling pathway, retinoic acid catabolism-related genes could promote the differentiation and proliferation of MCs; however, PDGFB gene might have a negative regulatory effect on the growth of MCs.
本研究旨在通过混合皮肤细胞的体外培养，筛选参与人黑素细胞（MCs）分化和增殖的关键基因及可能的信号通路。这将有助于进一步研究白癜风等色素相关疾病的发病机制及治疗策略。
通过消化分离正常人包皮组织获得混合皮肤细胞。从分选后的细胞中提取核糖核酸（RNA），并在不同培养时间点进行高通量转录组测序。通过比较不同培养时间点基因的表达丰度获得差异表达基因（DEGs）。然后通过实时定量聚合酶链反应（qPCR）试验筛选并验证参与MCs分化和增殖的关键基因及信号通路。
最终筛选出21个DEGs进行进一步的qPCR验证，主要涉及4条信号通路。Wnt通路中Wnt5A、Wnt5B、FZD2和FZD3的表达持续上调，Wnt4基因的表达持续下调，但上述结果均未通过qPCR验证。磷脂酰肌醇3-激酶（PI3K）通路中COL5A2、COL6A3、ITGB1、ITGA4、ITGAV、AKT3、PIK3CD、PIK3R1和PIK3R2的表达持续上调，其中PIK3CD、PIK3R2、COL5A2、ITGA4、ITGAV和AKT3通过qPCR验证。血小板衍生生长因子（PDGF）通路中PDGFB和GRB2基因表达下调，而PDGFRB持续上调，其中PDGFB和PDGFRB通过验证。视黄醇代谢通路中的DHRS3、DHRS9、RDH10和SDR16C5基因持续下调并通过qPCR验证。
我们认为，Wnt/β-连环蛋白经典通路中的Wnt5A基因、通过PI3K信号通路与细胞外基质结合的整合素、视黄酸分解代谢相关基因可促进MCs的分化和增殖；然而，PDGFB基因可能对MCs的生长具有负调控作用。
