转录辅激活因子Mediator 1调控小鼠皮肤毛发再生的作用机制研究

Regulatory role of the transcriptional coactivator Mediator 1 in skin hair regeneration and its mechanisms

目的探讨转录辅激活因子Mediator 1(Med1)对小鼠皮肤毛发再生的影响及可能机制。方法将C57BL/6J品系来源的Med1flox/flox小鼠与K14-Cre小鼠交配,通过Cre-Loxp系统获得Med1表皮特异性敲除小鼠,即表达K14-Cre的Med1flox/flox小鼠(敲除组),不表达K14-Cre的Med1flox/flox小鼠即为对照组,每组3只,共同饲养8周左右行背部脱毛实验,连续12 d观察毛发再生情况。12 d后,处死两组小鼠并切取其背部脱毛和未脱毛皮肤组织,提取组织总RNA,实时定量PCR检测毛发角蛋白基因、维生素D受体/β联蛋白通路相关基因、毛囊增殖与静息状态维持相关基因的表达。制备小鼠背部脱毛和未脱毛皮肤组织石蜡切片,免疫荧光染色检测小鼠皮肤毛囊隆突部位干细胞数量。组间比较采用两独立样本t检验。结果脱毛后0~12 d,与对照组相比,敲除组小鼠脱毛区皮肤毛发再生延迟。实时定量PCR检测显示,敲除组脱毛皮肤组织中毛发角蛋白基因Ha1、Krt2-16及维生素D受体/β联蛋白通路相关基因S100a3、Dlx3、Tubb3和毛囊增殖与静息状态维持相关基因Lhx2、Sox9、Nfatc1 mRNA相对表达量(22.09±12.32、2.07±0.20、0.02±0.01、12.36±2.12、1.75±0.46、0.39±0.02、4.42±0.76、0.44±0.07)均显著低于对照组(70.53±9.46、7.76±0.49、0.05±0.01、26.16±2.96、2.60±0.14、0.71±0.09、11.93±0.42、0.75±0.04;t值分别为5.40、18.64、3.89、6.57、3.04、6.10、15.03、6.18,均P<0.05)。免疫荧光染色显示,无论在脱毛还是未脱毛皮肤组织中,敲除组毛囊隆突部位CD34+K15+毛囊干细胞数量均远低于对照组。结论Med1基因敲除可能通过下调维生素D受体/β联蛋白通路下游基因及毛囊增殖与静息状态维持相关基因Sox9、Nfatc1、Lhx2的表达,减少毛囊干细胞数量,导致毛囊分化障碍,毛发再生延迟。

Objective To investigate the effect of the transcriptional coactivator Mediator 1(Med1)on mouse hair regeneration,and to explore potential mechanisms.Methods Med1flox/flox C57BL/6J mice were mated with K14-Cre mice,and the mice with epidermis-specific knockout of Med1 gene,namely K14-Cre-expressing Med1flox/flox mice(knockout group),were obtained by using the Cre-Loxp system,while Med1flox/flox mice without K14-Cre expression served as control group.Mice in the two groups(3 mice in each group)were raised together for 8 weeks followed by dorsal hair removal.Hair regeneration was observed for 12 consecutive days after hair removal.After 12 days,all mice in the two groups were sacrificed,their depilated and non-depilated dorsal skin tissues were resected,and total RNA was extracted from the tissues.Real-time quantitative PCR was performed to determine the mRNA expression of hair keratin genes,vitamin D receptor/β-catenin pathway-related genes,and genes associated with maintenance of hair follicle stem cell proliferation and quiescence.Paraffin-embedded sections of depilated and non-depilated mouse skin tissues were prepared,and immunofluorescence staining was conducted to determine the number of stem cells in the hair follicle bulge.Two-independent-sample t test was used for comparisons between two groups.Results From days 0 to 12 after depilation,hair regeneration was delayed in the depilated skin area in the knockout group compared with the control group.Real-time quantitative PCR showed significantly decreased mRNA relative expression levels of hair keratin genes Ha1 and Krt2-16,vitamin D receptor/β-catenin pathway-related genes S100a3,Dlx3 and Tubb3,and genes associated with maintenance of hair follicle stem cell proliferation and quiescence including Lhx2,Sox9 and Nfatc1 in the depilated skin tissues in the knockout group(22.09±12.32,2.07±0.20,0.02±0.01,12.36±2.12,1.75±0.46,0.39±0.02,4.42±0.76,0.44±0.07,respectively)compared with the control group(70.53±9.46,7.76±0.49,0.05±0.01,26.16±2.96,2.60±0.14,0.71±0.09,11.93±0.42,0.75±0.04,respectively;t=5.40,18.64,3.89,6.57,3.04,6.10,15.03,6.18,respectively,all P<0.05).Immunofluorescence staining showed that the number of CD34+K15+hair follicle stem cells in the hair follicle bulge in both depilated and non-depilated skin tissues was significantly lower in the knockout group than in the control group.Conclusion Med1 gene knockout may down-regulate the expression of downstream genes of the vitamin D receptor/β-catenin pathway and genes associated with maintenance of hair follicle stem cell proliferation and quiescence(Sox9,Nfatc1 and Lhx2),and reduce the number of hair follicle stem cells,leading to hair follicle differentiation disorder and hair regeneration delay.