不同黑素细胞群在有色毛囊重建中作用的研究

Role of different melanocyte populations in the reconstitution of pigmented hair follicles

目的观察不同种属小鼠的毛囊细胞能否嵌合重建有色毛囊,探讨不同黑素细胞群在小鼠有色毛囊重建中的作用。方法选取C57BL/6J、BALB/C胎鼠或乳鼠皮肤,分离出表皮细胞群、毛囊上皮细胞群和真皮细胞群,培养、纯化从表皮细胞群获得的表皮黑素细胞。实验包含三部分,①乳鼠C57BL/6J毛囊重建实验:分为表皮细胞+毛囊上皮细胞组、真皮细胞组;②嵌合毛囊重建实验:分为乳鼠C57BL/6J真皮细胞组、乳鼠BALB/C真皮细胞组、乳鼠BALB/C真皮+乳鼠C57BL/6J真皮细胞组、胎鼠BALB/C真皮细胞+胎鼠C57BL/6J真皮细胞组;③有色毛囊重建实验:分为乳鼠BALB/C真皮细胞+乳鼠C57BL/6J表皮细胞组、乳鼠BALB/C真皮细胞+乳鼠C57BL/6J毛囊上皮细胞组、乳鼠BALB/C真皮细胞+培养的C57BL/6J表皮黑素细胞组。采用小室移植法将不同细胞接种于裸鼠的背部,每组4只。于移植后4周和8周,通过大体观察、组织学及免疫荧光评估毛囊重建情况。结果移植后4周和8周,乳鼠C57BL/6J毛囊重建实验中(本部分实验2组共8只BALB/C裸鼠,7只存活,1只因创面感染死亡),表皮细胞+毛囊上皮细胞组(3只)未见毛发生长,混有上皮成分的真皮细胞组(4只)长出正常毛发;嵌合毛囊重建实验中(本部分实验4组共16只BALB/C裸鼠,14只存活,2只因创面感染死亡),乳鼠和胎鼠嵌合组(2组共7只)均长出棕灰色毛发;有色毛囊重建实验中(本部分实验3组共12只BALB/C裸鼠,10只存活,2只因创面感染死亡),BALB/C真皮细胞+培养的C57BL/6J表皮黑素细胞组(3只)仅长出白色毛发,免疫荧光检测未见毛囊黑素细胞,其余组均长出棕灰色毛发。结论间质细胞和上皮细胞的相互作用是毛囊重建的必要条件;不同种属小鼠的毛囊细胞可嵌合重建有色毛囊;毛囊黑素细胞和表皮黑素细胞均能参与形成有色毛囊,但分化后的黑素细胞不具有形成有色毛囊的能力。

Objective To observe whether hair follicle cells from mice of different species can integrate to generate new pigmented hair follicles,and to explore the role of different melanocyte populations in pigmented hair follicle reconstruction in mice.Methods The epidermal cell population,hair follicle epithelial cell population and dermal cell population were isolated from the skin of fetal or neonatal C57BL/6J and BALB/C mice,and epidermal melanocytes were obtained by culture and purification of the epidermal cell population.The experiments were divided into 3 parts:(1)hair follicle reconstruction experiment in neonatal C57BL/6J mice,which included 2 groups:epidermal cells+hair follicle epithelial cells group and dermal cells group;(2)chimeric hair follicle reconstruction experiment,which included 4 groups:dermal cells of neonatal C57BL/6J mice group,dermal cells of neonatal BALB/C mice group,dermal cells of neonatal BALB/C mice+dermal cells of neonatal C57BL/6J mice group,and dermal cells of fetal BALB/C mice+dermal cells of fetal C57BL/6J mice group;(3)pigmented hair follicle reconstruction experiment,which included 3 groups:dermal cells of neonatal BALB/C mice+epidermal cells of neonatal C57BL/6J mice group,dermal cells of neonatal BALB/C mice+hair follicle epithelial cells of neonatal C57BL/6J mice group,and dermal cells of neonatal BALB/C mice+cultured C57BL/6J epidermal melanocytes group.Different cells were implanted into dorsal skin fold chambers of the nude mice,and there were 4 mice in each group.At weeks 4 and 8 after inoculation,hair follicle reconstruction was assessed by gross observation,histological examination and immunofluorescence assay.Results Among the 8 BALB/C nude mice in the 2 groups in the hair follicle reconstruction experiment,7 survived and 1 died of wound infections on week 4 after inoculation;at weeks 4 and 8 after inoculation,no hair growth was observed in the epidermal cells+hair follicle epithelial cells group(3 mice),while normal hair grew out in the dermal cells group(4 mice)mixed with epithelial components.Among the 16 BALB/C nude mice in the 4 groups in the chimeric hair follicle reconstruction experiment,14 survived and 2 died of wound infections on week 4 after inoculation;at weeks 4 and 8 after inoculation,brown-grey hair grew well in the dermal cells of neonatal BALB/C mice+dermal cells of neonatal C57BL/6J mice group(4 mice),and dermal cells of fetal BALB/C mice+dermal cells of fetal C57BL/6J mice group(3 mice).Among the 12 BALB/C nude mice in the 3 groups in the pigmented hair follicle reconstruction experiment,10 survived and 2 died of wound infections on week 4 after inoculation;at weeks 4 and 8 after inoculation,only white hair grew out in the dermal cells of neonatal BALB/C mice+cultured C57BL/6J epidermal melanocytes group(3 mice),and no hair follicle melanocytes were observed by immunofluorescence assay,while brown-grey hair grew well in the dermal cells of neonatal BALB/C mice+epidermal cells of neonatal C57BL/6J mice group(4 mice),and dermal cells of neonatal BALB/C mice+hair follicle epithelial cells of neonatal C57BL/6J mice group(3 mice).Conclusions The interaction between mesenchymal cells and hair follicle epithelial cells is a necessary condition for hair follicle reconstruction.The hair follicle cells from different species of mice can integrate to generate new pigmented hair follicles.Both hair follicle melanocytes and epidermal melanocytes can participate in the formation of pigmented hair follicles,but differentiated melanocytes have no such ability.