胎儿头皮毛囊的黑素细胞定位及精细结构观察

Location and fine structure of melanocytes in human fetal scalp hair follicles

目的探讨胎儿毛囊黑素细胞的定位及精细结构。方法6个月胎儿因宫内发育畸形而引产、死亡后，取其带毛头皮，一部分常规包埋切片，分别用NKI／beteb、HMB-45、酪氨酸酶、酪氨酸酶相关蛋白1（TRP1）单抗染色。另一部分无菌处理后，0．1g／L的胶原酶Ⅱ和胰酶消化获得毛囊细胞，培养并传代后，透射电镜和原子力显微镜观察。结果胎儿头皮毛囊NKI／beteb阳性细胞位于外根鞘，而在毛球内许多细胞HMB-45、酪氨酸酶、TRP1单抗染色阳性。在毛囊细胞的体外培养中，除去成纤维细胞和角质形成细胞后，可见两种黑素细胞，一种数目极少，色素很多，传代后消失；另一种数目较多，开始无色素，但增殖很快。传第3代后，几乎所有细胞NKI／beteb染色阳性。扫描电镜和原子力显微镜下，多数细胞为双极梭形，偶尔有3个树突。细胞体呈圆形或卵圆形，极突上无明显的分支，其内有少数散在的黑素体。结论胎儿头皮毛囊外根鞘的黑素细胞推测为成黑素细胞和（或）其子代细胞。在早期的体外培养中，细胞增殖很快，但形态及功能上不成熟。

Objective To investigate the location, fine structure of melanocytes in human fetal scalp hair follicles. Methods The scalp with hair follicles was obtained from a dead fetus of 6 months of age, and divided into two parts. One part was embedded in paraffin, tissue sections were prepared with a width of 7 μm and stained with NKI/beteb, monoclonal antibodies to HMB-45, tyrosinase and tyrosinase-related protein 1 （TRP1）, respectively. The other part with hair follicles was treated with collagenase type Ⅱ 0.1 g/L and trypsin,then, cell suspension was collected and cultured. After 14-day culture, follicle melanocyte cells （FMC） were separated from keratinocytes by differential trypsinization, and fibroblasts were removed with geneticin. Following three times of pure passage, FMC were seeded and fixed on mica for scanning electron microscopy （SEM） and atomic force microscope （AFM） scanning. Results Histopathological examination showed that NKI/beteb positive cells located at the outer root sheath of human hair follicles, and these cells stained negatively for HMB-45, tyrosinase and TRP1 antibodies. However, in the hair bulb, lots of cells expressed HMB-45, tyrosinase and TRP1 antigens. After fibroblasts and keratinocytes were removed, two kinds of melanocytes remained in the culture： one was small in number and showed abundant melanin, which was lost after subsequent passage; the other was large in number and had no melanin initially, but proliferated very rapidly. After three passages, almost all the melanocytes were positive for NKI/beteb. As SEM and AFM showed, most cultured melanocytes appeared fusiform with two （rarely three） dendrites, and the cell body was round or oval with a few melanosomes scattered in but no clear secondary branches on the dendrites. Conclusions The melanocytes in outer root sheath of hair follicles from the fetal scalp are presumed as melanocyte stem cells or their progenies. In vitro, these cells proliferate very rapidly during early phases, but the morphology and function of them still remain immature, which is unfavorable for melanosome transport.