人胎儿毛囊隆突细胞的培养方法及其向皮脂腺细胞诱导分化的初步研究

Primary study on the culture of human fetal follicle bulge cells and their differentiation into sebaceous gland in vitro

目的探讨简单快捷地获得大量人毛囊隆突细胞并保持其干细胞特性的方法,观察其向皮脂腺细胞分化的可行性。方法用传统的器械分离法(简称传统法)和改进的消化分离法(简称改良法)对人胎儿毛囊隆突细胞进行分离培养,比较两种方法的细胞获得效率和细胞生长特性。在对毛囊隆突细胞进行诱导培养后,行抗上皮膜抗原(EMA)抗体免疫组织化学染色,以检测细胞EMA的表达情况。噻唑蓝(MTT)法检测细胞克隆形成率。用亲和素-生物索复合物(ABC)标记法行毛囊隆突细胞角蛋白19(K19)染色。结果传统法每小时可获得8～10个毛囊隆突,贴壁48h后有细胞长出,贴壁率为40%～50%,培养14d左右传代；改良法每小时可获得毛囊隆突100个左右,接种后12h即可贴壁并有细胞长出,贴壁率30%,细胞生长迅速,7d左右即可传代。毛囊隆突细胞诱导培养7d后,细胞体积变大,随着时间延长,细胞进一步变大,细胞质中有脂滴样颗粒围绕在核的周围,细胞形状不规则。诱导培养14d后,抗EMA抗体免疫组织化学染色呈阳性表达。改良法的细胞克隆形成率为(18．2±2．1)%,明显高于传统法[(12．7±3．4)%,P＜0．05]。毛囊隆突细胞K19免疫组织化学染色呈阳性,其细胞分布满视野,细胞质内含有大量棕色颗粒。结论改良法可以获得大量毛囊隆突细胞并保持其干细胞特性,在体外诱导条件下,具有向皮脂腺细胞分化的潜能。

Objective To develop a rapid and reproducible method for the culture of human fetal hair follicle bulge cells, and observe the plasticity of its differentiation into sebaceous gland in vitro. Methods The bulge cells isolated from fetal human hair follicles by enzymatic digestion （ digestion method ） and manual microdissection （conventional method） were cultured and passaged respectively, the efficiency and biological features of cells were investigated , the clone forming efficiency was assayed by MTT, and the expression of K19 was further compared by immunocytochemistry （ABC）. The morphological change and the expression of EMA of bulge cells were also observed after induction. Results By conventional method, 8 - 10 bulges were harvested in one hour, 40% - 50% of their cells were found to adhere to the culture plate after culturing for 48h, and they became confluent after 14 days. In comparison, about 100 bulges were harvested in one hour by digestion method, the adherence efficiency of their cells was 30% after cultivation for 12h and became confluent after 7 days. The cells grew larger with time, with irregular shape and droplets of lipid around the nucleus. The clone forming efficiency of bulge cells cultured by digestion method was（ 18.2 ± 2. 1 ）% , which was much higher than that of ceils obtained by conventional method[ （ 12.7 ± 3.4） % , P 〈 0.05 ]. Immunocytochemistry staining showed that positive staining of K19 was observed in most of the bulge cells, with a large amount of brown granules in the cytoplasm. Conclusion Human hair follicle bulge cells can be efficiently cultured and multiplied in vitro, and they retained the characteristics of stem cells. And they have the potential to differentiate into sebaceous glands by induction in vitro.