人毛囊无色素黑素细胞培养方法的改良及色素生成相关酶表达的研究

Culture of Amelanotic Melanocytes in Outer Root Sheath From Human Hair Follicles

目的进一步改良人毛囊无色素黑素细胞(amelanotic melanocytes,AMMC)培养的方法,并研究了AMMC内黑素生成相关酶的表达:酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白-1(tyrosinase related protein-1,TRP-1)和酪氨酸酶相关蛋白-2(tyrosinase related protein-1,TRP-2)。方法采用1%分离酶(dispase)消化分离毛囊,选用含干细胞因子(stem cell factor,SCF)、内皮素-3(endothelin-3,ET-3)、碱性成纤维细胞生长因子(basicfibroblast grow factor,bFGF)和霍乱毒素(cholera toxin,CT)的成黑素细胞培养基培养AMMC,同时培养表皮黑素细胞(melanocytes,MC)作为对照,采用免疫组化、多巴染色和透射电镜对细胞进行鉴定,蛋白印记法分析TYR、TRP-1和TRP-2的表达。结果1%分离酶消化24h可以容易获得游离的毛囊,结合我们所用的培养基完全排除了成纤维细胞的污染。所用成黑素细胞培养基促AMMC增殖明显,细胞可传5代以上。免疫组化结果显示,AMMC表达gp100、酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白-1(tyrosinase related protein-1,TRP-1)和酪氨酸酶相关蛋白-2(tyrosinase related protein-1,TRP-2),证明培养的细胞为黑素细胞。AMMC的多巴染色呈阴性,并且透射电镜发现AMMC胞质中仅含大量的I期、II期黑素小体,未见III、IV期黑素小体,因此说明AMMC处于未分化状态。TYR和TRP-1在MC中的表达明显强于AMMC,TRP-2的表达在两种细胞间没有明显差别,进一步说明AMMC与MC具有异质性。结论我们成功培养了完全纯化、快速增殖的AMMC,并且培养的细胞不能生成黑素,生物学性状更接近活体中的状态,其与表皮黑素细胞具有异质性。

Objective To improve the method for eulutring amelanotie melanocyte（AMMC） in out root sheath deriving from human hair follicles, and to study the expressions of tyrosinase（TYR）, tyrosinase related protein （TRP）-1 and TRP-2. Methods Hair follicles in the remaining dermis were isolated by incubation with 1% dispase for 24 h. Hair follicle cell suspensions were seeded into culture plates with culture medium containing some factors, including stem cell factor （SCF）, basic fibroblast growth faetor（bFGF）, endothlin （ET）-3 and cholera toxin（CT）. After two passages, we purified AMMC. And next confirmed cultured AMMC were melanoblasted by immunohisehemical method, 3,4-dihydroxy-phenylalanine （DOPA） staining and transmission electron microscope. Furthermore, we observed the expressions of TYR, TRP-1 and TRP-2 through western blot method. Results Individual follicles were easily released from the human scalp by incubation with 1% dispase, and the method decreased the contamination of fibroblasts significantly. The culture medium for melanoblasts promoted rapid proliferation of AMMC. Our cultured AMMC were unreactive to DOPA, indicating lack of active TYR and consistent with the cells having predominantly stage I and II melanosomes as seen by transmission electron microscopy. Immunocytoehemical and western blot results showed both AMMC and MC expressed TYR, TRP-1 and TRP-2, but AMMC show weaker expression of TYP and TRP-1 than MC. Conclusion We have successfully improved the method for culturing AMMC from hair follicles, and furthermore demonstrate the biological heterogeneity between AMMC and MC.