小鼠皮肤表皮和真皮细胞共移植诱导毛囊再生实验研究

CO-TRANSPLANTATION OF MOUSE EPIDERMIS AND DERMIS CELLS IN INDUCING HAIR FOLLICLE REGENERATION

目的探讨绿色荧光蛋白(green fluorescent protein,GFP)标记的C57小鼠皮肤表皮和真皮细胞共移植诱导裸鼠毛囊重建的实验研究。方法取C57-GFP新生及成年小鼠分离表皮与真皮,分别制备成年小鼠原代真皮-表皮混合细胞(按1∶1比例混合)、成年小鼠原代真皮细胞、新生小鼠第3代真皮细胞,荧光显微镜观察细胞激发绿色荧光情况,免疫细胞化学检测培养的新生小鼠第3代真皮细胞毛囊干细胞标志。取Balb/c裸鼠,分别将成年小鼠原代真皮-表皮混合细胞(A组)、成年小鼠原代真皮细胞(B组)、新生小鼠第3代真皮细胞(C组)及生理盐水(D组)移植至裸鼠皮下,术后4、5、6周行大体观察,6周时取移植部位皮肤行GFP免疫组织化学染色观察各组毛囊形成情况。结果荧光显微镜观察示,分离、培养的C57-GFP新生及成年小鼠表皮及真皮细胞均能激发绿色荧光;免疫细胞化学检测培养的第3代真皮细胞中毛囊干细胞标志,显示波形蛋白、α-平滑肌肌动蛋白强阳性,表明细胞多为真皮鞘细胞;部分细胞表达毛乳头细胞标志CD133、多能聚糖及毛囊隆突部标志角蛋白15。裸鼠皮下移植实验:大体观察示A组可见接种部位皮下呈灰黑色,但未见毛发穿出皮肤表面;B、C、D组裸鼠皮肤均无着色。免疫组织化学染色示,A组形成新的、完整的毛囊结构或参与形成毛囊;B组GFP阳性细胞多表达于毛囊真皮鞘、外根鞘;C组GFP阳性细胞主要分布于毛囊真皮鞘、外根鞘、毛乳头,在汗腺中也有表达;D组GFP染色呈阴性。结论小鼠皮肤的表皮和真皮细胞在裸鼠体内相互作用可形成完整的毛囊结构,而仅移植新鲜分离或培养的真皮细胞则主要参与毛囊形成,无法形成完整的毛囊结构。

Objective To investigate the co-transplantation of C57-green fluorescent protein（GFP） mouse epidermis and dermis cells subcutaneously to induce the hair follicle regeneration. Methods C57-GFP mouse epidermis and dermis were harvested for isolation the mouse epidermis and dermis cells. The morphology of epidermis and dermis mixed cells at ratio of 1 ∶ 1 of adult mouse, dermis cells of adult mouse, cultured 3rd generation dermis cells were observed by fluorescence microscope. Immunocytochemistry staining was used to detect hair follicle stem cells markers in cultured 3rd generation dermis cells from new born C57-GFP mouse. And then the epidermis and dermis mixed cells of adult mouse（group A）, dermis cells of adult mouse（group B）, cultured 3rd generation dermis cells of new born mouse（group C）, and saline（group D） were transplanted subcutaneously into Balb/c nude mice. The skin surface of nude mice were observed at 4, 5, 6 weeks of transplantation and hair follicle formation were detected at 6 weeks by immunohistochemistry staining. Results The isolated C57-GFP mouse epidermis and dermis cells strongly expressed the GFP under the fluorescence microscope. Immunocytochemistry staining for hair follicle stem cells markers in cultured 3rd generation dermis cells showed strong expression of Vimentin and α-smooth muscle actin, indicating that the cells were dermal sheath cells; some cells expressed CD133, Versican, and cytokeratin 15. After transplanted for 4-6 weeks, the skin became black at the injection site in group A, indicating new hair follicle formation. However, no color change was observed in groups B, C, and D. Immunohistochemical staining showed that new complete hair follicles structures formed in group A. GFP expression could be only observed in the hair follicle dermal sheath and outer root sheath in group B, and it could also be observed in the hair follicle dermal sheath, outer root sheath, dermal papilla cells, and sweat gland in group C. The expression of GFP was negative in group D. Conclusion Co-transplantation of mouse epidermis and dermis cells can induce the hair follicle regeneration by means of interaction of each other. And transplantation of isolated dermis cells or cultured dermis cells individually only partly involved in the hair follicles formation.