含小鼠表皮干细胞的真皮替代物体内移植追踪实验

Following the fate of murine epidermal stem cells in a syngeneic dermal equivalent in vivo

目的追踪种植于真皮替代物中的小鼠表皮干细胞(epidermal stem cells,ESCs)移植到同基因受者体内后的转归。方法在体外诱导胚胎干细胞(embryonic stem cells,ES细胞)分化为ESCs,采用荧光染料Hoechst33342染色后,种植到含有成纤维细胞的胶原-明胶海绵真皮替代物内,移植到与ES细胞同基因的129/J小鼠皮下,采用HE染色、免疫组化和Van Gieson染色等观察这些细胞的存活和分化情况。结果移植后至少3周内仍可以在荧光显微镜下清晰观察到ESCs。这些细胞能够存活并分化形成毛囊样、腺管样结构,形成与天然真皮相似的结构。在这些结构中有CD29(整合素β1亚单位)和细胞角蛋白18等表皮干细胞特异性标志物的表达。在移植后至少10周内未观察到明显排斥反应或严重毒副反应。结论ESCs能够在所构建的真皮支架内存活并分化形成毛囊样和腺管样结构,提示这些来源于ES细胞的ESCs可以作为种子细胞,用于研究制备含有皮肤附属器的真皮替代物。

Objective To follow the fate of murine epidermal stem cells （ESCs） seeded in a syngeneic dermal equivalent implanted in vivo. Methods Embryonic stem （ES） cells were induced in vitro to differentiate into ESCs. After stained with a fluorescent dye Hoeehst 33342, these ESCs were seeded into a fibroblast- collagen-gelatin sponge complex, functioning as a dermal equivalent model, and implanted subcutaneously into 129/J mice, which were syngeneic to these stem cells. The fate of these cells was observed with HE staining, immunocytochemical staining or Van Gieson＇s staining. Results These ESCs were clearly visible in the implant by fluorescent microscopy 3 weeks or longer after implantation. These cells remained viable, differentiated into hair follicle-like structure, glandular structure, and gave rise to additional structures displaying features resembling native dermis. A number of markers were expressed in the differentiated structures, including CD29 （integrin β1 subunit） and cytokeratin 18 （CK18）. No apparent rejection or severe side effects were observed at least during the 10 weeks following implantation. Conclusion Now that ESCs could survive in vivo in this dermal equivalent model, and differentiate into hair follicle-like structures as well as glandular structures, it is feasible to use these cells as seed cells in the study to fabricate dermal equivalent having the potential to develop dermal appendages.