SV40TAg和Cre/loxP系统诱导的正常人可逆性转化黑素细胞在豚鼠体内的存活能力和复色实验

目的 研究SV40TAg基因和Cre/loxP系统在体外诱导正常人黑素细胞的可逆性转化以及转化细胞在豚鼠体内的存活能力和复色效果。方法 用Cre-ERT2病毒上清感染经SV40TAg基因转化的正常人黑素细胞（MCT），诱导Cre重组酶表达（MCTC）；过氧化氢法建立黄褐色雄性豚鼠白癜风动物模型，按黑素细胞移植方法进行原代黑素细胞和MCTC移植，观察MCTC在豚鼠体内的存活能力及复色效果。结果 MCTC细胞移植实验结果显示，1个月左右移植区即可见色素沉着，连续观察3个月，可见0.5 ～ 1 cm的黑色斑片或褐色斑片形成，移植成功率为82.5%，原代黑素细胞移植成功率为76.7%。病理观察移植区有明显黑素沉积，部分毛囊内也可见黑素沉积。结论 联合应用SV40TAg基因和Cre/loxP系统可以成功地诱导具有良好生物学安全性的人源性可逆性永生化黑素细胞，具有与原代黑素细胞相似的在体存活率，具备在体黑素合成功能，可以达到良好的复色效果。

Survival and melanogenic potential of reversibly immortalized human melanocytes mediated by SV40T antigen gene and Cre/loxP system in Guinea pigs

Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of melanocyte distribution and melanin deposition in epidermis. Results Repigmentation started 4 weeks after the transplantation, and dark or brown patches, which ranged in size from 0.5 to 1 cm, were observed in the implanted area 3 months after the transplantation. The repigmentation rate was of no significant difference between pigs transplanted with MCTC and those with primary melanocytes (82.5% vs 76.7%, P > 0.05). Pathological examination revealed melanin deposition in the basal layer of epidermis and some hair follicles in transplanted area. Conclusions SV40T antigen gene combined with Cre/loxP site-specific recombinase system can induce the reversible immortalization of human melanocytes, and the immortalized melanocytes have a favorable profile of biological safety and similarity in survival rate and melanogenic potential to primary melanocytes.