甲殼素薄膜誘導黑色素細胞成球用於黑色素細胞移植之探討

Abstract

本實驗是透過裸鼠當模型進行人類黑色素細胞移植，藉以建立一個以PUVA為移植部位準備方法之黑色素細胞移植流程，並測試甲殼素薄膜為載體之黑色素細胞移植之可行性。 本實驗採用週齡八週以上之裸鼠，在身體左右兩側敷上光敏感藥物-美舒沙寧(methoxsalen)，並給以劑量8J的UVA照射全身，等待兩天後，以除毛貼片移除表皮。人類之黑色素細胞由頭皮檢體提取，實驗組採用甲殼素薄膜為生醫材料來移植黑色素細胞，對照組是以培養基回溶黑色素細胞並滴於裸鼠真皮上。分別透過照相及顯微鏡觀察黑色素細胞移植後貼附之情形，並且以切片方式分析黑色素細胞移植到皮膚中的位置。結果顯示，我們所建立之裸鼠模型可以作為人類黑色素細胞移植之測試平台。黑色素細胞在移植到裸鼠之真皮上，會被修復的表皮所覆蓋，而且能黑色素細胞可以存活在裸鼠背上長達二十一天。人類之黑色素細胞會與裸鼠之角質細胞作用，使表皮變黑，並且會移動進入裸鼠之毛囊，使裸鼠長出黑色毛髮。 在經由甲殼素薄膜誘導黑色素細胞成球的移植實驗中，有觀察到黑色素細胞有擴散移動的情形，因此希望藉由體外培養的人造表皮模型；觀察黑色素細胞的移動方式，藉以模擬白斑治療過程中，黑色素細胞的移動，希望能夠了解其移動機制，以達到更有效率的移植黑色素細胞。透過人造表皮觀察黑色素細胞的移動模式，黑色素細胞有傾向平均分散的趨勢，並且不同型態之黑色素細胞移動模式不同，以較未分化的黑色素細胞移動能力較好。 總結來說，裸鼠以PUVA移除表皮之模型可作為黑色素細胞移植之臨床前測試；可藉由人造表皮觀察黑色素細胞移動，藉以模擬表皮形成初期黑色素細胞的移動模式，亦可應用於藥物測試誘導黑色素細胞移動。

Vitiligo is a skin disorder characterized by white spot on appearance; and cellular grafting method is an option for vitiligo treatment. However, the success rate is low due to the administration of treatment in the cell suspension form would reduce the cell survive rate. The aim of our experiment is to establish a nude mice model for chitosan-based melanocytes patch transplant. In our experiment, 8 weeks old female nude mice were applied with methoxsalen for 30mins and exposed to 8J ultraviolet A irradiation, as a recipient sites preparation. This PUVA exposure would induce sunburn blisters within two days, and the blister roof can be peeled off by a waxing patch. Our control group consist of direct transplant of melanocytes suspension. The result showed that melanocytes successfully attached to the dermis within 24 hours; and epidermis coverage was observed within 72 hours with melanocytes stay at the basal layer. In the experimental group transplant by chitosan membrane, melanocytes spheroids were successfully transplanted onto nude mice. Turns out that human melanocytes can survival on nude mice epidermis and migration into hair follicle. In the in vivo setting, melanocytes migration were absent; and hence we constructed a in vitro model to study a melanocytes migration through pigmented epidermis equivalent. Melanocytes were fluorescence labelled and the migration of these cells were captured using time lapse camera for 24 hours. The results revealed that melanocytes would actively migrate toward the regions which consisted of keratinocytes and regions of low melanocytes density. Additionally, dark colored melanin were also successfully transferred to surrounding keratinocytes. In conclusion, the nude mice model were successfully demonstrated as a preclinical model for the melanocyte transplantation based on the chitosan based melanocyte spheroid patch; with the migration process clearly observable in the in vitro model. Potentially, this could lead to a better understanding in the treatment for depigmentation.