壳聚糖膜转载黑素细胞动物实验研究

Transport of melanocytes by chitosan membrane： an animal experiment

目的 探讨壳聚糖膜搭载、转运黑素细胞的可行性,探索利用壳聚糖膜改进黑素细胞移植技术.方法 使用电镜、MTT法、NaOH裂解法等技术观察黑素细胞在壳聚糖膜上贴附、生长、合成黑素情况.进行动物移植实验,免疫组化结合激光共聚焦显微镜技术检测壳聚糖膜上搭载的黑素细胞向动物皮 肤创面转移情况.结果 扫描电镜和倒置显微镜观察到黑素细胞在壳聚糖膜上分布均匀,贴附良好.利用MTT法测定的增殖曲线显示,壳聚糖膜能够支持黑素细胞正常生长;黑素合成量（A值为0.087±0.027）与培养皿所培养细胞（0.101±0.036）相比差异无统计学意义（P〉0.05）.激光共聚焦显微镜观察裸鼠移植部位,可以见到黑素细胞;在移植部位活检取材,用melan-A单克隆抗体免疫组化染色,结果阳性.结论 壳聚糖膜能够搭载黑素细胞贴附、生长,并支持黑素细胞向动物移植部位转移,同时保持黑素细胞活性.

Objective To study the feasibility of using chitosan membrane to carry and transport melanocytes, in order to refine the technique for melanocyte transplantation with chitosan membrane. Methods Melanocytes were inoculated onto chitosan membrane and cultured for a period of time, then, electron microscopy,MTT assay and NaOH assay were carried out to estimate the adherence, growth and melanogenesis of the melanocytes. Skin wound surface was prepared in 12 nude mice, which were equally divided into 3 groups, test group inoculated with melanocytes on chitosan membrane, negative control group I treated with chitosan membrane without melanocytes, and negative control group II directly dressed immediately after the preparation of wound surface. On day 10 and 20 after the transplantation, confocal laser microscopy and immunohistochemistry were performed to observe the migration of melanocytes into the skin wound surface. Results Scanning electron microscopy and inverted microscopy showed that melanocytes were evenly distributed on and adhered well to the underlying chitosan membrane. As the growth curve of melanocytes demonstrated, chitosan membrane could support the normal growth of melanocytes, and no significant difference was observed in the synthesized melanin content between melanocytes cultured on the chitosan membrane and those in culture disks （0.087 ± 0.027 vs. 0.101 ± 0.036, t = 0.79, P 〉 0.05）. Melanocytes were seen at the transplantation sites by confocal laser microscopy, and biopsy specimens from the transplantation sites stained positive for antimelan-A monoclonal antibody. Conclusions Melanocytes can adhere to and grow on the chitosan membrane,which can facilitate the migration of melanocytes to the transplantation sites in animals with the maintenance of biological activity of melanocytes.