^14C放射性核素标记硫脲嘧啶掺入法测定黑素小体转移

Assessment of melanosome transfer by selective incorporation of ^14C-thioumcil into nascent melanin

目的建立用^14C放射性核素标记的硫脲嘧啶（^14C—TU）掺入定量测定黑素小体转移的方法。方法^14C—TU被黑素细胞摄入后能迅速与黑素生成中间产物特异性地结合而掺入新生黑素中，用经^14C-TU预标记过夜的melan—a小鼠黑素细胞（MC）与SP-1小鼠角质形成细胞（KC）建立共培养体系，并在共培养后的不同时间，采用二次差异胰酶消化法再将两种细胞分离，用液体闪烁计数仪测定KC内被转移的放射活性量以示黑素小体转移量。同时，应用此方法初步观察毛喉素（一种PKA激动剂）和烟酰胺对黑素小体转移的影响。结果①相同数目（2．5×10^5）的MC和Kc分别用1μCi ^14C-TU标记12h或48h，放射活性测定显示，MC的放射活性（c／min）分别是KC的80或66倍，表明^14C—TU能高度特异地标记新生黑素，是一理想示踪黑素小体转移的标记物。②与未处理对照组相比，20μmol／L毛喉素能增加黑素小体转移近2．3倍，而1g／L烟酰胺能抑制黑素小体转移达0．67倍。③MC与KC共培养8～12h，MC树突伸展良好，并已有黑素小体发生转移，此时尚未观察到毛喉素诱导的MC增殖。二次差异胰酶消化法用于共培养体系中的KC分离能获得满意的效果（KC纯度约为84．5％）。结论建立体外的MC和KC共培养结合^14C—TU掺入法能定量观察毛喉素和烟酰胺对黑素小体转移的影响。

Objective To establish a method to quantitatively assess melanosome transfer with incorporation of ^14C- thiouracil （TU） into nascent melanin. Methods To characterize whether ^14C-TU was exclusively incorporated into melanin-producing cells, the same number of mouse melan-a or SP-1 keratinocytes were labeled with ^14C-TU for 12 hours and 48 hours, respectively, followed by the measurement of radioactivity. Mouse melan-a melanocytes were pre-labeled with 1 Ci/mL ^14C-TU, and cocultured with mouse SP1 keratinocytes to develop an assay system for melanosome transfer to keratinocytes. Following co-culture, the keratinocytes with transferred radioactivity were separated from melanocytes at different time points via two times of differential trypsinization. Transferred radioactivity in keratinocytes, denoting the amount of melanosome transfer, was measured with liquid scintillation counting. Meanwhile, the effects of forskolin, a PKA activator, and nicotinamide on melanosome transfer were also investigated with this assay system. Results The incorporated radioactivity in melan-a cells was 66- or 80-fold as high as that in SP-1 cells, indicating that ^14C-TU would be a suitable tracer for melanosome transfer in co-culture with keratinocytes. A purity of 84.5% was achieved for keratinocytes with transferred radioactivity by twice differential trypsinization. As shown by this assay, there was an approximately 0.67-fold decrease in melanosome transfer with the treatment of 1 g/L nicotinamide and 2.3-fold increase with 20 μmol/L forskolin treatment. After coculture with SP1 cells for 8-12 hours, melan-a cells developed well-extending dendrites with detectable melanosome transfer, while no proliferation of melan-a cells induced by forskolin was seen. Conclusion An optimized protocol for selective incorporation of ^14C-TU into nascent melanin has been successfully applied to the quantitative measurement of melanosome transfer from melanocytes to keratinocytes induced by forskolin or nicotinamide.