Li C-Y, Gao T-W, Wang G, Han Z-Y, Shen Z, Li T-H, Liu Y-F
Department of Dermatology, Xijing Hospital, Fourth Military Medical University. Xi'an 710033, China.
Br J Dermatol. 2004 Jun;150(6):1081-90. doi: 10.1111/j.1365-2133.2004.05929.x.
Tyrosinase-related proteins (TRPs) include tyrosinase, TRP-1 and TRP-2. The functions of tyrosinase and TRP-2 have been determined, but the biological role of TRP-1 is still controversial and is not well known in humans.
To study further the biological role of the human TRP-1 gene in melanocytes and melanoma cells.
TRP-1 cDNA was subcloned into eukaryotic expression vector pcDNA3.1 in the reverse direction, and antisense recombinant vector was transfected into melanocytes and a melanoma cell line using Lipofectamine 2000. Positive cells were selected by geneticin. TRP-1 mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR), and TRP-1 protein level by Western blot. Cell cycles were determined by flow cytometry, and the activity of tyrosinase was evaluated by L-DOPA reaction. Light microscopy, electron microscopy and flow cytometry were used to observe cell morphology and apoptosis. For in vivo assays, the antitumour activity of antisense TRP-1 against the malignant melanoma (MM) cell line, Libr, was evaluated in an animal-tumour model of subcutaneous tumours.
Positive transfected cells steadily expressed TRP-1 antisense RNA. RT-PCR and Western blot showed a low level of TRP-1 mRNA and TRP-1 protein, respectively. Cell cycles were blocked in the G1 stage, and the activity of tyrosinase decreased significantly (P < 0.01). Light and electron microscopy showed abnormal cell morphology, and apoptosis was detected. The neoplasia activity of antisense TRP-1-transfected MM cells was significantly lower than that of MM cells (P < 0.01).
TRP-1 plays an important role in the proliferation, morphology and tyrosinase activity of melanocytes and melanoma cells.
酪氨酸酶相关蛋白（TRPs）包括酪氨酸酶、TRP-1和TRP-2。酪氨酸酶和TRP-2的功能已明确，但TRP-1的生物学作用仍存在争议，在人类中也尚未完全清楚。
进一步研究人TRP-1基因在黑素细胞和黑色素瘤细胞中的生物学作用。
将TRP-1 cDNA反向亚克隆至真核表达载体pcDNA3.1，使用Lipofectamine 2000将反义重组载体转染至黑素细胞和一株黑色素瘤细胞系。用遗传霉素筛选阳性细胞。通过逆转录-聚合酶链反应（RT-PCR）检测TRP-1 mRNA水平，用蛋白质免疫印迹法检测TRP-1蛋白水平。通过流式细胞术测定细胞周期，用L-多巴反应评估酪氨酸酶活性。用光镜、电镜和流式细胞术观察细胞形态和凋亡情况。在皮下肿瘤动物模型中评估反义TRP-1对恶性黑色素瘤（MM）细胞系Libr的抗肿瘤活性。
阳性转染细胞稳定表达TRP-1反义RNA。RT-PCR和蛋白质免疫印迹法分别显示TRP-1 mRNA和TRP-1蛋白水平较低。细胞周期阻滞于G1期，酪氨酸酶活性显著降低（P < 0.01）。光镜和电镜显示细胞形态异常，并检测到凋亡。反义TRP-1转染的MM细胞的肿瘤形成活性显著低于MM细胞（P < 0.01）。
TRP-1在黑素细胞和黑色素瘤细胞的增殖、形态及酪氨酸酶活性方面发挥重要作用。
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