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目的:探讨分泌性的糖蛋白Wnt5a对永生化小鼠黑素细胞系melan-a细胞产黑素的影响。方法:用带有Wnt5a基因的腺病毒及对照组腺病毒作为载体感染体外培养的melan-a黑素细胞;MTT法测定细胞增殖率;体外氧化DOPA反应法测定酪氨酸酶活性;NaOH法测定黑素含量;RT-PCR方法检测melan-a细胞中MITF的表达。结果:与AdGFP对照组相比,AdWnt5a处理组melan-a细胞的增殖率明显降低(P<0.01);DOPA反应法和NaOH法检测结果发现,Wnt5a能显著降低melan-a细胞内酪氨酸酶活性(P<0.01)以及黑素含量(P<0.05);RT-PCR结果表明,Wnt5a显著下调melan-a细胞内MITF的表达(P<0.01)。结论:以上结果显示,AdWnt5a处理组melan-a细胞的增殖率、酪氨酸酶活性、产黑素的量及MITF的表达均有所下降。实验结果提示,Wnt5a能有效抑制melan-a细胞产黑素的能力,并且其作用机制可能与下调MITF的表达有关。
Objective: To investigate the effect of secretory glycoprotein Wnt5a on the melanogenesis of immortalized mouse melanocyte line melan-a. Methods: Melan-a melanocytes were cultured in vitro with adenovirus carrying Wnt5a gene and control adenovirus. Cell proliferation rate was measured by MTT assay. Tyrosinase activity was determined by oxidation reaction of DOPA in vitro. Melanin content. The expression of MITF in melan-a cells was detected by RT-PCR. Results: Compared with AdGFP control group, the proliferation rate of melan-a cells was significantly decreased in AdWnt5a treated group (P <0.01). The results of DOPA reaction assay and NaOH assay showed that Wnt5a could significantly reduce the intracellular metytasine tyrosinase (P <0.01) and melanin content (P <0.05). RT-PCR results showed that Wnt5a significantly down-regulated the expression of MITF in melan-a cells (P <0.01). Conclusion: The above results showed that proliferation, tyrosinase activity, the amount of melanin and the expression of MITF in melan-a cells of AdWnt5a-treated group decreased. The experimental results suggest that Wnt5a can effectively inhibit melan-a cell melanogenesis, and its mechanism may be related to the down-regulation of MITF expression.