黑色素细胞中过量表达Pax6^(10Neu)基因对MITF和TYR的影响

The Influences of Over-Expressing Pax6^(10Neu) on MITF and TYR in Melanocytes

【目的】MITF和β-catenin是黑色素生成通路中重要的调节基因,而Pax6通过调控MITF和β-catenin来调控黑色素细胞的黑色素生成,通过对只含有正常Pax6 paird domain的前102个氨基酸的Pax6^(10Neu)的研究,来探究Pax6^(10Neu)是否还有生物学功能,以及Pax6对其下游基因的作用机理。【方法】根据NCBI查找到的Pax6^(10Neu)基因序列设计PCR引物,克隆Pax6^(10Neu),收集所得基因片段送华大基因测序确认。后通过对Pax6^(10Neu)和慢病毒表达载体的序列分析来筛选合适的酶切位点,通过分析选取Sal I和Xba I作为酶切位点,设计含有Sal I和Xba I酶切位点的PCR引物,大量克隆Pax6^(10Neu)基因,从而通过胶回收得到含有Sal I和Xba I酶切位点的Pax6^(10Neu)基因。将含有酶切位点的目的片段与T载体相连,并送华大基因测序。将与T载体连接成功的质粒导入感受态细胞使其大量扩增,提取质粒,双酶切后,胶回收,得到大量含有Sal I和Xba I酶切位点的Pax6^(10Neu)基因片段,将其与含有小鼠tyrp2特异性启动子和绿色荧光蛋白(GFP)的慢病毒表达载体相连接,送华大基因测序确认。将连接好的慢病毒表达载体导入感受态细胞使其大量扩增,通过质粒中提试剂盒获得大量去内毒素的Pax6^(10Neu)慢病毒真核表达载体。将慢病毒真核表达载体通过细胞转染导入到培养的绵羊黑色素细胞中,使其过量表达。收集细胞,分别通过观察绿色荧光蛋白和使用RT-PCR来检测转染效率,使用RT-PCR和Western blot来检测MITF和TYR在m RNA和蛋白水平的变化,同时检测黑色素细胞中黑色素生成量的变化。【结果】与正常组相比,在m RNA水平,MITF显著升高3.2倍(P<0.05),TYR升高1.31倍,由此得出,Pax6^(10Neu)可以有效促进MITF m RNA的产生,对TYR m RNA产生的影响不是太显著;在蛋白质水平,MITF显著升高8.24倍(P<0.001),TYR显著升高2.09倍(P<0.001),由此得出,Pax6^(10Neu)可以显著提高MITF、TYR蛋白的产生;同时黑色素细胞产生黑色素的量显著升高1.22倍(P<0.05),由此得出Pax6^(10Neu)可以有效促进黑色素细胞黑色素的生成。【结论】只含有正常Pax6 paird domain前102个氨基酸的Pax6^(10Neu),仍然可以在黑色素细胞中与MITF相互作用,同时间接调控TYR的表达,从而使黑色素细胞黑色素的生成量发生改变。

【Objective】 MITF and β-catenin are important regulatory genes of the melanin system. Pax6 regulates the production of melanin indirectly, through regulates MITF and TYR. In order to explore the function of Pax6^（10Neu） and the mechanism of Pax6 with its target genes, we explored the function of Pax6^（10Neu）, which only included 102 N-terminalanimo acids of the paird domain. 【Method】 According to the sequence of Pax6^（10Neu） in NCBI, the primers of Pax6^（10Neu） were designed and synthesized by BGI. The coding sequences of Pax6^（10Neu） were PCR amplified and then confirmed by sequencing. Sal I and Xba I restriction sites were selected by analyzing the sequences of Pax6^（10Neu） and the mammalian expression vector. The Pax6^（10Neu） was cloned into the T-Vector, meanwhile, confirmed by sequencing. The fragment was then subcloned into a mammalian expression vector, resulting in a construction that contained a specific mouse tyrp2 promoter driving the expression of the green fluorescent protein（GFP） and the target fragment with Sal I and Xba I restriction sites. The plasmid vector was confirmed by sequencing. The expression vector was amplified by competent cells and obtained without endotoxin by the plasmid midiprep system. Then, the sheep melanocytes were transfected with the vector using Liposome 2000. There were three methods used in the results test which were quantitatively real-time PCR, western blot, and melanin content measurement. 【Result】 The results showed that the MITF m RNA, MITF and TYR protein, melanin content were significantly increased. MITF m RNA was significantly increased to 3.2 times（P〈0.05） and TYR m RNA was increased to 1.31 times, compared with control group, MITF protein was significantly increased to 8.24 times（P〈0.001） and TYR protein was significantly increased to 2.09 times（P〈0.001）. Meanwhile, the melanin content was significantly increased to 1.22 times（P〈0.05）. 【Conclusion】 We demonstrated that the Pax6^（10Neu） still interacts with MITF, and then regulated TYR indirectly, meanwhile changing the production of melanin of melanocytes.