GPNMB通过调控MITF下游色素相关基因表达影响黑色素细胞色素的生成

摘要

[目的]探究GPNMB是否会通过调控MITF及其下游色素相关基因的表达来影响黑色素细胞中色素的生成,为进一步阐明GPNMB对黑色素细胞中色素生成的具体机制提供理论依据.[方法]首先对小鼠GPNMB核酸序列进行检索,通过对GPNMB和慢病毒表达载体序列分析来筛选出合适的酶切位点,在此选取Sa1Ⅰ和Xba Ⅰ作为酶切位点.并对GPNMB基因序列设计含有Sa1Ⅰ和Xba Ⅰ酶切位点的全长引物,克隆GPNMB基因全长序列,将含有酶切位点的GPNMB基因片段与T载体进行连接,送华大基因测序.将构建成功的T载体提取质粒,双酶切后将其与含有绿色荧光蛋白(GFP)的慢病毒表达载体相连接,送华大基因进一步测序确认.使用质粒中提试剂盒获得大量去内毒素的GPNMB慢病毒真核表达载体.通过体外培养小鼠黑色素细胞,选择细胞对数生长期利用细胞转染技术过量表达GPNMB.观察转染后黑色素细胞形态特征变化和绿色荧光蛋白的表达量,收集黑色素细胞,并对其进行转染效率验证,黑色素含量进行测定,以及经Real-time PCR和Western blot检测转染后PMEL、MITF、TYR、TYRP1、TYRP2和OA1的mRNA和蛋白的表达量.[结果]与正常组(Control)相比试验组(Vector-GFP-GPNMB)、空载组(Vector-GFP)中黑色素细胞的形态特征无明显变化,并且试验组、空载组的绿色荧光蛋白表达量显示5 μg DNA/孔转染效率最高.相对于空载组和正常组,试验组的GPNMB mRNA和蛋白水平都显著的升高(P＜0.01).与空载组相比,试验组黑色素含量升高1.34倍,差异显著(P＜0.01).Real-time PCR检测结果显示,MITF mRNA显著降低2.25倍(P＜0.01);PMEL mRNA显著升高1.59倍(P＜0.05);TYRP1 mRNA显著升高2.35倍(P＜ 0.01);TYRP2 mRNA显著升高1.60倍(P＜ 0.01);TYR mRNA升高1.65倍和OA1 mRNA升高1.5倍,但变化不明显.Western blot检测结果显示,MITF蛋白显著降低1.59倍(P＜0.01);TYR蛋白显著升高1.23倍(P＜0.01);TYRP2蛋白显著升高4.35倍(P＜0.01);OA1蛋白显著升高1.31倍(P＜ 0.05);TYRP1蛋白升高1.05倍,但变化不明显.[结论]GPNMB过量表达使MITF下游基因PMEL、TYR、TYRP1、TYRP2和OA1的表达量增加,却使MITF的表达量降低,由此表明GPNMB可能受非MITF调控路径调节PMEL、TYR、TYRP1、TYRP2和OA1的表达,从而影响黑色素的生成.%[Objective] The objective of this study is to explore whether GPNMB affects melanin synthesis in the melanocytes by regulating the levels of MITF and its downstream pigmentation-related genes,further clarify the specific mechanism of GPNMB in the melanin genesis in the melanocytes to provide a theoretical foundation.[Method] Firstly,the mouse GPNMB nucleic acid sequences was searched from NCBI.After analysis of the sequences of GPNMB and the lentiviral expression vector,the Sal Ⅰ and Xba Ⅰ restriction sites was selected.Then,the GPNMB full-ength primers containing SalⅠ and XbaⅠ restriction sites was designed and the GPNMB full-length sequence containing the restriction site was cloned,and finally,the T vector was ligated and sequenced.The constructed T vector plasmid was extracted and doubly digested,followed by ligating with the lentiviral expression vector containing green fluorescence protein (GFP) and then sequenced.A large number of endotoxin-free GPNMB lentivirus eukaryotic expression vectors were collected according to the instruction of the plasmid midiprep system.Furthermore,mouse melanocytes was cultured in vitro and transfected using Lipofectamine 2000 reagent according to the manufacturer's guidelines when it was in the logarithmic growth phase.Forty-eight hours after transfection,the morphological changes and green fluorescent protein expression levels of melanocytes was observed.Then,the cells were harvested and the transfection efficiency and melanin content of melanocytes were tested.The relative expression levels of PMEL,MITF,TYR,TYRP 1,TYRP2 and OA 1 were qualitatively and quantitatively analyzed by real-time quantitative PCR (qRT-PCR) and Western blot.[Result] The morphological characteristics of melanocytes in the experimental group and empty vector group had no obvious change compared with the normal group.The results of green fluorescent protein (GFP) in both the experimental group and the empty vector group showed that the transfection efficiency in 5 μg DNA / well was the highest.GPNMB mRNA and protein levels in the experimental group were extremely significantly higher than the empty vector group and normal group.Compared with the empty vector group,the melanin content in the experimental group increased by 1.34 times (P＜0.01).The qRT-PCR results showed that the relative expression levels of PMEL,TYRP1 and TYRP2 mRNA in experimental group was 1.59 (P＜0.05),2.35 (P＜0.01) and 1.60 times (P＜0.01) of that in empty vector group,respectively;However,the relative expression level of MITF mRNA was significantly reduced by 2.25 times (P＜0.01);TYR and OA1 mRNA increased 1.65 and 1.50 times,respectively,but not significantly.Furthermore,the western blot results showed that TYRP2 protein was significantly increased by 4.35 times (P＜0.01);TYR protein was significantly increased by 1.23 times (P＜0.01);OA1 protein was significantly increased by 1.31 times (P＜0.05);By contrast,MITF protein was significantly reduced by 1.59 times (P＜0.01);TYRP1 protein had no significantly change.[Conclusion] Over-expression of GPNMB increased the expression of PMEL,TYR,TYRP1,TYRP2 and OA1,but reduced the expression of MITF.It was speculation that GPNMBmay affect the melanin synthesis by regulating the expression of PMEL,TYR,TYRP 1,TYRP2 and OA1 in a MITF-independent fashion.