nicastrin基因沉默的斑马鱼模型构建及其色素异常机制的初步研究

Construction of a nicastrin gene-silenced zebrafish model and a primary study on the mechanism of abnormal pigmentation

目的探讨nicastrin(nct)基因对斑马鱼黑素细胞生物学功能的影响。方法利用吗啉代寡核苷酸(MO)技术针对斑马鱼nct基因mRNA设计nct-MO序列,同时设计对照MO序列(ctrl-MO),并构建5′端为MO靶序列的增强绿色荧光蛋白(EGFP)mRNA,通过显微注射的方式将nct-MO或ctrl-MO与EGFP mRNA共注射入斑马鱼胚胎中,验证nct-MO的沉默效率,观察其表型变化。以野生型斑马鱼作为空白对照组,实时荧光定量PCR检测黑素合成notch信号通路相关基因mitfa、tyr、tyrp1a、tyrp1b、dct、pmela、notch1a、notch1b、hey1 mRNA表达水平的变化。多组间均数比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果斑马鱼受精后8 h,ctrl-MO+EGFP mRNA组斑马鱼胚胎中均有绿色荧光表达,而nct-MO+EGFP mRNA组及空白对照组胚胎均未见绿色荧光。受精后48 h,nct-MO组幼鱼尾部色素沉着区面积占比(0.169±0.083)低于ctrl-MO组(0.258±0.042,t=3.202,P=0.005),且nct-MO组色素分布紊乱。实时荧光定量PCR显示,nct-MO组、ctrl-MO组、空白对照组间pmela、tyrp1a、hey1基因mRNA相对表达量差异有统计学意义(均P<0.05),mitfa、tyr、tyrp1b、dct、notch1a、notch1b mRNA相对表达差异无统计学意义(均P>0.05);nct-MO组pmela、tyrp1a相对表达水平(0.708±0.028、0.558±0.136)低于ctrl-MO组(1.023±0.142、1.016±0.134,均P<0.05)。结论nct基因可能通过调控斑马鱼黑素合成影响黑素细胞生物学功能。

Objective To evaluate the effect of nicastrin(nct)gene on the biological functions of melanocytes in zebrafish.Methods By using a morpholino oligonucleotide(MO)technology,a nct-MO sequence targeting the zebrafish nct mRNA was designed,so was a MO control(ctrl-MO)sequence.Then,the enhanced green fluorescent protein(EGFP)mRNA with MO target sequence at its 5′end was synthesized,and co-microinjected with the nct-MO or ctrl-MO sequence into the zebrafish embryos to verify the silencing efficiency of nct-MO and observe changes in developmental phenotypes in zebrafish.With wild-type zebrafish as a blank control group,real-time fluorescence-based quantitative PCR(RT-PCR)was conducted to determine the mRNA expression of melanin synthesis-and notch signaling pathway-related genes,including mitfa,tyr,tyrp1a,tyrp1b,dct,pmela,notch1a,notch1b and hey1 genes.One-way analysis of variance was used for the comparison of means among multiple groups,and least significant difference(LSD)-t test for multiple comparisons.Results Eight hours after zebrafish fertilization,green fluorescence was observed in the zebrafish embryos in the ctrl-MO+EGFP mRNA group,but not in the nct-MO+EGFP mRNA group or blank control group.Forty-eight hours after fertilization,the proportion of pigmented area among the whole area of the tail of zebrafish larvae was significantly lower in the nct-MO group(0.169±0.083)than in the ctrl-MO group(0.258±0.042,t=3.202,P=0.005),and disorderly pigment distribution in the tails was observed in the nct-MO group.RT-PCR revealed significant differences in the mRNA expression of pmela,tyrp1a and hey1 genes among the nct-MO group,ctrl-MO group and blank control group(all P<0.05),but no significant difference was observed in the mRNA expression of mitfa,tyr,tyrp1b,dct,notch1a or notch1b genes among the 3 groups(all P>0.05);the relative expression levels of pmela and tyrp1a mRNAs were significantly lower in the nct-MO group(0.708±0.028,0.558±0.136,respectively)than in the ctrl-MO group(1.023±0.142,1.016±0.134,respectively,both P<0.05).Conclusion The nct gene may affect biological functions of melanocytes by regulating melanin synthesis in zebrafish.