MMP21基因表达下调对斑马鱼心脏环化的影响

目的 探讨基质金属蛋白酶21(matrix metallopeptidase 21,MMP21)基因表达下调对斑马鱼胚胎发育的影响.方法 在斑马鱼胚胎1～2细胞期采用显微注射吗啡啉反义寡核苷酸(morpholino antisense oligonucleotides,MO)的方法下调MMP21基因的表达.10体节期用RT-PCR方法检测MMP21-MO的有效性.设置0.5、0.75、1 mmol/L和1.5 mmol/L 4个不同浓度梯度的MMP21-MO注射组,以标准对照吗啡啉反义寡核苷酸(Con-MO)注射组和野生型(WT)组为对照.体视显微镜下观察各组斑马鱼胚胎发育情况,统计分析不同注射浓度斑马鱼胚胎死亡以及畸形数量.整胚原位杂交检测心脏特异性标志物(cardiac myosin light chain 2,cmlc2)在受精后28 h(hours post fertilization,hpf)及受精后48 h的表达,体视显微镜下观察受精后72 h斑马鱼胚胎心脏,分析各时间点斑马鱼心脏环化情况.通过计数各组斑马鱼胚胎受精后24、48及72 h心率和测量各组胚胎受精后72 h时心室收缩分数(ventricular shortening fraction,VSF),评价MMP21基因表达下调对斑马鱼心脏功能的影响.结果 MMP21-MO能够有效抑制MMP21基因的表达.MMP21-MO对斑马鱼胚胎发育的影响存在一定的剂量反应关系.1 mmol/L MMP21-MO注射组胚胎畸形率最高,死亡率较低,畸形主要表现为心前区水肿、心脏环化异常、心脏搏动减弱和心率减慢.整胚原位杂交结果显示MMP21基因的表达下调导致心脏特异标志物cmlc2 mRNA在受精后28 h和受精后48 h时期异常表达,提示心脏环化前期阶段(cardiac jogging)和心脏环化(cardiac looping)过程异常.受精后72 h体视显微镜下观察斑马鱼心脏形态同样发现MMP21-MO注射组斑马鱼心脏环化异常.与对照组比较MMP21-MO注射组胚胎心率减慢,心室收缩分数下降.结论 MMP21基因表达下调导致斑马鱼心脏环化异常及心功能受损,MMP21基因可能在斑马鱼心脏环化过程中发挥重要作用.

Effect of matrix metallopeptidase 21 knockdown on cardiac looping in zebrafish

Objective To determine the effect of down-regulation of matrix metallopeptidase 21 (MMP21) on the development of zebrafish embryos.Methods The expression of MMP21 gene was downregulated by injecting morpholino antisense oligonucleotides (MO) in zebrafish embryos at 1 ～ 2 cell stage.The effectiveness of MMP21-MO was detected by RT-PCR at 10 somite stage.Four different concentration gradients of MMP21-MO injection groups,that is,0.5,0.75,1 and 1.5 mmol/L,and 2 control groups (Con-MO injection group and wild-type group) were set up in this study.The development of zebrafish embryos was observed with stereoscopic microscopy,and the deaths and malformations in different groups were observed.Whole-mount in situ hybridization was used to examine the expression of cardiac myosin light chain 2 (cmlc2) at 24 and 48 hours post fertilization (hpf).Cardiac morphology of the living embryos was observed at 72 hpf for cardiac looping,and embryonic heart rate at 24,48 and 72 hpf were counted and the ventricular contraction fraction (VSF) in each group at 72 hpf were measured to identify the effect of down-regulation of MMP21 on cardiac function in zebrafish.Results MMP21-MO microinjection effectively knockdown the expression level of MMP21 in a dose-dependent manner.The rate of malformation was the highest and the mortality was low in the group of 1 mmol/L MMP21-MO.The main manifestations of malformation were edema of the anterior region of the heart,abnormal cardiac looping,weakened heart beat and slow heart rate.The results of whole-mount in situ hybridization showed abnormal phenotypes of cmlc2 at 24 and 48 hpf,which suggesting abnormal cardiac jogging and cardiac looping.At 72 hpf,zebrafish cardiac morphology also showed abnormal cardiac looping in the MMP21-MO injection group.Compared with the control groups,the heart rate was slowed down,and the VSF was decreased in the MMP21-MO injection group.Conclusion Knockdown of MMP21 gene results in abnormal cardiac looping and cardiac dysfunction.MMP21 may play an important role in the cardiac looping.