MMP21基因表达下调对斑马鱼心脏环化的影响

发布时间：2018-10-31 15:01

【摘要】：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。
[Abstract]:Objective to investigate the effect of down-regulation of matrix metalloproteinase 21 (matrix metallopeptidase 21 (MMP21) gene expression on embryonic development of zebrafish. Methods the expression of MMP21 gene was down-regulated by microinjection of morphine antisense oligodeoxynucleotide (morpholino antisense oligonucleotides,MO) in zebrafish embryonic 1 / 2 cell line, and the effectiveness of MMP21-MO was detected by RT-PCR in 10 body phase. Four MMP21-MO injection groups with different concentration gradient of 0.5 mmol/L and 1.5 mmol/L were established. The standard control group was injected with morphine antisense oligonucleotide (Con-MO) and the wild type (WT) group. The embryonic development of zebrafish in each group was observed under stereoscopic microscope. The death of zebrafish embryos and the number of deformities of zebrafish at different injection concentrations were statistically analyzed. In situ hybridization was used to detect the expression of cardiac specific marker (cardiac myosin light chain _ 2 (cmlc2) at 28 h (hours post fertilization,hpf after fertilization and 48 h after fertilization. The heart of zebrafish embryos at 72 h after fertilization was observed under stereoscopic microscope. The heart cyclization of zebrafish at different time points was analyzed. The effects of down-regulation of MMP21 gene expression on heart function of zebrafish were evaluated by counting the heart rate at 24 h and 72 h after fertilization and measuring ventricular contraction fraction (ventricular shortening fraction,VSF) at 72 h after fertilization. Results MMP21-MO could effectively inhibit the expression of MMP21 gene. There was a dose-response relationship between MMP21-MO and embryonic development of zebrafish. 1 the embryo malformation rate was the highest and the death rate was lower in the mmol/L MMP21-MO injection group. The main deformities were edema in precardiac area, abnormal cardiac cyclization, decreased heart beat and slow heart rate. The results of in situ hybridization showed that down-regulation of MMP21 gene resulted in abnormal expression of cardiac specific marker cmlc2 mRNA at 28 h after fertilization and 48 h after fertilization, suggesting that (cardiac jogging) and (cardiac looping) were abnormal in the early stage of cardiac cyclization. At 72 h after fertilization, zebrafish heart morphology was observed under stereoscopic microscope, and the cyclization of zebrafish heart was also found in MMP21-MO injection group. Compared with the control group, the fetal heart rate and ventricular systolic fraction decreased in MMP21-MO injection group. Conclusion down-regulation of MMP21 gene expression leads to abnormal cardiac cyclization and impaired cardiac function in zebrafish. MMP21 gene may play an important role in the cyclization of zebrafish heart.
【作者单位】：重庆医科大学附属儿童医院麻醉科儿童发育疾病研究教育部重点实验室儿童发育重大疾病国家国际科技合作基地儿科学重庆市重点实验室;
【基金】：国家自然科学基金青年基金项目(81200440) 国家临床重点专科建设项目(2013544)~~
【分类号】：R614

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