【摘要】：目的探討基質金屬蛋白酶21(matrix metallopeptidase 21,MMP21)基因表達下調對斑馬魚胚胎發育的影響。方法在斑馬魚胚胎1~2細胞期采用顯微注射嗎啡啉反義寡核苷酸(morpholino antisense oligonucleotides,MO)的方法下調MMP21基因的表達。10體節期用RT-PCR方法檢測MMP21-MO的有效性。設置0.5、0.75、1 mmol/L和1.5 mmol/L 4個不同濃度梯度的MMP21-MO注射組,以標準對照嗎啡啉反義寡核苷酸(Con-MO)注射組和野生型(WT)組為對照。體視顯微鏡下觀察各組斑馬魚胚胎發育情況,統計分析不同注射濃度斑馬魚胚胎死亡以及畸形數量。整胚原位雜交檢測心臟特異性標志物(cardiac myosin light chain 2,cmlc2)在受精后28 h(hours post fertilization,hpf)及受精后48 h的表達,體視顯微鏡下觀察受精后72 h斑馬魚胚胎心臟,分析各時間點斑馬魚心臟環化情況。通過計數各組斑馬魚胚胎受精后24、48及72 h心率和測量各組胚胎受精后72 h時心室收縮分數(ventricular shortening fraction,VSF),評價MMP21基因表達下調對斑馬魚心臟功能的影響。結果 MMP21-MO能夠有效抑制MMP21基因的表達。MMP21-MO對斑馬魚胚胎發育的影響存在一定的劑量反應關系。1 mmol/L MMP21-MO注射組胚胎畸形率最高,死亡率較低,畸形主要表現為心前區水腫、心臟環化異常、心臟搏動減弱和心率減慢。整胚原位雜交結果顯示MMP21基因的表達下調導致心臟特異標志物cmlc2 mRNA在受精后28 h和受精后48 h時期異常表達,提示心臟環化前期階段(cardiac jogging)和心臟環化(cardiac looping)過程異常。受精后72 h體視顯微鏡下觀察斑馬魚心臟形態同樣發現MMP21-MO注射組斑馬魚心臟環化異常。與對照組比較MMP21-MO注射組胚胎心率減慢,心室收縮分數下降。結論 MMP21基因表達下調導致斑馬魚心臟環化異常及心功能受損,MMP21基因可能在斑馬魚心臟環化過程中發揮重要作用。
[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.
【作者單位】： 重慶醫科大學附屬兒童醫院麻醉科 兒童發育疾病研究教育部重點實驗室 兒童發育重大疾病國家國際科技合作基地 兒科學重慶市重點實驗室;
【基金】：國家自然科學基金青年基金項目(81200440) 國家臨床重點專科建設項目(2013544)~~
【分類號】：R614

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