斑马鱼mmp21基因在胚胎左右不对称发育中作用的初步研究

发布时间：2018-03-24 00:11

本文选题：基质金属蛋白酶21　切入点：左右不对称发育　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨敲低基质金属蛋白酶21(matrix metallopeptidase 21,mmp21)基因对斑马鱼胚胎左右不对称发育的影响。方法:在斑马鱼胚胎1~2细胞期显微注射吗啉反义寡核苷酸(morpholino antisense oligonucleotides,MO)敲低mmp21基因的表达。(1)逆转录PCR(reverse transcription PCR,RT-PCR)检测mmp21 MO的有效性。(2)利用整胚原位杂交(whole mount RNA in situ hybridization,WISH)检测心脏和心外器官(肠道、脑)标志物以评价斑马鱼胚胎左右不对称发育情况。(3)通过观察活体胚胎心脏、计数心率和测量心室收缩分数(ventricular shortening fraction,VSF)评价心脏功能的改变。(4)体视显微镜下观察活体胚胎库氏囊泡(Kupffer's vesicle,KV)并拍照,利用Image J测量KV面积。(5)通过整胚原位免疫荧光(whole mount in situ immunofluorescence)对乙酰化微管蛋白(acetylated-tubulin,AC-tubulin)染色以评价纤毛变化。(6)通过WISH检测中线屏障标志物以观察中线屏障的改变。结果:mmp21 MO能有效敲低mmp21基因。敲低mmp21基因导致斑马鱼胚胎心脏、肠道以及脑部标志物左右随机分布,随着mmp21MO浓度的增高,异常比例增高;活体胚胎心脏结构异常,表现为心房、心室左右随机分布;心脏功能损害,表现为心率减慢、心室收缩分数减少(p0.05);KV面积变小(p0.05),囊泡内运动纤毛数量减少、长度变短(p0.05);中线分子屏障lefty1部分或全部缺失。结论:敲低mmp21基因导致斑马鱼心脏和其他心外结构(肠道、脑部)左右不对称发育异常以及器官功能损害,其可能与左右不对称发育关键结构KV和纤毛缺陷,以及中线分子屏障缺失有关。本研究以斑马鱼为模式动物初步探讨mmp21基因在胚胎左右不对称发育中的作用,为今后深入研究mmp21基因奠定了基础,同时也为mmp21基因在人类胚胎发育中的作用带来新的启示。
[Abstract]:Objective: to investigate the effect of knockout of 21(matrix metallopeptidase 21 mmp21) gene on left and right asymmetrical development of zebrafish embryos. Methods: morpholino antisense oligonucleotides moths were microinjected into zebrafish embryo 1 / 2 cell phase to knockdown the mmp21 gene. Expression of 1) reverse transcriptase PCR(reverse transcription PCR RT PCR was used to detect the effectiveness of mmp21 MO.) whole mount RNA in situ hybridization was used to detect heart and extracardiac organs (intestinal tract). Brain) markers to evaluate the left and right asymmetrical development of zebrafish embryos. Counting heart rate and measuring ventricular shortening fractionation (VSFs) to evaluate the changes of cardiac function. The changes of cardiac function were evaluated under stereoscopic microscope. Kupfferger's vesicular vesicle (KV) of living embryo was observed and photographed. Acetylated-tubulin AC-tubulin) staining of acetylated-tubulin AC-tubulin using Image J) whole mount in situ fluorescence staining of whole embryo) was used to evaluate the cilium changes. (6) the markers of midline barrier were detected by WISH to observe the changes of midline barrier. Results: mmp21. MO can effectively knock down the mmp21 gene. Knocking down the mmp21 gene leads to the zebrafish embryonic heart. The intestinal and brain markers are randomly distributed, with the increase of mmp21MO concentration, the abnormal proportion increases; the structure of the fetal heart in vivo is abnormal, showing the random distribution of left and right atrium and ventricle; the heart function is impaired, the heart rate is slow down, The area of KV decreased, the number of motor ciliates in vesicles decreased, and the length of ciliates became shorter, and the midline molecular barrier lefty1 was partially or completely absent. Conclusion: knockout of the mmp21 gene leads to zebrafish heart and other extracardiac structures (intestinal tract). Brain) abnormal development of left and right asymmetrical development and damage of organ function, which may be related to the key structure of left and right asymmetrical development of KV and ciliated defects, In this study, the role of mmp21 gene in the development of left and right asymmetrical embryos was preliminarily discussed, which laid a foundation for further study of mmp21 gene. At the same time, the role of mmp21 gene in human embryonic development has brought new enlightenment.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R321

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