本文選題：基質(zhì)金屬蛋白酶21　切入點(diǎn)：左右不對(duì)稱發(fā)育　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討敲低基質(zhì)金屬蛋白酶21(matrix metallopeptidase 21,mmp21)基因?qū)Π唏R魚胚胎左右不對(duì)稱發(fā)育的影響。方法:在斑馬魚胚胎1~2細(xì)胞期顯微注射嗎啉反義寡核苷酸(morpholino antisense oligonucleotides,MO)敲低mmp21基因的表達(dá)。(1)逆轉(zhuǎn)錄PCR(reverse transcription PCR,RT-PCR)檢測(cè)mmp21 MO的有效性。(2)利用整胚原位雜交(whole mount RNA in situ hybridization,WISH)檢測(cè)心臟和心外器官(腸道、腦)標(biāo)志物以評(píng)價(jià)斑馬魚胚胎左右不對(duì)稱發(fā)育情況。(3)通過觀察活體胚胎心臟、計(jì)數(shù)心率和測(cè)量心室收縮分?jǐn)?shù)(ventricular shortening fraction,VSF)評(píng)價(jià)心臟功能的改變。(4)體視顯微鏡下觀察活體胚胎庫氏囊泡(Kupffer's vesicle,KV)并拍照,利用Image J測(cè)量KV面積。(5)通過整胚原位免疫熒光(whole mount in situ immunofluorescence)對(duì)乙酰化微管蛋白(acetylated-tubulin,AC-tubulin)染色以評(píng)價(jià)纖毛變化。(6)通過WISH檢測(cè)中線屏障標(biāo)志物以觀察中線屏障的改變。結(jié)果:mmp21 MO能有效敲低mmp21基因。敲低mmp21基因?qū)е掳唏R魚胚胎心臟、腸道以及腦部標(biāo)志物左右隨機(jī)分布,隨著mmp21MO濃度的增高,異常比例增高;活體胚胎心臟結(jié)構(gòu)異常,表現(xiàn)為心房、心室左右隨機(jī)分布;心臟功能損害,表現(xiàn)為心率減慢、心室收縮分?jǐn)?shù)減少(p0.05);KV面積變小(p0.05),囊泡內(nèi)運(yùn)動(dòng)纖毛數(shù)量減少、長(zhǎng)度變短(p0.05);中線分子屏障lefty1部分或全部缺失。結(jié)論:敲低mmp21基因?qū)е掳唏R魚心臟和其他心外結(jié)構(gòu)(腸道、腦部)左右不對(duì)稱發(fā)育異常以及器官功能損害,其可能與左右不對(duì)稱發(fā)育關(guān)鍵結(jié)構(gòu)KV和纖毛缺陷,以及中線分子屏障缺失有關(guān)。本研究以斑馬魚為模式動(dòng)物初步探討mmp21基因在胚胎左右不對(duì)稱發(fā)育中的作用,為今后深入研究mmp21基因奠定了基礎(chǔ),同時(shí)也為mmp21基因在人類胚胎發(fā)育中的作用帶來新的啟示。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R321

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