lmo4a基因在斑馬魚(yú)內(nèi)耳發(fā)育中的功能研究
發(fā)布時(shí)間：2018-09-10 16:04
【摘要】：目的：世界范圍內(nèi)約有2.78億人患有中度以上的聽(tīng)力損失,在中國(guó)聽(tīng)力語(yǔ)言殘疾患者達(dá)2780萬(wàn)人。聾病防治主要包括傳導(dǎo)性耳聾和感音神經(jīng)性耳聾兩個(gè)方面。半個(gè)世紀(jì)以來(lái),傳導(dǎo)性耳聾已經(jīng)取得成熟的防治經(jīng)驗(yàn),感音神經(jīng)性耳聾的預(yù)防和治療是聾病基礎(chǔ)研究的熱點(diǎn)與難點(diǎn)。臨床上,很多感音神經(jīng)性耳聾為內(nèi)耳發(fā)育異常所引起。內(nèi)耳畸形和耳聾病程復(fù)雜,功能為不可逆缺失,治療方法相對(duì)局限。尋找更多的內(nèi)耳畸形、耳聾致病基因,探索致畸致聾機(jī)制,并篩選針對(duì)發(fā)病機(jī)制特異性治療制劑,已成為耳科學(xué)基礎(chǔ)與臨床研究者的共同責(zé)任。LM04基因編碼一種轉(zhuǎn)錄調(diào)節(jié)因子,小鼠耳泡中組織特異性敲除LM04,可見(jiàn)半規(guī)管發(fā)育異常、半規(guī)管嵴發(fā)育不良。因此LM04得到更多學(xué)者的關(guān)注。斑馬魚(yú)內(nèi)耳發(fā)生機(jī)制以及前庭形態(tài)、功能保守,胚胎透明、易于操作,是研究?jī)?nèi)耳發(fā)育畸形和耳聾的優(yōu)勢(shì)模型。本實(shí)驗(yàn)主要研究lmo4基因?qū)Π唏R魚(yú)內(nèi)耳發(fā)育的影響,并探索影響機(jī)制,為內(nèi)耳畸形、感應(yīng)神經(jīng)性耳聾的防治奠定理論基礎(chǔ)。 方法：本文主要采用斑馬魚(yú)胚胎內(nèi)注射寡核苷酸Morpholino (MO)和mRNA的方法構(gòu)建lmo4基因低表達(dá)和過(guò)表達(dá)模型；通過(guò)表型觀察、原位雜交、免疫組化等方式研究lmo4基因?qū)Π唏R魚(yú)內(nèi)耳發(fā)育的重要作用；通過(guò)篩選與內(nèi)耳結(jié)構(gòu)發(fā)生密切相關(guān)的基因表達(dá)變化研究lmo4基因影響內(nèi)耳發(fā)育的機(jī)制；并針對(duì)機(jī)制篩選可治療挽救表型的制劑。 結(jié)果：斑馬魚(yú)中l(wèi)mo4的兩個(gè)同源基因分別是lmo4a和lmo4b,兩者均為母源性基因。10hpf,lmo4a主要在中胚葉及神經(jīng)板中表達(dá)；14hpf開(kāi)始表達(dá)于耳基板、耳泡及周圍基質(zhì)；隨著耳泡發(fā)育,lmo4a表達(dá)范圍與毛細(xì)胞區(qū)域部分重疊,并標(biāo)記與半規(guī)管發(fā)生緊密相關(guān)的3個(gè)半規(guī)管嵴及半規(guī)管突起。lmo4b在耳泡、周圍基質(zhì)、及側(cè)線中持續(xù)表達(dá)。通過(guò)Morpholino低表達(dá)lmo4b后未觀察到內(nèi)耳表型,我們后期的研究主要集中于lmo4a在內(nèi)耳的功能。通過(guò)Morpholino低表達(dá)lmo4a后發(fā)現(xiàn)前基板發(fā)育受阻；耳泡大小、毛細(xì)胞數(shù)目、聽(tīng)平衡神經(jīng)節(jié)內(nèi)細(xì)胞數(shù)目呈等比減少；同時(shí)伴有特異的半規(guī)管發(fā)育異常(僅水平半規(guī)管突起形成)；與半規(guī)管發(fā)育緊密相關(guān)的bmp4,bmp2b明顯上調(diào),同時(shí)下調(diào)BMP信號(hào)可很大程度挽救低表達(dá)lmo4a誘導(dǎo)的半規(guī)管不發(fā)育表型。 結(jié)論：低表達(dá)lmo4a誘導(dǎo)的耳泡大小、內(nèi)耳毛細(xì)胞和神經(jīng)節(jié)的數(shù)量減小可歸因于lmo4a對(duì)前基板早期發(fā)育的影響；而半規(guī)管畸形是由于BMP信號(hào)上調(diào)導(dǎo)致；耳泡內(nèi)注射DM (AMP-activated protein kinase inhibitor, Dorsomorphin)小分子可下調(diào)BMP信號(hào),促進(jìn)半規(guī)管發(fā)育,挽救畸形。本實(shí)驗(yàn)探討了lmo4a基因在斑馬魚(yú)內(nèi)耳發(fā)育的重要作用和作用機(jī)制,耳泡內(nèi)注射小分子DM可促進(jìn)半規(guī)管發(fā)育,挽救半規(guī)管畸形。
[Abstract]:Objective: there are about 278 million people with moderate or more hearing loss worldwide and 27.8 million people with hearing and speech disability in China. The prevention and treatment of deafness mainly include conductive deafness and sensorineural deafness. The prevention and treatment of sensorineural deafness has been a hot and difficult point in the basic research of deafness since half a century, and the prevention and treatment of sensorineural deafness have gained mature experience. Clinically, many sensorineural deafness is caused by abnormal development of the inner ear. The course of inner ear deformity and deafness is complicated, the function is irreversible, and the treatment method is relatively limited. Searching for more genes causing deafness and deafness, exploring the mechanism of deafness, and screening specific therapeutic agents for the pathogenesis of deafness have become the common responsibility of the basis of otorology and clinical researchers to encode a transcriptional regulatory factor of .LM04 gene. The abnormal development of semicircular canal and dysplasia of semicircular canal cristae were observed in LM04, with tissue specific knockout in mouse ear vesicles. So LM04 gets more scholars' attention. Zebrafish internal ear formation mechanism, vestibular morphology, conserved function, transparent embryo, easy to operate, is the dominant model for the study of deformity and deafness of the inner ear. In this study, the effect of lmo4 gene on the development of zebrafish inner ear was studied, and the mechanism was explored to lay a theoretical foundation for the prevention and treatment of deformity of inner ear and sensorineural deafness. Methods: a low expression and overexpression model of lmo4 gene was constructed by injecting oligonucleotide Morpholino (MO) and mRNA into zebrafish embryos. The important role of lmo4 gene in the development of inner ear of zebrafish was studied by immunohistochemistry, and the mechanism of lmo4 gene affecting the development of inner ear was studied by screening the gene expression changes closely related to the structure of inner ear. And to screen the mechanism of the treatment of phenotypic agents. Results: the two homologous genes of lmo4 in zebrafish, lmo4a and lmo4b, were both maternal genes. 10hpflmo4a was mainly expressed in mesoderm and nerve plate. With the development of auricle, the expression range of lmo4a overlapped with that of hair cells, and the expression of three semicircular canal crest and semicircular canal protrusions, which were closely related to semicircular canal development, was continuously expressed in auricle, peripheral matrix and lateral line. No phenotype of inner ear was observed after low expression of lmo4b in Morpholino. Our later studies focused on the function of lmo4a in inner ear. After the low expression of lmo4a in Morpholino, it was found that the development of anterior basal plate was blocked, the size of ear follicles, the number of hair cells and the number of cells in auditory balanced ganglion decreased, and the abnormal development of semicircular canal (only horizontal semicircular canal processes) was observed. Bmp4,bmp2b closely related to semicircular canal development was upregulated, and down-regulation of BMP signal could save the dysplastic phenotype of semicircular canal induced by low expression of lmo4a to a great extent. Conclusion: the decrease in the number of hair cells and ganglion of inner ear induced by low expression of lmo4a can be attributed to the effect of lmo4a on the early development of anterior basal plate, while the semicircular canal malformation is caused by upregulation of BMP signal. Intravesicular injection of DM (AMP-activated protein kinase inhibitor, Dorsomorphin) small molecule) can down-regulate BMP signal, promote semicircular canal development and save malformation. In this study, we investigated the important role and mechanism of lmo4a gene in the development of zebrafish inner ear. Injection of small molecule DM into ear vesicle could promote semicircular canal development and save semicircular canal malformation.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764
本文編號(hào)：2234935
[Abstract]:Objective: there are about 278 million people with moderate or more hearing loss worldwide and 27.8 million people with hearing and speech disability in China. The prevention and treatment of deafness mainly include conductive deafness and sensorineural deafness. The prevention and treatment of sensorineural deafness has been a hot and difficult point in the basic research of deafness since half a century, and the prevention and treatment of sensorineural deafness have gained mature experience. Clinically, many sensorineural deafness is caused by abnormal development of the inner ear. The course of inner ear deformity and deafness is complicated, the function is irreversible, and the treatment method is relatively limited. Searching for more genes causing deafness and deafness, exploring the mechanism of deafness, and screening specific therapeutic agents for the pathogenesis of deafness have become the common responsibility of the basis of otorology and clinical researchers to encode a transcriptional regulatory factor of .LM04 gene. The abnormal development of semicircular canal and dysplasia of semicircular canal cristae were observed in LM04, with tissue specific knockout in mouse ear vesicles. So LM04 gets more scholars' attention. Zebrafish internal ear formation mechanism, vestibular morphology, conserved function, transparent embryo, easy to operate, is the dominant model for the study of deformity and deafness of the inner ear. In this study, the effect of lmo4 gene on the development of zebrafish inner ear was studied, and the mechanism was explored to lay a theoretical foundation for the prevention and treatment of deformity of inner ear and sensorineural deafness. Methods: a low expression and overexpression model of lmo4 gene was constructed by injecting oligonucleotide Morpholino (MO) and mRNA into zebrafish embryos. The important role of lmo4 gene in the development of inner ear of zebrafish was studied by immunohistochemistry, and the mechanism of lmo4 gene affecting the development of inner ear was studied by screening the gene expression changes closely related to the structure of inner ear. And to screen the mechanism of the treatment of phenotypic agents. Results: the two homologous genes of lmo4 in zebrafish, lmo4a and lmo4b, were both maternal genes. 10hpflmo4a was mainly expressed in mesoderm and nerve plate. With the development of auricle, the expression range of lmo4a overlapped with that of hair cells, and the expression of three semicircular canal crest and semicircular canal protrusions, which were closely related to semicircular canal development, was continuously expressed in auricle, peripheral matrix and lateral line. No phenotype of inner ear was observed after low expression of lmo4b in Morpholino. Our later studies focused on the function of lmo4a in inner ear. After the low expression of lmo4a in Morpholino, it was found that the development of anterior basal plate was blocked, the size of ear follicles, the number of hair cells and the number of cells in auditory balanced ganglion decreased, and the abnormal development of semicircular canal (only horizontal semicircular canal processes) was observed. Bmp4,bmp2b closely related to semicircular canal development was upregulated, and down-regulation of BMP signal could save the dysplastic phenotype of semicircular canal induced by low expression of lmo4a to a great extent. Conclusion: the decrease in the number of hair cells and ganglion of inner ear induced by low expression of lmo4a can be attributed to the effect of lmo4a on the early development of anterior basal plate, while the semicircular canal malformation is caused by upregulation of BMP signal. Intravesicular injection of DM (AMP-activated protein kinase inhibitor, Dorsomorphin) small molecule) can down-regulate BMP signal, promote semicircular canal development and save malformation. In this study, we investigated the important role and mechanism of lmo4a gene in the development of zebrafish inner ear. Injection of small molecule DM into ear vesicle could promote semicircular canal development and save semicircular canal malformation.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764
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