【摘要】：研究目的 SOX (Sry related high mobility group box)基因家族是一類由SRY相關(guān)基因構(gòu)成的基因家族,最初是由Gubbay. J等在小鼠Y染色體中發(fā)現(xiàn)的起性別決定作用的轉(zhuǎn)錄調(diào)控因子,其編碼的蛋白都包含保守型較高的高速泳動(dòng)蛋白(high mobility group proteins, HMG)結(jié)構(gòu)域。在個(gè)體的早期發(fā)育過程中,sox基因廣泛參與調(diào)控性別決定、軟骨形成、神經(jīng)系統(tǒng)發(fā)育、晶狀體形成及內(nèi)胚層發(fā)育等過程。 Soxl9b基因是斑馬魚SOXB1家族中重要成員之一,為母源性表達(dá),囊胚期之前為泛表達(dá)；合子型表達(dá)開始于75%下包時(shí)期,主要在預(yù)定神經(jīng)外胚層區(qū)域表達(dá)：原腸運(yùn)動(dòng)后期,soxl9b在神經(jīng)板區(qū)域特異性的表達(dá)；24h時(shí)期,ox19b的表達(dá)主要集中于中樞神經(jīng)系統(tǒng)及脊索區(qū)域；48h時(shí)期,主要表達(dá)在間腦、端腦、中腦和延腦區(qū)域,其表達(dá)模式與小鼠中sox2基因的表達(dá)基本一致。上述研究結(jié)果提示soxl9b基因可能在神經(jīng)系統(tǒng)發(fā)育的過程中發(fā)揮重要的調(diào)節(jié)作用。 本研究主要是通過抑制soxl9b基因的表達(dá),探索soxl9b基因?qū)τ谏窠?jīng)系統(tǒng)發(fā)育過程的調(diào)控作用及機(jī)制。目前,對(duì)于神經(jīng)系統(tǒng)發(fā)育相關(guān)疾病的發(fā)病機(jī)制及治療方法尚不成熟。因此,研究soxl9b基因在神經(jīng)系統(tǒng)發(fā)育過程的調(diào)控機(jī)制將為臨床治療神經(jīng)系統(tǒng)發(fā)生相關(guān)的疾病提供新的線索和理論依據(jù)。 研究方法 本研究主要是利用斑馬魚為模式生物,通過顯微注射嗎啉寡聚核苷酸(Morpholinos, MO)的方法抑制soxl9b基因的表達(dá),通過整封原位雜交、RT-PCR、組織切片HE染色、BrdU摻入及吖啶橙染色等方法,研究soxl9b基因在斑馬魚神經(jīng)系統(tǒng)發(fā)育過程中的調(diào)控作用及機(jī)制。 研究結(jié)果 1、soxl9b基因在斑馬魚胚胎早期發(fā)育過程中時(shí)空表達(dá)模式的建立 首先,我們通過RT-PCR檢測(cè)soxl9b基因在斑馬魚胚胎發(fā)育不同時(shí)期的表達(dá)量,然后通過胚胎整封原位雜交檢測(cè)soxl9b基因在斑馬魚胚胎發(fā)育不同時(shí)期的表達(dá)情況。結(jié)果顯示,soxl9b基因?yàn)槟冈葱员磉_(dá)的基因,囊胚期之前為泛表達(dá)；神經(jīng)外胚層形成之后,soxl9b主要在神經(jīng)外胚層區(qū)域表達(dá)；原腸運(yùn)動(dòng)后期,主要表達(dá)在神經(jīng)板區(qū)域；神經(jīng)分化結(jié)束時(shí),soxl9b基因主要表達(dá)在中樞神經(jīng)系統(tǒng)以及脊索區(qū)域。 2、soxl9b基因?qū)Π唏R魚胚胎早期神經(jīng)系統(tǒng)發(fā)育的調(diào)節(jié)作用 抑制soxl9b基因表達(dá)之后,斑馬魚胚胎的神經(jīng)系統(tǒng)發(fā)育過程受到影響。神經(jīng)誘導(dǎo)階段,原位雜交的檢測(cè)結(jié)果發(fā)現(xiàn),神經(jīng)外胚層的標(biāo)記基因sox3的表達(dá)區(qū)域明顯減小,神經(jīng)板的標(biāo)記基因otx2和dlx3b基因的表達(dá)也明顯減少。神經(jīng)發(fā)生階段,中前腦的標(biāo)記基因otx2及原神經(jīng)基因neurogl基因的表達(dá)也明顯降低。通過檢測(cè)神經(jīng)干細(xì)胞sox2及神經(jīng)前體細(xì)胞ascl1b基因的表達(dá)情況,結(jié)果顯示其表達(dá)也明顯減弱。通過BrdU摻入及吖啶橙染色等方法檢測(cè)胚胎細(xì)胞增殖及凋亡的情況,實(shí)驗(yàn)結(jié)果顯示,抑制sox19b基因的表達(dá),會(huì)導(dǎo)致中樞神經(jīng)系統(tǒng)區(qū)域細(xì)胞凋亡增加,細(xì)胞增殖減弱。 3、soxl9b基因?qū)Π唏R魚胚胎眼睛發(fā)育的調(diào)節(jié)作用 抑制soxl9b基因的表達(dá)會(huì)導(dǎo)致斑馬魚胚胎眼睛發(fā)育受到抑制,包括小眼或眼睛缺失,視網(wǎng)膜分層及晶狀體的結(jié)構(gòu)異常。視網(wǎng)膜的形成包括視網(wǎng)膜祖細(xì)胞的增殖,細(xì)胞命運(yùn)決定,神經(jīng)元及膠質(zhì)細(xì)胞形成等過程。通過原位雜交及RT-PCR進(jìn)一步研究發(fā)現(xiàn),參與調(diào)控眼區(qū)發(fā)育的眼區(qū)轉(zhuǎn)錄因子(eye field transcription factors, EFTFs) pax2a、vsx2及rx3的表達(dá)區(qū)域及表達(dá)量都明顯減弱或降低。 4、soxl9b基因在斑馬魚胚胎早期神經(jīng)系統(tǒng)發(fā)育過程中作用機(jī)制的研究 脊椎動(dòng)物神經(jīng)系統(tǒng)發(fā)育過程依靠外胚層細(xì)胞的胞內(nèi)信號(hào)通路之間的相互協(xié)調(diào)來實(shí)現(xiàn)的,包括BMP、FGF以及Wnt信號(hào)通路等,這些信號(hào)通路之間的協(xié)調(diào)作用使得機(jī)體神經(jīng)系統(tǒng)和非神經(jīng)系統(tǒng)分化得以實(shí)現(xiàn)。抑制soxl9b基因的表達(dá)后,通過RT-PCR檢測(cè)出BMP及FGF信號(hào)通路中部分配體及下游靶基因的表達(dá)都明顯降低。 研究結(jié)論 1、soxl9b基因調(diào)節(jié)神經(jīng)誘導(dǎo)及神經(jīng)發(fā)生的過程。 2、soxl9b基因參與調(diào)節(jié)神經(jīng)干細(xì)胞及神經(jīng)前體細(xì)胞的增殖和凋亡過程。 3、soxl9b基因調(diào)控斑馬魚胚胎眼睛的發(fā)育。4、soxl9b基因參與調(diào)控BMP及FGF信號(hào)通路。
[Abstract]:research objective
SOX (Sry related high mobility group box) gene family is a kind of gene family composed of SRY related genes. Originally, SOX (Sry related high mobility group box) gene family is a sex-determining transcription regulator found in Y chromosome of mice by Gubbay. Structural domains. During the early development of an individual, Sox genes are widely involved in the regulation of sex determination, cartilage formation, nervous system development, lens formation and endodermal development.
Soxl9b gene is one of the important members of the SOXB1 family in zebrafish, which is maternal expression and is ubiquitously expressed before blastocyst stage; zygotic expression begins at 75% subcapsular stage and is mainly expressed in the predetermined neuroectodermal region: soxl9b is locally expressed in the late gastrointestinal motility; ox19b expression is mainly concentrated in the middle during 24h. The expression pattern of soxl9b gene in the mesencephalon, telencephalon, mesencephalon and medulla oblongata was similar to that in mice. These results suggest that soxl9b gene may play an important regulatory role in the development of the nervous system.
The purpose of this study is to explore the regulatory role and mechanism of soxl9b gene in the development of nervous system by inhibiting the expression of soxl9b gene. At present, the pathogenesis and treatment of nervous system development-related diseases are still not mature. Therefore, the study of the regulation mechanism of soxl9b gene in the development of nervous system will be the clinical therapeutic spirit. It provides new clues and theoretical basis for diseases related to systemic diseases.
research method
In this study, zebrafish were used as model organisms to inhibit the expression of soxl9b gene by microinjection of Morpholinos (MO). The regulation of soxl9b gene during the development of zebrafish nervous system was studied by in situ hybridization, RT-PCR, HE staining, BrdU incorporation and acridine orange staining. Function and mechanism.
Research results
1, the establishment of spatio-temporal expression pattern of soxl9b gene in zebrafish embryos during the early stage of development.
Firstly, we detected the expression of soxl9b gene in different stages of zebrafish embryo development by RT-PCR, and then detected the expression of soxl9b gene in different stages of zebrafish embryo development by in situ hybridization. After maturation, soxl9b was mainly expressed in the neuroectodermal region, in the late gastrointestinal motility, mainly in the nerve plate region, and at the end of nerve differentiation, soxl9b gene was mainly expressed in the central nervous system and notochord region.
2, the regulation of soxl9b gene on the nervous system development of zebrafish embryos at the early stage.
After inhibiting the expression of soxl9b gene, the development of zebrafish embryonic nervous system was affected. In the neuroinduction stage, the expression of Sox3 gene in the neural ectoderm was significantly decreased, and the expression of Otx2 and dlx3b gene in the neural plate was also significantly decreased. The expression of labeled gene Otx2 and neurogl was also decreased. The expression of Sox2 and ascl1b in neural stem cells and neural precursor cells was also decreased. BrdU incorporation and acridine orange staining were used to detect the proliferation and apoptosis of embryonic cells. The expression of sox19b gene can increase cell apoptosis and weaken cell proliferation in the central nervous system region.
3, the regulation of soxl9b gene on the development of zebrafish embryos' eyes.
Inhibition of soxl9b gene expression may result in inhibition of eye development in zebrafish embryos, including the absence of microphthalmos or eyes, retinal stratification and lens structural abnormalities. Retinal formation includes the proliferation of retinal progenitor cells, cell fate determination, neuronal and glial formation and other processes. Further studies were carried out by in situ hybridization and RT-PCR. It was found that the expression of pax2a, VSX2 and RX3 in the ocular transcription factors (EFTFs) involved in the regulation of ocular development were significantly decreased or decreased.
4, the role of soxl9b gene in the development of nervous system during early stage of zebrafish embryos.
The development of the vertebrate nervous system depends on the coordination of intracellular signaling pathways in ectodermal cells, including BMP, FGF and Wnt signaling pathways. The coordination of these signaling pathways enables the differentiation of the nervous system and the non-nervous system. After inhibiting the expression of soxl9b gene, the expression of soxl9b is detected by RT-PCR The expression of some ligands and downstream target genes in BMP and FGF signaling pathway decreased significantly.
research conclusion
1, the soxl9b gene regulates neural induction and neurogenesis.
2, soxl9b gene is involved in regulating the proliferation and apoptosis of neural stem cells and neural precursor cells.
3, soxl9b gene regulates.4 development in zebrafish embryo eyes, and soxl9b gene is involved in regulating BMP and FGF signaling pathways.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R338;Q75

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本文編號(hào)：2211917