本文選題：嗅球　切入點(diǎn)：MicroRNAs　出處：《浙江大學(xué)》2016年博士論文

【摘要】：嗅覺系統(tǒng)是屬于邊緣系統(tǒng)(limbic system)的一部分,由嗅覺上皮,主嗅球和嗅皮質(zhì)組成。嗅覺在動(dòng)物尋找食物,識(shí)別危險(xiǎn),尋找配偶和社會(huì)交往具有重要的意義。成體室下區(qū)(SVZ)是哺乳動(dòng)物成體大腦神經(jīng)元發(fā)生區(qū)域之一。從SVZ神經(jīng)母細(xì)胞(neuroblast)沿嘴側(cè)遷移流(RMS)遷移到OB和分化成顆粒細(xì)胞(GCs)和球周細(xì)胞(PGs),然后整合到嗅球的突觸環(huán)路。顆粒細(xì)胞屬于抑制性神經(jīng)元其數(shù)量占嗅球中間神經(jīng)元的95%。他們的包體在顆粒細(xì)胞層(GCL)中生長幾個(gè)短基底樹突,而頂樹突橫跨僧帽細(xì)胞層(MCL),延申到外叢狀層(EPL)。microRNA屬于Small ncRNAs,是單鏈的短RNA,長度在18-25個(gè)核苷酸分子左右。microRNA是轉(zhuǎn)錄后基因表達(dá)的主要調(diào)節(jié)分子。microRNA以序列互補(bǔ)的方式與特異靶mRNA結(jié)合,通過降解靶基因mRNA或抑制其蛋白翻譯調(diào)控靶基因的表達(dá)。microRNA在神經(jīng)元發(fā)育、腦退行性疾病中發(fā)揮重要作用。miR-124是一種腦特定的miRNA,在小鼠大腦的分化和成熟的神經(jīng)元中占所有大腦的miRNA的25-48%。miR-124其表達(dá)的水平,在發(fā)育的小鼠的中樞神經(jīng)系統(tǒng)(CNS)中是逐漸增加的。miR-124決定成體室下區(qū)(SVZ)神經(jīng)元前體的命運(yùn),并調(diào)節(jié)成年神經(jīng)元在SVZ的再生。miR-124重要功能是調(diào)控神經(jīng)元發(fā)育以及在神經(jīng)退行性疾病中發(fā)揮重要作用。小G蛋白(Small G Protein),因分子量只有20～30KD而得名,是一類位于膜內(nèi)側(cè)的偶聯(lián)蛋白,通過與膜受體胞漿區(qū)結(jié)合,將膜受體與配體結(jié)合的刺激與下游的效應(yīng)分子偶聯(lián)起來。RhoGTPases家族通過調(diào)控肌動(dòng)蛋白細(xì)胞骨架,在突觸可塑性神經(jīng)元的樹突棘的形態(tài)發(fā)生中發(fā)揮重要的作用。通過miRNA芯片方法揭示了嗅球的miRNA表達(dá)譜,發(fā)現(xiàn)miR-124是嗅球中最高表達(dá)的miRNA。通過靶基因預(yù)測以及功能網(wǎng)絡(luò)富集,我們了解到miR-124參與調(diào)控細(xì)胞骨架重要功能。通過構(gòu)建miR-124過表達(dá)和抑制慢病毒載體,并通過腦立體定位注射進(jìn)小鼠的RMS,我們證明了 miR-124在小鼠嗅球顆粒細(xì)胞發(fā)育后期調(diào)控樹突形態(tài)發(fā)育以及樹突棘密度。在miR-124調(diào)控細(xì)胞骨架靶基因富集組分中我們發(fā)現(xiàn)小G蛋白R(shí)hoG可能具有重要作用。通過構(gòu)建了 RhoG的過表達(dá)載體,RhoG的組成性激活突變,RhoG的組成性失活載體以及RhoG的shRNA慢病毒載體。通過腦立體定位注射到小鼠的RMS,我們觀察RhoG是否能促進(jìn)嗅球顆粒細(xì)胞樹突形態(tài)發(fā)育以及樹突棘密度。結(jié)果顯示RhoG是通過下游Rac1的激活以及PAK和confilind的磷酸化發(fā)揮作用的。RhoG的表達(dá)模式與miR-124很相似。這種看似矛盾但是可以理解為RhoG有可能參與調(diào)控遷移作用,所以RhoG在神經(jīng)母細(xì)胞從SVZ到嗅球首先可能發(fā)揮遷移作用,但是這種功能受到miR-124的調(diào)控。所以推測miR-124可能通過RhoG調(diào)控嗅球顆粒細(xì)胞形態(tài)合適的發(fā)育水平。
[Abstract]:Olfactory system is a part of limbic system, which consists of olfactory epithelium, main olfactory bulb and olfactory cortex.Smell plays an important role in finding food for animals, identifying dangers, and finding spouses and social contacts.Adult subventricular area (SVZ) is one of the neuronal regions in mammalian adult brain.SVZ neuroblastcells migrated to OB and differentiated into granulosa cells (GCs) and peribulbar cells (PGS), and then integrated into the synaptic loop of olfactory bulb.Granulosa cells belong to inhibitory neurons which account for 95% of olfactory bulb intermediate neurons.Their inclusions grow several short basal dendrites in the granular cell layer (GCLs).The apical dendrites straddle the lamina of mitral cells and extend to the outer plexiform layer of EPLN. MicroRNAs belong to Small ncRNs, which are short RNAs with a length of 18-25 nucleotides. MicroRNAs are the major regulatory molecules of post-transcriptional gene expression. MicroRNAs bind to specific mRNA in a sequence complementary manner.The expression of target gene was regulated by degradation of target gene mRNA or inhibition of protein translation.Brain degenerative disease. MiR-124 is a brain-specific miRNAs that account for the 25-48%.miR-124 expression level of miRNA in all brain differentiated and mature neurons in mice.In the central nervous system (CNS) of developing mice, the increasing number of .miR-124 determines the fate of SVZ) neurons in the adult subventricular area.The important function of regulating the regeneration of adult neurons in SVZ. MiR-124 is to regulate the development of neurons and play an important role in neurodegenerative diseases.Small G protein, named for its molecular weight of only 20~30KD, is a class of conjugated proteins located on the medial side of the membrane that bind to the cytosolic domain of the membrane receptor.The membrane receptor ligand binding stimuli are coupled with downstream effector molecules. The RhoGTPases family plays an important role in the morphogenesis of dendritic spine in synaptic plasticity neurons by regulating actin cytoskeleton.The miRNA expression profile of olfactory bulb was revealed by miRNA chip method. It was found that miR-124 was the most expressed miRNA in olfactory bulb.Through target gene prediction and functional network enrichment, we understand that miR-124 plays an important role in regulating cytoskeleton.By constructing miR-124 overexpression vector and inhibiting lentivirus vector, and injecting miR-124 into mouse by stereotactic localization, we demonstrated that miR-124 regulates dendritic morphology and dendritic spine density in the later stage of mouse olfactory bulb granulosa cell development.We found that small G protein RhoG may play an important role in the regulation of cytoskeleton target gene enrichment by miR-124.The constitutive inactivation vector of RhoG and the shRNA lentivirus vector of RhoG were constructed.We observed whether RhoG could promote dendritic development and dendritic spinous density of olfactory bulb granulosa cells by stereotaxic injection of RMS in mice.The results showed that RhoG was activated by downstream Rac1 and phosphorylation of PAK and confilind. The expression pattern of. RhoG was similar to that of miR-124.This seems paradoxical but it can be understood that RhoG may be involved in the regulation of migration, so RhoG may first play a role in migration from SVZ to olfactory bulb in neuroblastocytes, but this function is regulated by miR-124.Therefore, it is speculated that miR-124 may regulate the morphology of olfactory bulb granulosa cells by RhoG.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R339.12

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