【摘要】：[研究背景] SLC33A1(Solute carrier family33, member1)是在研究神經(jīng)節(jié)苷脂O-乙�；倪^(guò)程中被發(fā)現(xiàn)的一個(gè)基因,位于人類染色體3q25,因?yàn)槠淇梢詫⒓?xì)胞質(zhì)中的乙酰輔酶A轉(zhuǎn)運(yùn)至高爾基體,為神經(jīng)節(jié)苷脂O-乙�；峁┮阴；�,所以又名AT-1(the acetyl-Co A transporter1)。研究發(fā)現(xiàn)很多內(nèi)質(zhì)網(wǎng)蛋白可以被SLC33A1調(diào)節(jié),包括β位淀粉樣前體蛋白裂解酶1(β-site APP cleaving enzyme1, BACE1)。淀粉樣蛋白前體(amyloid protein precursor, APP)和低密度脂蛋白受體(low density lipoprotein receptor, LDLR)等,這些蛋白質(zhì)在翻譯修飾后對(duì)于膜結(jié)構(gòu)的組裝和分泌蛋白的運(yùn)輸有重要作用。如果SLC33A1的表達(dá)異常,則會(huì)引起一系列神經(jīng)系統(tǒng)相關(guān)的疾病,如散發(fā)性肌萎縮側(cè)索硬化(amyotrophic lateral sclerosis, ALS)、晚發(fā)性阿爾茨海默病(Alzheimer's disease, AD)等。 本實(shí)驗(yàn)室2008年在山東青島地區(qū)發(fā)現(xiàn)了一個(gè)遺傳性痙攣性截癱(hereditary spastic paraplegia, HSP)大家系,呈常染色體顯性遺傳。采用全基因組掃查方法和定位候選策略確定人類SLC33A1基因是該家系的致病基因,患者均攜帶S113R錯(cuò)義突變,家系中的正常個(gè)體和群體中未檢測(cè)到該突變。斑馬魚實(shí)驗(yàn)顯示,抑制斑馬魚slc33al基因表達(dá)影響運(yùn)動(dòng)神經(jīng)元軸突生長(zhǎng),這種異常能被人類野生型SLC33A1基因拯救而不能被突變型拯救。由以上研究可知,SLC33A1對(duì)于維持神經(jīng)元『正常功能非常重要,但SLC33A1表達(dá)異常導(dǎo)致HSP的作用機(jī)制尚不明確,因此,有必要深入研究和探討SLC33A1蛋白的功能和作用機(jī)制。 [研究目的] 通過(guò)構(gòu)建slc33al突變基因敲入小鼠模型,檢測(cè)SLC33A1突變對(duì)小鼠表型的影響,以探討SLC33A1的功能。 [研究方法] 構(gòu)建slc33al突變基因敲入小鼠模型,并通過(guò)Slc33al突變基因敲入小鼠的雜合子與雜合子交配,檢測(cè)子代小鼠基因型是否符合預(yù)期分離比例。如果不能得到純合子新生小鼠,提示純合子小鼠在胚胎期致死,則通過(guò)解剖雜合子與雜合子交配的孕鼠分離得到不同胚胎時(shí)期的胎鼠(E3.5、E7.5、E18.5),利用單純PCR擴(kuò)增法和PCR-RFLP法對(duì)胎鼠進(jìn)行基因型鑒定。根據(jù)以上各個(gè)發(fā)育時(shí)期胚胎的基因型鑒定和分型結(jié)果,確定純合子小鼠的致死時(shí)間。 [研究結(jié)果] 成功構(gòu)建slc33a1突變基因敲入小鼠模型。對(duì)雜合子小鼠交配子代基因型分析結(jié)果顯示,可以獲得預(yù)期比例的雜合子,但不能得到純合子小鼠,提示純合子小鼠胚胎期致死。通過(guò)解剖與雜合子雄鼠交配的雜合子孕鼠,得到的E7.5和E18.5胚胎均無(wú)純合子,E3.5胚胎有少量純合子存活。 [結(jié)論] SLC33A1基因突變導(dǎo)致純合子小鼠胚胎早期致死,SLC33A1對(duì)早期胚胎發(fā)育有非常重要的作用。
[Abstract]:[background] SLC33A1 (Solute carrier family33, member1) is a gene found in the study of gangliosides O-acetylation, which is located on human chromosome 3q25. because it can transport acetylcoenzyme A from cytoplasm to Gore matrix and provide acetylgroup for gangliosides O-acetylation, it is also known as AT-1 (the acetyl-Co A transporter1). It has been found that many endoplasmic reticulum proteins can be regulated by SLC33A1, including 尾-site APP cleaving enzyme1, BACE1. Starch protein precursor (amyloid protein precursor, APP) and low density lipoprotein receptor (low density lipoprotein receptor, LDLR), these proteins play an important role in the assembly of membrane structure and the transport of secretory proteins after translation modification. If the expression of SLC33A1 is abnormal, it will cause a series of nervous system related diseases, such as sporadic amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), late onset Alzheimer's disease (Alzheimer's disease, AD) and so on. In 2008, our laboratory found a genetic spastic paraplegia (hereditary spastic paraplegia, HSP) family in Qingdao, Shandong Province, showing autosomal dominant inheritance. The whole genome scanning method and localization candidate strategy were used to determine that human SLC33A1 gene was the pathogenic gene of the pedigree. All the patients carried S113R missense mutation, and the mutation was not detected in normal individuals and populations in the family. Zebrafish experiments showed that inhibition of slc33al gene expression in zebrafish affected axonal growth of motor neurons, which could be saved by human wild type SLC33A1 gene but not by mutant. From the above studies, it can be seen that SLC33A1 is very important to maintain the "normal function of neurons, but the mechanism of HSP caused by abnormal expression of SLC33A1 is not clear. Therefore, it is necessary to further study and explore the function and mechanism of SLC33A1 protein. [objective] to detect the effect of SLC33A1 mutation on the phenotype of mice by constructing the mouse model of slc33al mutant gene knockout in order to explore the function of SLC33A1. [methods] the mouse model of slc33al mutant gene knockout was established, and the heterozygote and heterozygote of Slc33al mutant gene knockout mice were mated to detect whether the genotype of the offspring mice was in accordance with the expected segregation ratio. If the homozygotic neonatal mice could not be obtained, suggesting that the homozygotic mice died in the embryonic stage, the fetal mice (E3.5, E7.5, E18.5) were isolated from the pregnant mice mated with heterozygote and dissected. The genotyping of the fetal mice was identified by simple PCR amplification and PCR-RFLP. According to the genotypic identification and typing of embryos at each developmental stage, the lethal time of homozygous mice was determined. [results] the mouse model of slc33a1 mutant gene knockout was successfully constructed. The results of genotypic analysis of gametophyte generation in heterozygous mice showed that the expected proportion of heterozygotes could be obtained, but the homozygotic mice could not be obtained, suggesting that the embryo death of homozygous mice. By dissecting the heterozygotic pregnant mice mated with heterozygous male mice, the E7.5 and E18.5 embryos had no homozygote, and a small amount of homozygote survived in E3.5 embryos. [conclusion] SLC33A1 gene mutation leads to early embryo death in homozygous mice. SLC33A1 plays a very important role in early embryonic development.
【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)】：R363

【共引文獻(xiàn)】

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本文編號(hào)：2506505