生殖細(xì)胞特異性基因的表達(dá)受甲基化和轉(zhuǎn)錄因子的雙重調(diào)控
發(fā)布時(shí)間：2018-07-15 22:34
【摘要】：在體內(nèi)原始生殖細(xì)胞(PGC, primordial germ cell)的數(shù)目是極其有限的,最終約26,000個(gè),且不易分離得到；目前已知在PGC形成初期特異性表達(dá)Blimp1、中期Stella和Nanos3,后期表達(dá)Vasa、Dazl及減數(shù)分裂時(shí)期表達(dá)Nanos2和Stra8外,其他已知的PGC特異性標(biāo)志基因很少；另外,目前人類對(duì)PGC形成的機(jī)理尚不清楚�；谏鲜鲈�,我們利用傳統(tǒng)的EB (embryonic body)體外分化體系來(lái)模擬PGC的分化形成,通過(guò)基因的篩選希望能找到促進(jìn)ES (embryonic stem cell)細(xì)胞分化形成PGC的基因,并進(jìn)而發(fā)現(xiàn)PGC細(xì)胞新的標(biāo)志性基因,從而對(duì)PGC的形成機(jī)理有更深入的了解。 本論文包括兩部分內(nèi)容：第一部分是能促進(jìn)胚胎干細(xì)胞向原始生殖細(xì)胞分化的基因的篩選。通過(guò)傳統(tǒng)的EB分化體系、SSEA1染色和AP染色,檢測(cè)了基因Sohlh1、Sohlh2、 Fgd1和Fkbp3對(duì)原始生殖細(xì)胞形成的影響。發(fā)現(xiàn)基因Sohlh1對(duì)PGC的形成可能有促進(jìn)作用,基因Sohlh2、Fgdl和Fkbp3對(duì)PGC的形成無(wú)明顯影響。第二部分內(nèi)容是生殖細(xì)胞特異性基因的表達(dá)受轉(zhuǎn)錄因子和DNA甲基化的雙重調(diào)節(jié)�；诒緦�(shí)驗(yàn)室的其他研究發(fā)現(xiàn)生殖細(xì)胞特異性基因As1對(duì)ES向原始生殖細(xì)胞的分化起促進(jìn)作用,在研究其作用機(jī)理的過(guò)程中發(fā)現(xiàn)轉(zhuǎn)錄因子Nrf1(Nuclear respiratory factor1)結(jié)合在它的啟動(dòng)子上,對(duì)其表達(dá)有一定的調(diào)節(jié)作用,通過(guò)熒光素酶報(bào)告基因技術(shù)(luciferase assay)的進(jìn)一步證明了Nrf1對(duì)Asz1的表達(dá)起到促進(jìn)作用,而且Nrfl的結(jié)合位點(diǎn)富含CG,結(jié)果表明Nrf1的結(jié)合受到DNA甲基化的影響。我們廣泛分析了一些生殖細(xì)胞特異性基因的啟動(dòng)子,發(fā)現(xiàn)基因Ddx25、Lin28上都有Nrf1的結(jié)合位點(diǎn),且基因Ddx25的啟動(dòng)子上也富含CG。通過(guò)熒光素酶報(bào)告基因技術(shù),我們證明了Nrf1能夠促進(jìn)基因Ddx25和Lin28的表達(dá),當(dāng)Ddx25被體外甲基化處理后,Nrf1的促進(jìn)作用消失。另外,我們發(fā)現(xiàn)許多生殖細(xì)胞特異性的基因的啟動(dòng)子上含有轉(zhuǎn)錄因子Oct1的結(jié)合位點(diǎn),并通過(guò)熒光素酶報(bào)告基因技術(shù)試驗(yàn)證明了Oct1能夠促進(jìn)基因Vasa的表達(dá)。我們認(rèn)為Oct1、Nrf1可能對(duì)生殖細(xì)胞特異性的基因的表達(dá)起著重要的調(diào)節(jié)作用,而這種調(diào)控受到甲基化的雙重影響。我們的研究,對(duì)生殖細(xì)胞特異性基因表達(dá)的表觀遺傳學(xué)修飾有一個(gè)初步的認(rèn)識(shí)。
[Abstract]:In vivo, the number of primordial germ cells (PGCs, primordial germ cell) is very limited, and it is not easy to isolate. It is known that Blimp1, Stella and Nanos3 are specifically expressed in the early stage of PGC formation, Stella and Nanos3 are expressed in the middle stage, VasaanDazl is expressed in the late stage, and Nanos2 and Stra8 are expressed in meiosis. Other known PGC specific marker genes are few, and the mechanism of PGC formation is still unclear. For the above reasons, we used the traditional EB (embryonic body) differentiation system to simulate the differentiation of es (embryonic stem cell) cells in vitro. Through gene screening, we hope to find the genes that promote the differentiation of es (embryonic stem cell) cells. Furthermore, a new signature gene was found in PGC cells, and the formation mechanism of PGC was further understood. This thesis consists of two parts: the first part is the screening of genes that can promote embryonic stem cells to differentiate into primitive germ cells. The effects of genes Sohlh1, Fgd1 and Fkbp3 on the formation of primordial germ cells were detected by traditional EB differentiation system, SSEA1 staining and AP staining. It was found that the gene Sohlh1 might promote the formation of PGC, while the genes Sohlh2Fgdl and Fkbp3 had no effect on the formation of PGC. The second part is that the expression of germ cell specific genes is regulated by transcription factors and DNA methylation. Based on other studies in our laboratory, the germ cell-specific gene As1 was found to promote the differentiation of es into primordial germ cells, and the transcription factor Nrf1 (Nuclear respiratory factor1) was found to bind to its promoter in the course of studying its mechanism. Through luciferase reporter gene technique, (luciferase assay) further proved that Nrf1 promoted the expression of Asz1, and the binding site of Nrfl was rich in CG. the results showed that the binding of Nrf1 was affected by methylation of Asz1. Some promoters of germ cell-specific genes were extensively analyzed. It was found that Nrf1 binding sites were found in the gene Ddx25hLin28, and the promoter of the gene Ddx25 was also rich in CG. By luciferase reporter gene technique, we have proved that Nrf1 can promote the expression of Ddx25 and Lin28, and the promotion of Nrf1 disappeared when Ddx25 was methylated in vitro. In addition, we found the binding sites of transcription factor Oct1 in the promoters of many germ cell-specific genes. Oct1 can promote the expression of Vasa by luciferase reporter gene technique. We believe that Oct1nrf1 may play an important role in regulating the expression of germ cell-specific genes, which is affected by methylation. We have a preliminary understanding of epigenetic modification of germ cell specific gene expression.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
[Abstract]:In vivo, the number of primordial germ cells (PGCs, primordial germ cell) is very limited, and it is not easy to isolate. It is known that Blimp1, Stella and Nanos3 are specifically expressed in the early stage of PGC formation, Stella and Nanos3 are expressed in the middle stage, VasaanDazl is expressed in the late stage, and Nanos2 and Stra8 are expressed in meiosis. Other known PGC specific marker genes are few, and the mechanism of PGC formation is still unclear. For the above reasons, we used the traditional EB (embryonic body) differentiation system to simulate the differentiation of es (embryonic stem cell) cells in vitro. Through gene screening, we hope to find the genes that promote the differentiation of es (embryonic stem cell) cells. Furthermore, a new signature gene was found in PGC cells, and the formation mechanism of PGC was further understood. This thesis consists of two parts: the first part is the screening of genes that can promote embryonic stem cells to differentiate into primitive germ cells. The effects of genes Sohlh1, Fgd1 and Fkbp3 on the formation of primordial germ cells were detected by traditional EB differentiation system, SSEA1 staining and AP staining. It was found that the gene Sohlh1 might promote the formation of PGC, while the genes Sohlh2Fgdl and Fkbp3 had no effect on the formation of PGC. The second part is that the expression of germ cell specific genes is regulated by transcription factors and DNA methylation. Based on other studies in our laboratory, the germ cell-specific gene As1 was found to promote the differentiation of es into primordial germ cells, and the transcription factor Nrf1 (Nuclear respiratory factor1) was found to bind to its promoter in the course of studying its mechanism. Through luciferase reporter gene technique, (luciferase assay) further proved that Nrf1 promoted the expression of Asz1, and the binding site of Nrfl was rich in CG. the results showed that the binding of Nrf1 was affected by methylation of Asz1. Some promoters of germ cell-specific genes were extensively analyzed. It was found that Nrf1 binding sites were found in the gene Ddx25hLin28, and the promoter of the gene Ddx25 was also rich in CG. By luciferase reporter gene technique, we have proved that Nrf1 can promote the expression of Ddx25 and Lin28, and the promotion of Nrf1 disappeared when Ddx25 was methylated in vitro. In addition, we found the binding sites of transcription factor Oct1 in the promoters of many germ cell-specific genes. Oct1 can promote the expression of Vasa by luciferase reporter gene technique. We believe that Oct1nrf1 may play an important role in regulating the expression of germ cell-specific genes, which is affected by methylation. We have a preliminary understanding of epigenetic modification of germ cell specific gene expression.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
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