【作者】 潘传英；

【导师】 陈宏； Colin E Bishop；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2010， 博士

【摘要】 本研究以转录因子为研究重点,利用PCR-SSCP、PCR-RFLP、DNA测序、细胞转染、蛋白表达纯化、体外转录等方法,以特定因子组合的基因、蛋白和RNA等三种方式,研究了特定转录因子在哺乳动物的生长、发育和体细胞重编程过程中的作用,获得了如下主要结果:1.垂体转录因子基因POU1F1和PROP1的遗传效应分析⑴利用SSCP和DNA测序,在8个中国地方牛品种中检测到了POU1F1基因第2外显子区域存在三个SNP位点,建立了一种TaqI PCR-RFLP的方法检测其中一个新的同义突变位点（NM₁74579:c.545G>A）;发现了POU1F1基因第6外显子区域存在一个新的错义突变位点（NM₁74579:c.1201C>T,p.S284F）,并建立了一种DdeI PCR-RFLP的检测方法。⑵首次检测到了5个中国地方品种PROP1基因5个新的SNPs。2.以SNL成纤维细胞为滋养层细胞,利用慢病毒载体携带诱导因子基因（Oct4、Sox2、Nanog和Lin28）诱导成纤维细胞IMR90获得了多潜能干细胞系（induced pluripotent stem cells, iPS）。这些iPS细胞具有高的碱性磷酸酶活性,表达干性标志基因如Nanog、Oct4和Tra-1-60等,并且检测发现这些细胞中Nanog和Oct4的DNA启动子区域呈高度的去甲基化,体外分化实验显示其具有分化为脂肪细胞的潜能。3.由于病毒的介入,限制了iPS在再生医学和临床治疗上的应用。据此,我们尝试了无外源DNA介入的,利用转录因子蛋白和mRNA直接重编程体细胞的研究。⑴本研究构建了来自HIV的跨膜结构区域TAT与诱导多潜能干细胞的转录因子的融合表达载体。利用TAT将转录因子蛋白转导进入到靶细胞内,发现转录因子蛋白在细胞内的积聚成浓度和时间依赖性。蛋白活性检测和定位检测发现:大部分的TAT-转录因子融合蛋白受制于包含体内而未到达细胞核,几乎无相应的生物学活性。下一步的研究,将通过体外复性解决蛋白的活性问题,从而实现TAT-转录因子蛋白直接介导的人的体细胞的重编程。⑵构建了体外转录mRNA的载体,利用体外转录的诱导因子Oct4、Sox2和SV40 T的mRNA,转染体细胞,获得了正确定位的有生物活性的诱导因子蛋白;并且发现这些外源的诱导因子组合的转入能够激活细胞内源的干性基因Nanog的表达,为进一步实现利用体外转录的mRNA重编程体细胞提供了理论和实验基础。更多还原

【Abstract】 In this study, PCR-SSCP, PCR-RFLP, DNA sequencing, protein expression and purification, cell transfection and in vitro transcription were applied to verify the functions of specific transcription factor in mammalian growth, development and the process of somatic cell reprogramming. At the meantime, 3 different levels （gene, protein and RNA） of specific induced factors cocktail were utilized in this dissertation. The main results were summarized as followings:1. The genetic variation of two Pituitary transcription factor genes bPOU1F1 and bPROP1 and their associations with economic traits in bovine.⑴Three novel single nucleotide polymorphisms （SNPs） in exon 2 and one novel missense （NM₁74579:c.1201C>T） mutation in exon 6 at the POU1F1 locus in 963 Chinese cattle belonging to eight breeds were reported. The missense results in p.S284F, namely, Ser （TCT） > Phe （TTT） at position 284 of the mature protein of POU1F1. No significant associations of these genetic variations with body weight and average daily gain for different growth periods （6, 12, 18, and 24 months old） were observed （P>0.05）, as well as for body sizes （P>0.05）.⑵Five novel single nucleotide polymorphisms （SNPs） were found in 5’flanking region, exon 1 and intron 1 of the prophet of PIT1 （PROP1） gene in 606 unrelated cattle which belonged to five cattle breeds in China.2. Human iPS cells were reprogrammed from somatic cells by lentiviral expressed transcription factors using SNL fibroblasts as feeders. These iPS cells expressed common pluripotency markers, displayed human embryonic stem cells （hES） morphology and unmethylated promoters of Nanog and Oct4. These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.3. For the purpose of human therapeutic applications, the integration of viral transgenes into the genome is unlikely to be accepted.⑴Recombinant transcription factor proteins in E. coli （Oct4, Sox2, c-Myc and Klf4） carrying the cell penetrating TAT domain from HIV1 were produced. The purified proteins were able to enter into mammalian cells when added to tissue culture medium but appeared not to translocate to the nucleus. Further investigation indicated that most of the protein was tied up in the endosomes and was unavailable for reprogramming.⑵In vitro transcribed induced factors （Oct4、Sox2 and SV40 T） mRNA were utilized to reprogram fibroblasts. 5’UTR and 3’UTR of Globin gene were used for stabilization in vitro transcribed mRNA following in vitro capping and poly（A） tailing. Target factors’expressions can be detected in 293 cells and IMR90 cells after mRNA transfection and all the expressed protein were localized in the nucleus. Endogenous Nanog expression was inducted by mRNA cock-tail transfection.更多还原