【作者】 殷慧群；

【导师】 章孝荣；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 猪诱导性多潜能干细胞的建立诱导性多潜能干细胞（iPS细胞）是指通过在分化的体细胞中表达特定的转录因子来诱导其发生重编程而获得的可不断自我更新和具有多向分化潜能的细胞。iPS细胞技术的产生和发展,是干细胞领域乃至整个生物学领域的一项重大发现,具有广阔的应用前景。本研究旨在应用限定因子建立猪iPS细胞诱导技术,为将来深入研究应用猪iPS细胞奠定基础。论文由三个试验组成,其内容如下:试验一:携带限定因子的慢病毒表达载体的构建以胎猪原始生殖嵴和囊胚为材料,用RT-PCR方法克隆出猪Sox2、Klf4和c-Myc基因的开放阅读框序列,人OCT4基因序列直接从质粒pMX-hOCT4中扩增获取。经限制性内切酶酶切,与逆转录病毒载体pLEGFP-N1连接,分别构建出限定因子EGFP融合蛋白逆转录病毒表达载体。再从重组逆转录病毒载体中扩增出CMV启动子,限定因子和EGFP序列,同时通过酶切从pLL3.7质粒中移除U6启动子、CMV启动子和EGFP序列,最后把扩增出的CMV启动子,限定因子和EGFP序列与改造后的pLL3.7进行重组,分别构建出限定因子EGFP融合蛋白慢病毒表达载体pLL-hOCT4/pSox2/pKlf4/pMyc -EGFP。重组慢病毒载体质粒转染293T细胞后,在荧光显微镜下观察限定因子表达情况。结果显示,猪Sox2、Klf4和c-Myc基因开放阅读框序列分别与已发表的猪Sox2、Klf4和c-Myc基因序列高度同源;重组慢病毒载体质粒转染293T细胞后表现为限定因子EGFP融合蛋白定位于细胞核表达,符合限定因子表达特点。这些结果证实,本试验的限定因子EGFP融合蛋白慢病毒载体构建正确。试验二:慢病毒载体法诱导猪iPS细胞采用组织块培养法从57d胎龄的杜洛克-长白-大约克三元杂交猪胎儿分离培养建立7株猪胎儿成纤维细胞系。通过磷酸钙法转染四种限定因子EGFP融合蛋白慢病毒表达载体质粒和包装辅助质粒,包装产生的四种限定因子慢病毒经浓缩后感染猪胎儿成纤维细胞,在含1000U/mL LIF和4ng/mL bFGF的干细胞培养液培养条件下进行培养传代,逐步分离培养出集落边缘界限清晰的细胞克隆,高倍显微镜下集落中的单个细胞呈现细胞核较大,核质比例较高。按1∶4比例每3⁴ d传代一次,细胞集落生长稳定,核型正常,AP检测为阳性,免疫细胞化学和RT-PCR检测有关键性多潜能相关基因Oct4和Nanog的表达。此外,细胞集落中SSEA-1蛋白表达检测阳性,SSEA-3/4和TRA-1-61/81检测阴性。将其注射到BALB/cA裸鼠背侧皮下可形成含有外胚层、中胚层和内胚层组织来源的畸胎瘤。以上结果证实,本试验所获得的猪的细胞克隆具有ES细胞样特征,即成功地从猪胎儿成纤维细胞诱导出iPS细胞。试验三:纯化蛋白穿膜法诱导猪iPS细胞由于慢病毒携带外源基因的随机稳定整合会带来潜在的细胞癌化畸变等危害,因此根据慢病毒介导外源限定因子EGFP融合蛋白的试验结果,构建携带细胞穿膜肽外源限定因子的融合蛋白用于猪iPS细胞的诱导,将会为猪iPS细胞的安全生产、应用奠定基础。将体外表达、纯化的携带有细胞穿膜肽——九聚精氨酸（R₉）的限定因子-R₉融合蛋白添加到培养的猪胎儿成纤维细胞培养液中尝试建立猪iPS细胞。结果显示,在荧光显微镜下可观察到限定因子-R₉原核蛋白能进入猪胎儿成纤维细胞,且蛋白能定位于细胞核;在干细胞培养液培养条件下同时用限定因子-R₉原核蛋白处理6个周期后,能逐渐分离培养出集落边缘界限清晰的细胞克隆,重构克隆中的单个细胞呈现细胞核较大,核质比例较高,AP为阳性,免疫细胞化学检测ES细胞特异性分子标记Oct4和Nanog蛋白表达阳性。这些结果证实本试验的限定因子-R₉原核蛋白表达载体构建正确;细胞穿膜肽R₉可转导限定因子重组蛋白进入细胞核;重组限定因子-R₉原核蛋白可使猪胎儿成纤维细胞发生重构,且重构细胞具有ES细胞样形态,可表达AP、Oct4和Nanog蛋白。综合以上三个试验的研究结果,可以得出如下结论:1.运用RT-PCR技术可以从胎猪的原始生殖嵴和囊胚细胞克隆出猪Sox2、Klf4和c-Myc限定因子基因的开放阅读框序列;用基因工程技术将这些限定因子基因序列（包括人Oct4基因序列）与逆转录病毒载体pLEGFP-N1连接,再进行基因重组,能成功地构建限定因子EGFP融合蛋白慢病毒表达载体。应用这些重组限定因子EGFP融合蛋白可有效追踪掌握限定因子在细胞重编程过程中的作用机制和特点。2.用限定因子EGFP融合蛋白慢病毒能诱导猪胎儿成纤维细胞重编程,建立猪iPS细胞。通过限定因子EGFP融合蛋白追踪发现,外源限定因子在猪iPS细胞集落中大多数不表达或低水平表达,少数细胞高表达,提示体细胞完全重编程形成iPS细胞后外源基因不表达,不完全重编程的体细胞外源基因仍处于高表达状态;不同的猪胎儿成纤维细胞可能需要不同的时间去完成重编程进程,或者不同的细胞可能是在不同的时间点上启动重编程进程。3.将携带有细胞穿膜肽——九聚精氨酸（R₉）的限定因子重组蛋白用于猪胎儿成纤维细胞的重编程,获得了具有ES细胞样形态、可表达AP、Oct4和Nanog蛋白的重构细胞,为建立诱导猪体细胞直接重编程的新方法提供了新的思路。本论文的创新点:首次应用含有猪源性限定因子EGFP融合蛋白慢病毒成功建立猪iPS细胞。首次尝试应用限定因子重组蛋白直接重编程猪胎儿成纤维细胞。更多还原

【Abstract】 Generation of Induced Pluripotent Stem Cells from Porcine Fetal Fibroblasts by Defined FactorsPluripotent cells, which derived from any differentiated cell type through ectopic expression of transcription factors, were designated as induced pluripotent stem （iPS） cells, exhibiting morphology and growth properties indistinguishible to ES cells and expressing ES cell marker genes. With the ability to differentiate into all types of cells, iPS cells technology has enormous potential values in generation of human disease models, drug screening, toxicology and regenerative medicine. The current research was attempted to generate porcine iPS by defined factors EGFP fusion protein, which would be of great help to better our understanding of nuclear reprogramming mechanisms, and enrich our means of examine safety and effectiveness of iPS cells in human, as well as to extend the applications of swine iPS cells.Experiment 1. To construct lentiviral expression vector with defined factors geneOpen reading frame （ORF） of porcine Sox2, Klf4 and c-Myc gene was successfully amplified respectively by RT-PCR from the primordial genital ridges and blastula. Human OCT4 gene was amplified directly by PCR from plasmid pMX-hOCT4. After confirmation by DNA sequencing, such correct sequences of defined factors were inserted individually into retroviral vector pLEGFP-N1. Recombinant retroviral vector pLEGFP-hOCT4/pSox2/pKlf4/pMyc was obtained respectively. Next, DNA sequences containing CMV promoter, defined factors and EGFP were amplified respectively from pLEGFP-hOCT4/pSox2/pMyc/pKlf4 by PCR. The lentiviral vector pLL3.7 was modified by removing U6 promoter, CMV promoter and EGFP sequences. Lastly, modified pLL3.7 was ligated to DNA sequence containing CMV promoter, defined factors and EGFP by T4 DNA ligase. Thus, recombinant lentiviral vector pLL-hOCT4/pSox2/pKlf4/pMyc-EGFP was constructed well respectively. Then 293T cells were transfected with recombinant lentiviral plasmids respectively. The results showed that the cloned sequences of pig Sox2, Klf4 and c-Myc gene were highly homologous to corresponding genes ORF published in genebank respectively. All the four fusion proteins hOCT4/pSox2/pKlf4/pMyc-EGFP was located in the nucleus of 293T cells as their natural localization. This confirmed that recombinant lentiviral vector pLL-hOCT4/pSox2/pKlf4/pMyc-EGFP was constructed correctly.Experiment 2. To generate pig iPS cells by lentiviral vector with defined factorsSeven porcine fetal fibroblasts （PFFs） cell lines were established from 7 porcine fetuses of Duroc-Landrace-Yorkshire crossbred at 57d of gestation by using explants-seeding method. Lentiviral plasmids harboring four defined factors and packaging helper plasmids were co-transfected into 293T cells by calcium phosphate transfection method. Four defined factors lentivirus generated by 293T were concentrated to reprogram PFFs. PPFs, expressed exogenous defined factors genes, were then sub-cultured in stem cell medium supplemented with 1000U/mL LIF and 4ng/mL bFGF, and as a consequence the clear-cut cell colonies were gradually derived. The cells in such colonies had large translucent nuclei and a high nucleocytoplasmic ratio. All of the cells grew at similar rates, requiring sub-culture at a roughly 1:4 ratio every 3⁴ days. They exhibited normal karyotype, and expressed alkaline phosphatase （AP）, Oct4 and Nanog. Unlike human ES cells, such porcine cell colonies were positive for SSEA-1, but negative for SSEA-3/4 and Tra-1-61/81. Teratomas containing tissues of ectoderm, mesoderm, and endoderm formed gradually after subcutaneous injection of these cells into BALB/cA nude mice dorsal skin. Taken together, our results demonstrated that these derived cell colonies can exhibit very similar morphology and characteristics to ES cells, could be termed as pig iPS cells.Experiment 3. To generate pig iPS cells by defined factors recombinant proteinsAs exogenous genes were randomly inserted into the target cells genome, it possibly brought the potential hazard of insertional mutagenesis. Therefore it is necessary to seek for some new methods to induce somatic cell reprogramming without virus. In view of above study results, we attempted to use defined factors recombinant proteins carring cell-penetrating peptide （CPP） for generation of porcine iPS cells, which would be of tremendous benefits for the safe applications of iPS cells. Defined factors genes were amplified by PCR with specific primers of 9 arginines （R₉） from recombinant plasmid pLL-hOCT4/pSox2/pMyc/pKlf4-EGFP and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the four recombinant plasmids were then transformed into BL21 （Escherichia coli） strain respectively. After IPTG induction, hOCT4/pSox2/pMyc/pKlf4-R₉-EGFP fusion proteins were purified using Novagen His-Bind kit and confirmed by SDS-PAGE respectively. Then defiend factors recombinant proteins were added into PFFs medium every 48h to generate pig iPS cells. Our results showed that such purified hOCT4/pSox2/pMyc/pKlf4-R₉-EGFP fusion proteins could enter into PFFs efficiently, and most of them located in nuclei. PPFs were sub-cultured in stem cell medium condition and treated with defined factors recombinant proteins for 6 cycles simultaneously; the clear-cut cell colonies were gradually derived. These cells had large translucent nuclei and a high nucleocytoplasmic ratio. They were positive for AP, Oct4 and Nanog. It confirmed that recombinant prokaryotic expression vector for the hOCT4/pSox2/pKlf4/pMyc-R₉-EGFP was constructed correctly. And the transduction activity of hOCT4/pSox2/pKlf4/pMyc-R₉-EGFP fusion protein to PFFs was proved respectively. Recombinants proteins hOCT4/pSox2/pKlf4/pMyc-R₉-EGFP can reprogram PFFs, and reprogrammed cells exhibited the morphology of ES cells and express ES cell-specific genes, such as AP, Oct4 and Nanog.Form above results, we concluded that:1. The open reading frame （ORF） of pig Sox2, Klf4 and c-Myc genes were obtained from the primordial genital ridges and blastula by RT-PCR. Lentiviral vectors of defined factors （including pig Sox2, Klf4 and c-Myc genes, human OCT4） EGFP fusion protein were constructed successfully by genetic engineering technology. The mechanism and characteristics of defined factors in cells reprogramming could be tracked effectively by constructed defined factors EGFP fusion protein.2. Pig iPS cells were generated successfully from PFFs by defined factors EGFP fusion proteins lentivirus. Some cells in pig iPS colonies expressed defined factors EGFP fusion proteins, the others showed there was no or weak expression of defined factors EGFP fusion proteins under fluorescence microscope. It suggested that different PFFs may require different time to complete reprogramming process, or different cells may start reprogramming process at different time points.3. Reprogrammed cells exhibited the morphology of ES cells and expressed ES cell marker genes, such as AP, Oct4 and Nanog, were generated from PFFs by defined factors recombinant proteins with cell penetrating peptide------R₉. This would provide new ideas for the induction of porcine somatic cells reprogramming.The innovation of the present study was for the first time, to our knowledge, to establish pig iPS cells by defined factors （including pig Sox2, Klf4 and c-Myc genes, human OCT4） EGFP fusion proteins lentivirus. Moreover, first attempt to reprogram directly PFFs by defined factors recombinant proteins.更多还原