【作者】 辛晓玲；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 博士

【摘要】 胚胎干细胞(ESCs)一般由胚胎的内细胞团分离而来,具有无限增殖和多向分化潜能,但人类ESCs的分离由于需要损坏早期胚胎,受到法律和伦理道德的约束。最近,应用几种转录因子诱导小鼠体细胞转变为诱导型多能干细胞(iPSCs)获得成功,这种iPS细胞与小鼠ES细胞在形态、增殖、基因表达和体内外分化等方面基本一致。人类体细胞随后也被成功诱导为iPS细胞,这种方法不需要破坏人类胚胎,这些人类iPS细胞可分化为患者自体的各种细胞,在细胞治疗和再生医学方面具有重大意义。之后,猕猴、大鼠、猪等物种也得到iPS细胞。动物iPS细胞除可用于建立动物疾病模型和药物筛选等医学领域,对于家畜遗传育种和基因改良等畜牧业生产亦意义重大,但牛iPS细胞的诱导研究迄今未见报道。本试验应用人和小鼠诱导iPS细胞的方法,将几种转录因子通过反转录病毒载体导入牛体细胞,诱导产生牛iPS细胞,并对其特性加以检测。经过试验,取得了如下结果：(1)本试验成功地从胎牛原始生殖嵴中克隆出963 bp的牛sox2基因开放阅读框序列,与发表的牛sox2基因序列(NM-001105463)比对,其核苷酸同源性为99.6%,对应氨基酸同源性为99.7%；以生长接触抑制的胎牛肾上皮细胞(MDBK)为材料,克隆出1434bp的牛Klf4基因开放阅读框序列,经测序并与发表的牛Klf4基因序列(NM-001105385)比对,其核苷酸与对应氨基酸序列同源性达99.9%。(2)将基因片段插入反转录病毒载体pMSCVneo,成功构建pMSCV-sox2 (pMS)和pMSCV-klf4 (pMK)真核表达载体。将pMS和pMK分别转染包装细胞PT67,分布得到稳定的产毒细胞株,MSCV-sox2病毒滴度达8.16×107CFU/mL, MSCV-klf4病毒滴度达7.16×107CFU/mL。电镜下观察病毒上清,可看到典型的反转录病毒颗粒形态。(3)本试验应用四转录因子组合Oct4、Sox2、c-Myc和Klf4,以反转录病毒载体为介导,诱导新生奶公牛成纤维细胞(NBF),以人羊膜(HAM)为饲养层,成功得到ESCs样的细胞集落,诱导所得到的细胞被命名为iPS-NBF细胞。(4)牛iPS-NBF细胞AKP染色阳性,Nanog、Oct4、Sox2、SSEA-3、SSEA-4、TRA1-60、TRA1-81、TERT的免疫荧光染色阳性,而SSEA-1免疫荧光染色为阴性。经实时定量PCR分析,牛iPS-NBF细胞的有关多能性基因nanog、oct4、sox2已被有效激活,表达量接近牛PGCs。经核型分析发现传代至第15代的牛iPS-NBF细胞仍保持正常的二倍体核型。(5)用类胚体介导方法,牛iPS-NBF细胞自发分化为多种形态的细胞,通过免疫荧光染色和RT-PCR分析,证明牛iPS-NBF细胞在体外可以分化为三个胚层类型的细胞。(6)将牛iPS-NBF细胞注入免疫缺陷鼠皮下成功得到畸胎瘤,HE染色显示,肿瘤包含有中胚层(肌肉、血管、软骨等)和内胚层(内脏上皮)组织,而外胚层组织在切片中未找到。(7)把10-15个牛iPS-NBF细胞注射入小鼠的8-细胞胚或桑椹胚内,成功得到6只成活的牛-鼠嵌合体小鼠,经牛特异性线粒体D-loop序列检测,证实牛iPS-NBF细胞参与了嵌合体小鼠多种组织的发育,包括心脏、血液、肺、胃肠道、胃、肾、神经组织、骨骼肌和性腺等。(8)采用Nanog、Oct4、Sox2、c-Myc、Klf4和Lin28六个转录因子,以不同的组合(2因子SO；3因子NOS；4因子OSCK和NOSL;5因子NOSKL;6因子NOSCKL)来诱导牛NBF细胞重编程,结果显示,几种组合都可以成功诱导体细胞重编程获得干细胞样集落,与4因子组比较,5因子和6因子组诱导效率显著提高,而2因子和3因子组诱导细胞集落少,且不典型,可能是体细胞未能被完全重编程。更多还原

【Abstract】 Embryonic stem cells (ESCs) commonly were derived from the inner cell mass (ICM) of mammalian blastocysts, and they remain the unique capacity to proliferate unlimitedly while maintain pluripotency. The study about human ESCs was restricted by law and ethics because of the use of human early embryos. Recently, The direct reprogramming of mouse somatic cells to induced pluripotent stem cells (iPSCs) was first succeeded through retroviral transduction of some transcription factors. The mouse iPSCs showed marked similarities to mouse ES cells in morphology, proliferation property, gene expression profiles and in vivo/ in vitro differentiation potential. Subsequently human iPSC lines were successfully established from human somatic cells, enabling the generation of patient-specific cells of any lineage without the use of human embryos. The generation of human iPSCs provides an invaluable resource for cell therapy and regenerative medicine. The iPSCs of other species, including rhesus monkey, rat and pig, were established from somatic cells in succession, which can be used in medicinal field such as establishing animal disease models and filtering medicine in vitro. Up to now the study about bovine iPSCs has not been reported. So in this study we attempt to generate the bovine iPSCs from bovine somatic cells through retroviral transduction of defined factors which used in induced process of mouse and human iPSCs. The bovine iPSCs has been established successfully and its properties similar to ES cells have been demonstrated with scientific test. The important results were listed below.(1) The ORF(open reading frame) sequence of bovine sox2 gene (963 bp) was cloned from the primodial genital ridges of fetal cattle by RT-PCR. Compare to the published sox2 gene sequence (NM-001105463), the nucleotide homology is 99.6% and the related amino acids homology is 99.7%. The ORF sequence of bovine klf4 gene (1434 bp) was cloned from MDBK cells in contact inhibition growth. Compare to the published klf4 gene sequence (NM-001105385), both the nucleotide and the related amino acids homology is 99.9%.(2) The two genes ORF sequence were respectively inserted into the restriction sites of retroviral vector pMSCVneo to construt recombinant retroviral expression vector pMSCV-sox2 (pMS) and pMSCV-klf4 (pMK). The pMS and pMK was transfected into packaging cell line(PT67). Then we successfully got stable virus producing cell strains which viral titer respectively was up to 8.16×107 CFU/mL(MSCV-sox2) and 7.16×107 CFU/mL(MSCV-klf4). The typical infectious retroviral particles in the supernatant can be found under electron microscope.(3) In this study the four transcription factors (Oct4, Sox2, c-Myc and Klf4) were transducted into new-born bovine fibroblasts(NBF) through the retroviral pMSCVnoe vector. The infected NBF cells were cultured on the cell-free human amniotic membrane (HAM) support. Subsequently ESCs-like cell colonies were gained successfully and were named as "iPS-NBF" cells.(4) The bovine iPS-NBF colonies were strongly positive for alkaline phosphatase (AKP). The results of immunocytochemistry stain showed that the bovine iPS-NBF colonies were negative for stage-specific embryonic antigen-1 (SSEA-1), while they were positive for SSEA-3, SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81, Nanog, Oct4, Sox2, and telomerase reverse transcriptase (TERT) proteins. The real-time quantitative polymerase chain reaction (qPCR) analysis showed that the related pluripotency genes, nanog, oct4 and sox2, have been effectively activated and their expression levels were comparable to the bovine PGCs. In addition, the bovine iPS-NBF cells about passage 15 retained normal diploid karyotypes by karyotype analysis.(5) The bovine iPS-NBF cells could differentiate randomly into cells with various morphologies through embryoid body (EB)-mediated differentiation. Immunocytochemistry stain and RT-PCR analysis showed that the bovine iPS-NBF cells could differentiate into all cell types of three germ layers in vitro.(6) The bovine iPS-NBF cells were injected subcutaneously into NOD/SCID mice and formed teratoma. Hematoxylin and eosin staining showed that the teratoma contained mesoderm tissues (muscle,blood vessel and cartilage) and endoderm tissue(viscus epithelium),but no ectoderm tissues.(7) 10～15 bovine iPS-NBF cells were microinjected into mouse 8-cell embryos or morula. Then six neonatal bovine-mouse chimeric mice were obtained. Detection of bovine specific D-loop region of mitochondrial DNA sequences demonstrated that the bovine iPS-NBDF cells contributed to the development of chimeric mice various organs, including heart, blood, lung, gastrointestinal tract, liver, kidney, neural tissues, skeletal muscle and gonad.(8) The six transcription factors(Nanog, Oct4, Sox2, c-Myc, Klf4 and Lin28) were splited into different combinations:2-factors group(SO),3-factors group(NOS),4-factors group(OSCK and NOSL),5-factors group(NOSKL) and 6-factors group(NOSCKL). The NBF cells were infected respectively with different fators combinations. The results showed all the six combinations could induced the NBF cells to ESCs-like colonies successfully. Comparing to the 4-factors group, the induced efficiency of 2-factors and 3-factors groups was lower, while the induced efficiency of 5-factors and 6-factors groups were increased significantly.更多还原