【作者】 覃敏；

【导师】 莫曾南；

【作者基本信息】 广西医科大学 ， 外科学， 2009， 博士

【摘要】 背景及目的人胚胎干细胞(embryonic stem cells, ES cells)是一种具多项分化潜能的细胞,可被诱导分化成各种特化细胞和组织,用来修复或替代人体丧失功能的组织和器官,这就是“干细胞治疗(stem cell therapy)”。干细胞治疗在疾病治疗和损伤修复领域中有潜在的优势,它可治疗包括脑、肾脏、骨髓及组织病变在内的多种疾病。但是,由于每个个体的组织相容性不同,同种异体胚胎干细胞用于临床会引起免疫排斥。解决这一难题的主要途径就是产生患者自己的胚胎干细胞。体细胞核移植法(somatic cell nuclear transfer, SCNT)即治疗性克隆(therapeutic cloning)和诱导产生多能性干细胞这两项技术已成功诱导出组织相容的多能干细胞,并且取得了重大进展。治疗性克隆是指将体细胞核移入去核的卵母细胞中,在理论上,体细胞的细胞核将被卵母细胞的胞质重编程,沉默表达体细胞基因同时激活干细胞基因。重构胚发育至囊胚阶段后,从囊胚内细胞团(inner cell mass, ICM)中分离出胚胎干细胞。随后胚胎干细胞发育分化产生携带患者自己基因的胚胎干细胞,这些胚胎干细胞及其衍生组织移植后就不会产生免疫排斥。但是,体细胞核移植技术仍不完善,成功的诱导核移植胚胎干细胞系需要依赖多种因素,包括受体细胞的类型、重编程的时间、激活方法、胚胎的培养系统等。此外,损伤小的重构的方法也是关键因素之一。目前,使用该项技术成功获得克隆囊胚的效率仍然很低。最近,有研究利用异位表达胚胎干细胞标志因子重编程小鼠成纤维细胞,使其产生组织相容胚胎干细胞,为干细胞治疗开辟了新的途径。这类诱导产生的多能干细胞(induced pluripotent stem Cell, iPS)是利用逆转录病毒载体在小鼠成纤维细胞中导入了4个与多能性有关的基因Oct4, Sox2, c-myc和Klf4诱导产生的。得到的iPS细胞在许多方面都与ES细胞十分相似,并且可以产生嵌合体。随后这四个因子又成功诱导人类成纤维细胞重编程为iPS细胞。这一技术的应用对产生组织相容多能干细胞及医学研究有着巨大的价值。但是,c-Myc逆转录病毒的活化增加了嵌合体和子代的致瘤性。此外,诱导产生多能干细胞的效率也不高,阻碍了它的临床应用。因此,为了提高干细胞治疗的效率,深入研究体细胞重编程的分子机制,本课题从体细胞核移植和诱导产生的多能干细胞两个方面研究了多种因素对其效率产生的影响。本论文分为6部分:第1部分不同核移植方法对小鼠体细胞核移植效率的影响目的研究不同核移植方法对小鼠体细胞核移植效率的影响,建立一种简单有效的核移植方法。方法以小鼠颗粒细胞作为体细胞核移植的供核细胞,采用四种去核方法(盲吸法、蔗糖辅助去核法、化学去核法、荧光染色去核法)研究卵母细胞去核率的差异,并比较了胞质内注射法和反向核移植法应用于小鼠体细胞核移植的效果。结果荧光染色法的去核率(88%)显著高于其他试验组(P<0.05);胞质内注射法的囊胚率和囊胚细胞数较反向核移植高(P<0.05)。结论荧光染色去核法和胞质内注射法构建体细胞核移植胚胎效率较高,可维持胚胎早期发育。第2部分单一和联合化学激活方法对小鼠体细胞核移植胚胎体外发育的影响目的探讨单一和联合化学激活方法对小鼠体细胞核移植胚胎体外发育的影响。方法构建小鼠核移植胚胎。分别使用单一激活剂钙离子载体(A23187)、乙醇(Eth)、氯化锶(SrCl2)及联合蛋白激酶抑制剂6-DMAP、CB激活。结果10mmol/L SrCl2处理6 d可获得较高卵裂率(68.9%)和囊胚率(7.2%)(P<0.05),也较A23187、Eth组高(P<0.05);联合激活方法中,Eth+6-DMAP+CB组(69.8%和46.05±2.62)、SrCl2 +CB组(71.9%和45.40±2.23)和A23187+6-DMAP+CB组(62%和39.75±1.15)卵裂率和囊胚细胞计数明显高于未联合组(P<0.05)。结论乙醇联合6-DMAP、CB组合,SrCl2联合CB组合均能较好地激活小鼠体细胞核移植胚胎。第3部分一步法和二步法培养系统对小鼠体细胞核移植胚胎体外发育的影响目的比较一步法和二步法两种培养系统对小鼠体细胞核移植胚胎体外发育的影响,确立一种有效的培养系统。方法构建小鼠核移植胚胎。激活后,随机分为两组。第1组,比较核移植胚胎在KSOMgAA , KSOMgAA / KSOMgAA , cleavage medium and cleavage medium/blastocyst medium中的发育情况;第2组,观察牛黄酸对核移植胚胎发育影响。结果核移植胚胎发育的囊胚率、孵化率和囊胚细胞计数一步法和两步法培养体系中没有显著差异(P>0.05)。牛磺酸加入KSOMgAA后能有效改善早期核移植胚胎体外发育能力。结论一步法和两步法培养体系均符合小鼠体细胞核移植胚胎不同发育阶段需求。第4部分小鼠体细胞核移植囊胚的微卫星DNA鉴定目的利用微卫星DNA技术进行体细胞核移植囊胚的鉴定。方法提取近交系小鼠体细胞核移植囊胚、供体BALB/c小鼠、受体C57BL/6小鼠及昆明小鼠的基因组DNA,使用巢式聚合酶链反应扩增4个微卫星位点DNA片段,即D3Mit28, D11Mit258,D12Mit136及D14Mit50。结果通过微卫星DNA序列的扩增,证明核移植囊胚的微卫星DNA与供体细胞完全相同,而与受体细胞或者对照细胞无亲缘关系。结论体细胞核移植囊胚基因组来源于供体BALB/c小鼠。第5部分体细胞核移植胚胎ES细胞样集落分离目的探讨小鼠体细胞核移植胚胎ES细胞样集落分离和培养方法,比较不同因子对建立核移植胚胎ES细胞样集落效率的影响。方法构建小鼠体细胞核移植胚胎。当核移植胚胎培养至早期囊胚阶段,移至小鼠胎儿成纤维细胞饲养层上,换成ES细胞培养液继续培养4～5d,分离消化ICM,重新接种。2 d后分离出ES样细胞集落,进行形态学观察。结果ES样细胞集落具有典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大。碱性磷酸酶染色呈阳性。体外可分化为拟胚体,体内可分化成上皮样细胞、腺体和肌肉组织。结论小鼠体细胞核移植胚胎中可以分离出ES细胞样集落。第6部分小鼠成纤维细胞诱导产生多能干细胞目的体外诱导小鼠成纤维细胞重编程为多能胚胎干细胞样集落。方法分别将Pou5f1, Sox2, c-Myc和Klf4真核表达载体转染NIH3T3细胞。分离小鼠成纤维细胞,移至小鼠胎儿成纤维细胞饲养层上,取转染后的上清液培养。3d后,加入G418,筛选克隆2～3w。结果筛选初期大部分克隆形态学为扁平状,仅4株克隆为ES细胞样生长,后期克隆数增长至11株。ES细胞样克隆具有岛屿状团状隆起结构。结论四个逆转录因子Pou5f1, Sox2, Klf4和c-Myc可诱导小鼠体细胞ES细胞集落样改变。更多还原

【Abstract】 Background Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types which serves as a sort of repair system for the body. The remarkable potential of stem cells makes it possible to treat patients by transplanting specialized healthy cells produced from them to repair damaged and diseased body-parts. This concept is known as“stem cell therapy”. Stem cell therapy is now emerging as a potentially revolutionary new way to treat disease and injury. Stem cell therapy has potential applications in treating a wide array of diseases and ailments of the brain, kidney, bone and many other tissues. However, the transplanted cells are grown from stem cells that are not genetically compatible with one patient, their immune system will reject the cells. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from the patient’s own cells. Diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and the generation of human iPS cells (induced pluripotent stem cells) from somatic cells, have been attempted to produce genetic compatible pluripotent stem cells. These approaches were in progress.Therapeutic cloning refers to the transfer of the nucleus of a somatic cell into an enucleated donor oocyte. In theory, the oocyte’s cytoplasm would reprogram the transferred nucleus by silencing all the somatic cell genes and activating the embryonic ones. The reconstructed embryos are induced embryonic developments and ES cells would be isolated from the inner cell (ICMs) of the cloned blastocysts. It is proposed that following directed cell differentiation, these cells which carried the nuclear genome of the patient could be transplanted without immune rejection for treatment of degenerative disorders among others. However, Somati cell nuclear transfer(SCNT) is still a developing technique. Success in the production of SCNT-ES cell line is attributed to optimization of several factors including the donor cell type, reprogramming time, activation protocol and use of sequential culture system. Furthermore, use of less-invasive enucleation method is suggested to be one of key factors. Today, the success rate of obtaining cloned blastocysts from this technique remains low.An approach toward the same end of the generation of“patient-specific”ES cells was recently described, in which murine fibroblasts were reprogrammed by ectopically expressing factors known to be highly expressed in ES cells. Such iPS cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells were indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. While the four transcription factors were used to reprogram human fibroblasts to iPS cells. Application of this approach in human cells would have enormous potential and generate patient-specific pluripotent stem cells to study and potentially ameliorate human diseases. However, reactivation of the c-Myc retrovirus increases tumorigenicity in the chimeras and progeny. Moreover, the efficiency of generating human iPS cells is low, hindering clinical application.Thus, to improved the efficiency of stem cell therapy and provided necessary theory for the further study of molecular mechenisms of somatic cell reprogram, I have studied some factors on the efficiency of somatic cell nuclear transfer and generating iPS cells from mouse somatic cells.This thesis divided into six parts as follows:Part1 Effects of Different Methods of Nuclear Transfer On the Efficiency of Somatic Cell Nuclear Transfer in MiceObjective To study the effects of different methods of nuclear transfer on the efficiency of somatic cell nuclear transfer in mice, and to establish a simple, efficient method for nuclear transfer.Methods Granulosa cell was used as a donor of mouse somatic nuclear transfer.Different enucleation methods(blind aspiration,sucrose pretreatment for enucleation, chemical induced enucleation, bisbenzimide stain method) and two methods of enucleation(intracytoplasmic nuclear injection and reverse nuclear transfer)were studied on enucleation rate of the oocyte.Results The enucleation rate(88 % ) was significantly the higher in bisbenzimide stain method than in other three groups(P<0.05). The intracytoplasmic nuclear injection had a higher rate of blastocyst development and more of total cell numbers per blastocyst than those of reverse nuclear transfer method(P<0.05).Conclusion Bisbenzimide stain method and intracytoplasmic nuclear injection are efficient procedures in mouse somatic cell nuclear transfer, these procedures have the ability to maintain early nuclear transfer embryos development.Part2 Effects of Single or Combined chemical activation methods on the Activation of Mouse Somatic Cell Nuclear Transfer EmbryosObjective To establish the optimum procedure of somatic cell nuclear transfer embryos activation in mice, and a comparison has been made of the development of mouse somatic cell nuclear transfer embryos activated by single or combined chemical activation methods.Methods mouse reconstructed embryos were constructed. The development of reconstructed embryos which have activated in different methods of activation including ethanol(Eth),calcium ionophore(A23187), strontium (SrCl2) and combined these single agent treatment with 6-dimethylaminopurine(6-DMAP), cytochalasin B(CB) were observed.Results①When NT embryos were treated with 10 mmol/L SrCl2 for 6h, the activation rate (68.9%) and the rate of blastulation (7.2%) were higher than other groups(P <0.05).②The cleavage rate and total cell numbers per blastocyst were significantly higher after treated with Eth + 6-DMAP + CB ( 69.8%;46.05±2.62), SrCl2+ CB(71.9%;45.40±2.23) or A23187+6-DMAP+CB (62%;39.75±1.15) than the control group(P <0.05).Conclusion The Eth+6-DMAP+CB and the SrCl2+CB combined chemical activation methods could better activate mouse somatic cell nuclear transfer embryos.Part3 Effect of one-step or two-step culture systems on in vitro development of mouse somatic cell nuclear transferred embryosObjective To select some culture media profitable for early mouse somatic cell nuclear transfer embryos in vitro development, a comparison has been made of the development of mouse somatic cell nuclear transfer embryos in either one-step or two-step culture systems.Methods Mouse reconstructed embryos were done. Following activation, NT embryos were allocated randomly to one of two treatment groups. Group 1, NT embryo development was compared in: KSOMgAA, KSOMgAA / KSOMgAA, cleavage medium and cleavage medium/blastocyst medium. Second group, NT embryos were cultured in medium KSOMgAA and KSOMgAA +Taurine. The development of NT embryos in one-step and two-step culture systems was observed, respectively.Results There were no significantly differences in the proportion of blastocysts, hatching rate, and total cell numbers between one-step and two-step culture systems. Development of early reconstructed and parthenogenetic embryos was significantly increased by adding taurine in KSOMgAA culture medium when compared to KSOMgAA media.Conclusion Both one-step and two-step culture systems were feasible mouse somatic cell nuclear transffer embryos culture method in vitro. Part4 Microsatellite DNA analysis of mouse somatic cell nuclear transfer blastocystsObjective To identify the source of the somatic cell nuclear transfer blastocysts, microsatellite DNA assay was developed.Methods DNA isolation from SCNT blastocyst,donor BALB/c mouse, recipient C57BL/6 mouse and KM mouse were tested by nested polymerase-chainreaction(PCR) analysis. Primers designed for four specific microsatellite locus (D3Mit28, D11Mit258, D12Mit136, D14Mit50) were employed.Results Microsatellite DNA analyses examining four loci confirm that all the SCNT blastocysts were genetically identical to the donor mouse. Additionally, the SCNT embryos were not genetically related to the respective recipient mouse. Furthermore sequences of SCNT blastocysts are diferent from the control KM mouse.Conclusion The results have proved that the nucleus of somatic cell nuclear transfer blastocysts come from somatic nucleus of donor BALB/c mouse.Part5 Isolation of Embryonic Stem Cell-like Colonies From Mouse Nuclear Transfer EmbryosObjective The aim of the experiment was to isolate and culture mouse ES cell-like colonies from mouse nuclear transfer embryos, and to compare some factors influencing the efficiency of establishing those stem cell-like colonies.Methods Mouse NT embryos were done. When NT embryos developed to the stage of early blastocyst,embryos were transferred to the feeder layer of mouse embryo fibroblast with cardiomyocyte media conditioned for 4-5 days,and then ICMs were digested and recultured. ES cell-like colonies were isolated after 2 days,and the morphology of this colonies were observed.Results Outcome of isolating ES cell-like colonies was good. And the ES cell-like colonies had its typical morphological characteristics such as nest-like colonies,round or elliptic,with clear edge and smooth surface,strong refraction,significant swelling growth, compactness of cell aggregation,and obscure distinction between two edges of neighbour cells within a colony,relatively bigger nucleus compared with little cytoplasm.ES cells showed strong AKP activity. The differentiation of ES cells is outgrowth from embryonic bodies in vitro, while glandular tissues, scalelike cylindrical epithelium and muscular tissue were formed from ES cells in vivo.Conclusions This result demonstrates that ES cell-like colonies can be isolated from mouse NT embryos.Part6 Induction of Pluripotent Stem Cells from Mouse Adult FibroblastObjective This present study aimed to reprogramm mouse fibroblasts into a pluripotent ES-cell-like state in vitro.Methods The NIH-3T3 cells were infected with Pou5f1-, Sox2-, c-Myc- and Klf4-expressing retroviral vectors respectly. The mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) were seeded on feeders and incubated in the virus/polybrene-containing supernatants. Three days after infection, we added G418. Clones were selected for 2 to 3 weeks. Results Most colonies had a flat morphology and 4 colonies were ES-like when selection was applied early, the number of ES-like colonies (11) that increased at later time points. ES-like colonies isolated were with island-like images.Conclusions This demonstrates that the activity of Pou5f1, Sox2, Klf4 and c-Myc can apparently turn mouse somatic cells into cells that characteristic morphology closely resemble ES cell colony.更多还原