【作者】 黄奔；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 1．从形态观察、碱性磷酸酶染色、表面抗原的免疫组化检测、多能性的标志基因表达和体外分化潜能等方面对水牛胚胎生殖样细胞（EG-likecells）的生物学特性进行了研究。结果发现，水牛EG样细胞形成多层结构的克隆，有明显的边界与饲养层分开，与小鼠的ES／EG克隆形态相似。通过组化染色发现，水牛EG样细胞表达AP、SSEA-1、SSEA-3、SSEA-4以及OCT-4。另外，RT-PCR结果显示，Fgf4除外，水牛EG样细胞均表达OCT-4、NANOG、SOX2、FOXD3、GP130、STAT3和HEB基因。将细胞延迟培养2个星期后，可以观察到成纤维样细胞，神经样细胞，肌样细胞和上皮样细胞的分化形成。悬浮培养的水牛EG样细胞可以形成简单拟胚体和复杂拟胚体（EB），复杂拟胚体仍然表达FOXD3、GP130、STAT3以及HEB基因，但已经无法检测出OCT-4、NANOG和SOX2这三个基因。此外，三个胚层标志基因的表达也在复杂拟胚体检测出来，分别是内胚层KERATIN-14（Endoderm），中胚层GATA4、ACTA2（Mesoderm）以及外胚层TUBB3（Ectoderm），表明这些水牛EG样细胞可在体外分化形成三个胚层。以上结果表明，水牛EG样细胞具有与人和小鼠ES／EG相似的生物学特性。2．探讨了水牛EG样细胞分离培养及传代的影响因素（性别、胎龄、分离方法、饲养层及细胞因子）。结果发现，雄性胎牛组的单位胎牛克隆数和克隆直径都显著高于雌性胎牛组（5.77±6.34 vs 1.03±2.64；227.76±83.84vs．102.34±40.59，P＜0.05）。80～90 dpc胎牛组的单位胎牛原代克隆形成数为1.60±2.63个，显著低于其他各组（P＜0.05），其形成的克隆直径（190.63±65.11），也显著低于除70～80 dpc组以外的其他各组（P＜0.05），并且80～90 dpc组的最高传代数仅为3代，而其他各组都达到或超过了6代。对于单位胎牛原代克隆形成数和克隆直径，机械组都显著高于胰酶消化组（8.087±6.935 vs 1.765±4.747；216.95±77.41 vs140±38.76，P＜0.05）。BEF饲养层组的各代克隆形成数都显著高于其它各组（P＜0.05），STO饲养层组的传代数仅为5代，而MEF（小鼠胎儿成纤维细胞）和BEF（水牛胎儿成纤维细胞）组为8代以上。在F₁代，添加20ng／mLLIF+20ng／mLbFGF+20ng／mLSCF或20ng／mL LIF+40ng／mL bFGF+40ng／mL SCF组的克隆形成数显著高于添加10ng／mLLIF+10ng／mL bFGF组和对照组；在F₂-F₈代，添加20ng／mLLIF组的克隆形成数显著高于添加10ng／mLLIF组。对照组的传代数仅为2代，添加10ng／mL LIF组为5代，而添加20ng／mLLIF组都为8代以上。以上结果表明，（1）胎牛的性别和胎龄以及分离方法对水牛EG样细胞的分离培养效果有显著影响，雄性胎牛优于雌性胎儿，小于80dpc的胎儿由于大于80dpc的胎儿，机械分离方法优于酶消化法；（2）BEF饲养层利于水牛EG样细胞的传代培养；LIF对水牛EG样细胞的传代培养有重要作用，而bFGF和SCF的作用不明显。3．对基因转染水牛胚胎生殖样细胞（EG-like cells）的影响因素及利用转GFP基因EG样细胞制备嵌合体囊胚的可行性探讨。结果发现，脂质体浓度为8μL／mL和12μL／mL的转染率显著高于2μL／mL和4μL／mL（33.6％，36.2％vs 13.3％，16.8％；P＜0.05）；质粒DNA 2μg／mL组的转染率显著高于质粒DNA 1μg／mL和4μg／mL组（28.8％vs 14.5％，15.2％；P＜0.05）；不同代数水牛EG样细胞（0、2、4、8）所获得的表达GFP基因克隆率之间差异不显著（19.8％vs 22.9％vs 25.8％vs 19.0％vs 16.9％；P＞0.05）；将转染GFP基因的阳性水牛EG样细胞注入到8-16细胞水牛胚胎后的囊胚发育率与注入转染GFP基因阳性水牛胎儿成纤维细胞（BEF）的囊胚发育率差异不显著（35.6％vs 33.3％；P＞0.05），但EG样细胞组获得10个表达GFP的囊胚，而BEF组则没有获得表达GFP的囊胚。以上结果表明，0-8代的水牛EG样细胞均可用于GFP基因转染研究，适宜的脂质体量和质粒DNA浓度分别为8μL／mL和2μg／mL；水牛EG样细胞具有胚胎嵌合能力，而成纤维细胞则无此能力。4．对不同代数水牛EG样细胞以及转染GFP基因的EG样细胞的核移植效果进行了研究。结果发现，不同代数水牛EG样细胞（0、2、4、8）以及胎儿成纤维细胞核移植后的融合卯率、分裂率和囊胚率差异都不显著（82.0％vs 84.5％vs 80.1％vs 78.7％vs 84.0％，67.6％vs 72.9％vs 72.0％vs 62.9％vs 63.1％，11.4％vs 12.4％vs 13.4％vs 10.0％vs 7.1％；P＞0.05）；GFP阳性水牛EG样细胞与未转染的水牛EG样细胞核移植后的融合卵率、分裂率和囊胚率亦无显著差异（79.0％vs 81.0％，62.4％vs 71.4％，11.0％vs 13.3％；P＞0.05）。以上结果表明，O-8代的水牛EG样细胞均可用于核移植，且其核移植重构胚的发育能力不受GFP基因转染的影响。更多还原

【Abstract】 1. The biological characteristic of buffalo embryonic germ-like cells （EG-like cells） hadbeen investigated in this study by morphology, AP staining, exterior antigensimmunohistochemical staining, pluripotent marker gene expressing, and differentiationpotential in vitro). Buffalo EG like cells grew in large, multilayered colonies which weredensely packed with an obvious border resembling the mouse embryonic stem cells （EScells） and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4and OCT-4. Undifferentiated swamp buffalo EG-like cells also expressed the OCT-4,NANOG, SOX2, and FOXD3, GP130, STAT3, HEB gene mRNA, but not Fgf4. Whenthese cells were cultured for more than 2 wk without passage, they could differentiate intoseveral types of cells including fibroblast-like, neuron-like, smooth muscle-like, andepithelium-like cells. Some cells formed simple embryoid bodies （EBs） and cystic EBs bysuspension culture. Cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA,but not OCT-4, NANOG, and SOX2 gene mRNA which could be detected in theundifferentiated buffalo EG-like cells. Meanwhile, the mRNA of KERATIN-14（Endoderm）, GATA4, ACTA2 （Mesoderm） and TUBB3 （Ectoderm） gene were alsodetected in cystic EBs, indicating that these cells are capable of forming three germ layersin vitro.. In conclusions, expression of OCT-4, NANOG and SOX2 may be the pluripotentstate mark of buffalo EG-like cells, and buffalo EG-like cells have morphology andcharacterization similar to those of the established EG/EG-like cells in mouse and human.2. Factors （gender, fetal age, dissociating methods, feeder, cytokines） affected the isolationand culture of the buffalo EG-like cells were investigated in this study. Male fetus yieldedsignificantly more buffalo EG-like colonies （5.77±6.34 vs 1.03±2.64 from each fetus） andlarger primary colonies （227.76±83.84μm vs 102.34±40.59μm, P＜0.05） in comparisonwith female fetus. The average number of primary colonies derived from 80～90dpc groupwas just 1.60±2.63, which was significantly （P＜0.05） lower than other groups. Thediameter of colony from 80～90dpc group is 190.63±65.11μm, which was also significantly（P＜0.05） lower than other groups with the exception of 70～80 dpc group. The EGlikeCells from 80～90 dpc group was just three passages in the maximal subculture, but other groups were more than six passages. The mechanical dissociation yielded significantlymore buffalo EG-like colonies （8.087±6,935 vs 1.765±4.747 from each fetus, P＜0.05） andlarger primary colonies （216.95±77.41μm vs140±38.76μm, P＜0.05） in comparison withtrypsin isolation. More colonies formed when the EG-like cells were cultured on the BEFfeeder group in comparioson with other groups（P＜0.05）, and the EG-like cells growing onMEF and BEF feeder were eight passages at least, but growing on STO feeder was justfive passages. In F₁ subculture, addition of 20ng/mLLIF+20ng/mL bFGF+20ng/mLSCFor 20ng/mL LIF+40ng/mL bFGF+40ng/mL SCF to the culture medium resulted insignificantly more EG-like cell colonies formed in comparison with the control group （nocytokines group） and 10ng/mL LIF+10ng/mL bFGF group （P＜0.05）. In F₂-F₈ subculture,addition of 20ng/mLLIF resulted in significantly more EG-like cell clones formed incomparison with 10ng/mLLIF group （P＜0.05）. The passages of EG-like cells in controlgroup was just two in the maximal subculture, and were increased to five and eight byaddition of 10ng/mL LIF and 20ng/mLLIF respectively. In conclusions, （1） the sex and ageof fetus, and dissociating method have a significant influence on the isolation and cultureof buffalo EG-like cells, male and younger than 80 dpc fetus are more suitable to theisolation and culture of buffalo EG-like cells, mechanical dissociating method is lessdamage to buffalo EG-like cells than trypsin digestion; （2） BEF feeder is benefit to thesubculture of buffalo EG-like cell, LIF, plays an important role in the subculture of buffaloEG-like cells, but bFGF and SCF do not.3. Factors （lipid and plasmid DNA consentration, EG-like cells passages） affecting GPFgene transfection of the buffalo EG-like cells were investigated, and the feasibility ofproducing chimeric embryos by introducing GFP-EG-like cells was explored. Thetransfection efficiency of buffalo EG-like cells was higher when 8μL/mL and 12μL/mL oflipid were used in comparison with 2μL/mL and 4μL/mL lipid （33.6%, 36.2% vs 13.3%,16.8%; P＜0.05）. The optimum concentration of plasmid DNA was proved to be 2μg/mL,in which higher transfection efficiency was achieved in comparison with 1μg/mL and4μg/mL group （28.8% vs 14.5%, 15.2%; P＜0.05）. There was not significantly differencein transfection efficiency among the different passage （0-8） EG-like cells （19.8%, 22.9%,25.8%, 19.0% and 16.9% respectively, P＞0.05）. The blastocyst yield of 8-16 cell buffaloembryos injected with GFP-EG-like cells was not significantly different from the 8-16 cellbuffalo embryos injected with GFP-BEF （35.6% vs 33.3%; P＞0.05）. However, ten ofsixteen blastocysts from GFP-EG-like cells group expressed GFP, but none of blastocystsfrom BEF group expressed GFP. In conclusions, 0-8 passages of buffalo EG-like cells canbe used for GFP gene transfection and the optimal concentration of lipid and plasmid DNAwas proved to be 8μL/mL and 2μg/mL respectively; buffalo EG-like cells can merge into the buffalo chimeric embryos, but fibroblasts do not.4. The influenCeS of different passages EG-like cells and GFP-EG-like cells on nucleartransfer were investigated in this study. There was not significantly difference amongdifferent passage （0-8） EG-like cells and fibroblast cells in the efficiency of fused, cleaved,and blastocyst developed after nuclear transfer （82.0% vs 84.5% vs 80.1% vs 78.7% vs84.0%, 6.7.6% vs 72.9% vs 72.0% vs 62.9% vs 63.1%, 11.4% vs 12.4% vs 13.4% vs 10.0%vs 7.1%; P＞0.05）, the efficiency of fused, cleaved, and blaStocyst developed after nucleartransfer were also similar between GFP-EG cells and non-transtected EG-like cells （79.0%vs 81.0%, 62.4% vs 71.4%, 11.0% vs 13.3%; P＞0.05）. In conclusions, buffalo EG-likecells （0-8 passages） can be used as donor cells for nuclear transfer, and their nucleartransfer efficiency is not affected by GFP gene transfection.更多还原