牛胚胎干细胞培养条件以及差异表达基因的研究
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摘要
胚胎干细胞(Embryonic stem cell,简称ES细胞),是从哺乳动物早期胚胎的内细胞团(Inner cell mass, ICM)或原始生殖细胞(Primordial germcell, PGCs)中分离出来,经体外分化抑制培养获得的具有无限增殖能力和保持未分化状态的细胞系。该细胞系具有胚胎细胞与普通培养细胞的双重特性,其具有正常的二倍体核型,具有发育的全能性和多能性,并可无限增殖、冷冻保存及进行基因遗传操作。牛作为家畜中重要的动物,其ES细胞的建立具有广阔的应用前景。如何克服牛ES细胞建系中存在的问题,找到适合牛ES细胞生长增殖的环境已成为现在亟待解决的问题。本研究比较了饲养层的种类和密度、胚胎的处理方法及不同培养液对牛类胚胎干细胞培养的影响,并采用DDRT-PCR和Western blotting技术从mRNA水平和蛋白质水平上观察了不同形态和不同代次牛类胚胎干细胞基因表达的差异,以便为建立牛胚胎干细胞培养体系提供实验依据。
一、饲养层对牛胚胎干细胞的影响
1.小鼠胎儿成纤维细胞和牛胎儿成纤维细胞做饲养层对牛类胚胎干细胞培养的影响
分别用小鼠胎儿成纤维细胞(MEF)和牛胎儿成纤维细胞(BEF)做饲养层对牛类胚胎干细胞进行培养,发现两种饲养层都可形成具有干细胞形态特征的克隆。在BEF饲养层培养条件下干细胞生长速度慢,干细胞传至第五代后克隆边缘细胞与饲养层细胞融合,界限模糊。而在MEF饲养层培养条件下干细胞生长速度快,干细胞传至第十代仍保持未分化状态。
2.饲养层密度对牛类胚胎干细胞的影响
比较三种不同密度的小鼠胎儿成纤维细胞做饲养层对牛类胚胎干细胞分离培养的影响。结果显示,在密度为1.25×105/ml的饲养层上囊胚贴壁时间短,贴壁率高,初代克隆形成率高,最适合牛类胚胎干细胞的分离培养。
二、囊胚的处理方法和接种密度对牛类胚胎干细胞分离的影响
1.囊胚处理方法对牛类胚胎干细胞分离的影响
本研究中分别采用链酶蛋白酶去除透明带的囊胚、自然孵化囊胚以及机械切割得到的内细胞团进行牛类胚胎干细胞分离培养。结果显示,自然孵化的囊胚贴壁时间短,贴壁率高,更有利于牛类胚胎干细胞的分离培养。
2.囊胚接种密度对牛类胚胎干细胞分离的影响
将自然孵化囊胚接种在24孔板中,每孔接种1-4枚囊胚。结果显示,每孔接种2枚囊胚的原代克隆率最高为76%。
三、血清和IGF1对牛类胚胎干细胞分离的影响
以密度为1.25×105/ml的胎鼠成纤维细胞为饲养层,24孔板每孔接种两枚自然孵化囊胚,用三种不同的培养液培养牛类胚胎干细胞,来确定血清和IGF1因子对牛类胚胎干细胞培养的影响。结果显示,普通血清和干细胞专用血清对牛类胚胎干细胞培养的影响不明显；添加10ng/mlIGF1的培养液,培养的牛类胚胎干细胞克隆集落生长最旺盛,形成的克隆最大,但传至五代后细胞明显分化,表明IGFl会促进牛类胚胎干细胞的生长但不利于干细胞多能性的维持。
四、牛类胚胎干细胞的鉴定
用DMEM/F12+15%FBS+0.1mMβ-巯基乙醇+O.1mM非必需氨基酸+双抗+10ng/ml LIF+10ng/ml bFGF培养液培养的牛类胚胎干细胞进行OCT-4、SSEA-1、SSEA-4、TRA-1-61的免疫荧光检测和Nanog、Oct-4和Sox-2的RT-PCR检测,结果均为阳性。并对不同形态和代次的牛类胚胎干细胞做了碱性磷酸酶检测,结果为阳性。证明了本实验所培养和收集的牛类胚胎干细胞具有干细胞特性。
五、不同形态牛类胚胎干细胞的mRNA差异显示与鉴定
1.差异基因的mRNA表达量检测
本研究利用DDRT-PCR筛选片状、块状与泡状三种形态牛类胚胎干细胞差异表达的基因,并用实时定量PCR(Real Time Quantitative PCR)分析差异基因在不同发育阶段的表达丰度。筛选得到6个差异片段,经测序和GenBank数据库比对分析,这6个片段与已知基因有较高的同源性,分别为RPL9、LOC100850994、AMP、RPL31、Erbb2ip、CLIP1。通过实时定量PCR检测这6个基因的表达量,并用SPSS统计分析软件对实时数据进行了统计,结果表明,RPL9在块状和泡状中的表达量分别是片状的0.67倍和1.46倍(P<0.01)；LOC100850994在块状和泡状中的表达量分别是片状的0.33和2.73倍(P<0.01)；RPL31在块状和泡状中的表达量分别是片状的0.71(P<0.05)和1.58倍(P<0.01)；AMP、Erbb2IP和CLIP1在片状与块状中的表达量无显著差异(P>0.05),而在泡状中的表达量分别为片状的2.11、1.43和1.61倍(P<0.05)。
2.差异基因的蛋白表达量检测
用Western blotting检测各差异基因在不同形态中蛋白表达情况,以片状牛类胚胎干细胞为校准组,以a-tubulin为内参基因。结果显示,RPL9在片状细胞中表达量最高,泡状次之,块状细胞中表达量最低(P<0.05)；RPL31在片状和泡状细胞中表达量基本相同(P>0.05),在块状细胞中表达量显著降低(P<0.05)；AMP和CLIP1在三种形态中的蛋白表达量没有显著差异(P>0.05)。
六、不同代次牛类胚胎干细胞的mRNA差异显示与鉴定
1.差异基因的]mRNA表达量检测
本研究利用DDRT-PCR筛选第一、五和十代不同代次牛类胚胎干细胞差异表达的基因,并用实时定量PCR(Real Time Quantitative PCR)分析差异基因在不同发育阶段的表达丰度。结果筛选得到7个差异片段,经测序和GenBank数据库比对分析,这7个片段与已知基因有较高的同源性,分别为IK、TKDP1、BZW、RPL31、PRL9、 RP42、IGBP1。通过实时定量PCR检测这7个基因的表达量,并用SPSS统计分析软件对实时数据进行了统计,结果表明,IK、TKDP1、 BZW、PRL9、RP42、IGBP1在第十代中的表达量较第一代均显著降低(P<0.05),仅RPL31表达量无显著差异(P>0.05)。
2.差异基因的蛋白表达量检测
用Western bloting检测各差异基因在不同代次中蛋白表达情况,以第一代牛类胚胎干细胞为校准组,以α-tubulin为内参基因。结果显示,各检测蛋白在第一代的表达量均显著高于第十代(P<0.05)。
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell masses (ICMs) of preimplantation embryos or primordial germcell (PGCs), capable of self-renewing, proliferating indefinitely and differentiating into a wide variety of cell types both in vitro and in vivo. This line of cells possess features of both embryo cells, with normal diploid karyotype and developmental toti-or pluripotency, and that of normal cultured cells, proliferating indefinitely, tolerant of cryopreservation and genetic operations. Cattle as one of the most important domestic animal, its ES cells have a broad application prospect. It has turned to be an urgent problem to resolve the difficulties confronted in the establishment of ES cell line and to define the suitable condition for the growth of ES cells. In this research, different types and densities of feeder cells, three kinds of treatment of embryos and multiple classes of culture media have been explored to investigate the effects on culture of bovine ES cells. The expression of differential expressed mRNA and protein in different morphology and passages of bovine ES cell were tested in the method of DDRT-PCR and Western blotting, so that to provide the experimental basis for establishing bovine ES cells culture system.
Effects of different feeder layer cells on bESC-like cell growth
1. Effects of murine embryonic fibroblasts (MEF) or bovine embryonic fibroblasts (BEF) as feeder layer cells on bESC-like cell growth
BEF and MEF were used as feeder layers respectively for bESC-like cells culture. Formation of stem cells clones with morphological characteristics could be observed on both kinds of feeder cells. However, bESC-like cells on BEF layer could not grow as fast as that on MEF layer, and the colonies could not attach tightly. Disaggregation occurred till the fifth passage and all the clones disappeared. Whereas, ES cell colonies on MEFs could maintain undifferentiated state untill the10th passage.
2. Effects of feeder layer density on embryo attachment rates and ES cell colony forming rates
Three densities of MEF feeder cells were used to compare the effects on embryo attachment and ES cell colony formation. The results showed that the best derivation and maintenance of bESC-like cells could be acquired at a feeder cell density of1.25×105cells/ml, with efficient attachment and the highest primary colony forming rate.
Effects of embryo treatment and inoculation density on bESC-like cell isolation and culture
1. Effects of embryo treatment on bESC-like cell isolation and culture
The ICM used for bESC-like cells isolation were acquired from zona pellucida free blastocysts treated by pronase, naturally hatched blastocysts and mechanical isolated ICM. The result showed that the most efficient adherence was obtained in naturally hatched blastocysts, indicating this method to be beneficial for isolation and culture of bESC-like cells.
2. Effect of inoculation density on bESC-like cell isolation and culture
Naturally hatched blastocysts were inoculated at a density of1-4embryos/well in24-well plates, the highest primary colony forming rates (76%) were acquired at the density of2embryos/well.
Effects of culture medium formula on bESC-like cells culture
To confirm the effects of serum and IGF1on bESC-like cells culture, three different kinds of culture media were investigated, with a MEF feeder cells density of1.25×105/ml and inoculation density of2embryos/well in24-well plate. The results showed that there was no significant difference on bESC-like cells culture between normal serum and stem cell-specific serum supplemented in the culture media. The bESC-like cells cultured in media with10ng/ml IGF1possessed the most vigorous clone colony growth and the largest formed clone. However, the cells showed a distinct differentiation at the fifth passage, suggesting that IGF1facilitated bESC-like cells growth, but was not in favor of maintenance of stem cells pluripotency.
Verification of bESC-like cell
Immunofluorescence assay of OCT-4, SSEA-1, SSEA-4and TRA-1-61and RT-PCT assay of Nanog, Oct-4and Sox-2has been taken in bESC-like cells cultured in DMEM/F12media with15%FBS,0.1mM β-mercaptoethanol,0.1mM non-essential amino acids, penicillin/streptomycin,10ng/ml LIF and10ng/ml bFGF, with a feeder cells density of1.25×105/ml and inoculation density of2embryos/well in24-well plate. These analysis all showed positive results. The alkaline phosphatase assay of bESC-like cells in different shapes and passages also showed positive results. These results confirmed that the cultured and collected bESC-like cells shared the features of stem cells.
mRNA DDR-PCR assay and identification of bESC-like cells of different shapes
1. mRNA expression detection of differential expression genes
In this research, differential expression genes of bESC-like cells in sheet, vesicular and lump shapes have been screened by DDRT-PCR assay, expression quantity was analyzed by Q-PCR. Six DNA fragment has been acquired and confirmed to be homologous to RPL9、 LOC100850994、AMP、RPL31、Erbb2ip and CLIP1, respectively after sequencing and comparative analysis with the GenBank database. The results of real time PCR detection and SPSS statistical analysis of the six differential genes showed that RPL9expression quantity in lump and vesicular shaped bESC-like cells were0.67(P<0.01) and1.46(P<0.01) times as that in sheet shaped cells, LOC100850994expression quantity in lump and vesicular shaped bESC-like cells were0.33(P<0.01) and2.73(P<0.01) times as that in sheet shaped cells, RPL31expression quantity in lump and vesicular shaped bESC-like cells were0.71(P<0.05) and1.58(P<0.01) times as that in sheet shaped cells,, respectively. There were no significant difference for AMP, Erbb2IP and CLIP1expression quantity between sheet shaped cells and lump shaped cells, whereas their expression quantity in vesicular cells were2.11(P<0.05),1.43(P<0.05) and1.61(P<0.05) times as that in sheet shaped cells respectively.
2. Protein expression detection of differential expression genes
Protein expression of differential expression genes were detected by Western blotting, with sheet shaped bESC-like cells as calibration and a-tubulin as internal reference. The results showed that sheet shaped cells possessed the highest protein expression level of RPL9, lump shaped cells possessing the lowest level (P＜0.05). RPL31expression was almost same (P>0.05) between sheet and vesicular shaped cells but descended significantly in lump shaped cells (P＜0.05). There was no significant difference for AMP and CLIP1protein expression in the three shaped cells(P>0.05).
mRNA DDR-PCR assay and identification of bESC-like cells of different passages
1. Detection of differential genes expression
Differential expression genes in bESC-like cells of the first, fifth and tenth passage were screened by DDRT-PCR assay, and the expression quantity was analyzed by Q-PCR. Seven DNA fragments had been acquired and confirmed to be homologous to IK、TKDP1、BZW、 RPL31、PRL9、RP42and IGBP1respectively after sequencing and comparative analysis with the GenBank database. The results of real time PCR detection and SPSS statistical analysis showed that the expression level of IK, TKDP1, BZW, PRL9, RP42and IGBP1in the cells of the tenth passage descend significantly (P<0.05) compared with that of the first passage, and there was no significant difference for RPL31expression among them (P>0.05).
2. Protein expression detection of differential expression genes
Protein expression of differential genes was detected by Western blotting, with sheet shaped bESC-like cells as calibration and a-tubulin as internal reference. The results suggested that there was a consistently significant higher expression level of all the detected genes in the cells of the first passage than that of the tenth (P＜0.05).
一、饲养层对牛胚胎干细胞的影响
1.小鼠胎儿成纤维细胞和牛胎儿成纤维细胞做饲养层对牛类胚胎干细胞培养的影响
分别用小鼠胎儿成纤维细胞(MEF)和牛胎儿成纤维细胞(BEF)做饲养层对牛类胚胎干细胞进行培养,发现两种饲养层都可形成具有干细胞形态特征的克隆。在BEF饲养层培养条件下干细胞生长速度慢,干细胞传至第五代后克隆边缘细胞与饲养层细胞融合,界限模糊。而在MEF饲养层培养条件下干细胞生长速度快,干细胞传至第十代仍保持未分化状态。
2.饲养层密度对牛类胚胎干细胞的影响
比较三种不同密度的小鼠胎儿成纤维细胞做饲养层对牛类胚胎干细胞分离培养的影响。结果显示,在密度为1.25×105/ml的饲养层上囊胚贴壁时间短,贴壁率高,初代克隆形成率高,最适合牛类胚胎干细胞的分离培养。
二、囊胚的处理方法和接种密度对牛类胚胎干细胞分离的影响
1.囊胚处理方法对牛类胚胎干细胞分离的影响
本研究中分别采用链酶蛋白酶去除透明带的囊胚、自然孵化囊胚以及机械切割得到的内细胞团进行牛类胚胎干细胞分离培养。结果显示,自然孵化的囊胚贴壁时间短,贴壁率高,更有利于牛类胚胎干细胞的分离培养。
2.囊胚接种密度对牛类胚胎干细胞分离的影响
将自然孵化囊胚接种在24孔板中,每孔接种1-4枚囊胚。结果显示,每孔接种2枚囊胚的原代克隆率最高为76%。
三、血清和IGF1对牛类胚胎干细胞分离的影响
以密度为1.25×105/ml的胎鼠成纤维细胞为饲养层,24孔板每孔接种两枚自然孵化囊胚,用三种不同的培养液培养牛类胚胎干细胞,来确定血清和IGF1因子对牛类胚胎干细胞培养的影响。结果显示,普通血清和干细胞专用血清对牛类胚胎干细胞培养的影响不明显；添加10ng/mlIGF1的培养液,培养的牛类胚胎干细胞克隆集落生长最旺盛,形成的克隆最大,但传至五代后细胞明显分化,表明IGFl会促进牛类胚胎干细胞的生长但不利于干细胞多能性的维持。
四、牛类胚胎干细胞的鉴定
用DMEM/F12+15%FBS+0.1mMβ-巯基乙醇+O.1mM非必需氨基酸+双抗+10ng/ml LIF+10ng/ml bFGF培养液培养的牛类胚胎干细胞进行OCT-4、SSEA-1、SSEA-4、TRA-1-61的免疫荧光检测和Nanog、Oct-4和Sox-2的RT-PCR检测,结果均为阳性。并对不同形态和代次的牛类胚胎干细胞做了碱性磷酸酶检测,结果为阳性。证明了本实验所培养和收集的牛类胚胎干细胞具有干细胞特性。
五、不同形态牛类胚胎干细胞的mRNA差异显示与鉴定
1.差异基因的mRNA表达量检测
本研究利用DDRT-PCR筛选片状、块状与泡状三种形态牛类胚胎干细胞差异表达的基因,并用实时定量PCR(Real Time Quantitative PCR)分析差异基因在不同发育阶段的表达丰度。筛选得到6个差异片段,经测序和GenBank数据库比对分析,这6个片段与已知基因有较高的同源性,分别为RPL9、LOC100850994、AMP、RPL31、Erbb2ip、CLIP1。通过实时定量PCR检测这6个基因的表达量,并用SPSS统计分析软件对实时数据进行了统计,结果表明,RPL9在块状和泡状中的表达量分别是片状的0.67倍和1.46倍(P<0.01)；LOC100850994在块状和泡状中的表达量分别是片状的0.33和2.73倍(P<0.01)；RPL31在块状和泡状中的表达量分别是片状的0.71(P<0.05)和1.58倍(P<0.01)；AMP、Erbb2IP和CLIP1在片状与块状中的表达量无显著差异(P>0.05),而在泡状中的表达量分别为片状的2.11、1.43和1.61倍(P<0.05)。
2.差异基因的蛋白表达量检测
用Western blotting检测各差异基因在不同形态中蛋白表达情况,以片状牛类胚胎干细胞为校准组,以a-tubulin为内参基因。结果显示,RPL9在片状细胞中表达量最高,泡状次之,块状细胞中表达量最低(P<0.05)；RPL31在片状和泡状细胞中表达量基本相同(P>0.05),在块状细胞中表达量显著降低(P<0.05)；AMP和CLIP1在三种形态中的蛋白表达量没有显著差异(P>0.05)。
六、不同代次牛类胚胎干细胞的mRNA差异显示与鉴定
1.差异基因的]mRNA表达量检测
本研究利用DDRT-PCR筛选第一、五和十代不同代次牛类胚胎干细胞差异表达的基因,并用实时定量PCR(Real Time Quantitative PCR)分析差异基因在不同发育阶段的表达丰度。结果筛选得到7个差异片段,经测序和GenBank数据库比对分析,这7个片段与已知基因有较高的同源性,分别为IK、TKDP1、BZW、RPL31、PRL9、 RP42、IGBP1。通过实时定量PCR检测这7个基因的表达量,并用SPSS统计分析软件对实时数据进行了统计,结果表明,IK、TKDP1、 BZW、PRL9、RP42、IGBP1在第十代中的表达量较第一代均显著降低(P<0.05),仅RPL31表达量无显著差异(P>0.05)。
2.差异基因的蛋白表达量检测
用Western bloting检测各差异基因在不同代次中蛋白表达情况,以第一代牛类胚胎干细胞为校准组,以α-tubulin为内参基因。结果显示,各检测蛋白在第一代的表达量均显著高于第十代(P<0.05)。
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell masses (ICMs) of preimplantation embryos or primordial germcell (PGCs), capable of self-renewing, proliferating indefinitely and differentiating into a wide variety of cell types both in vitro and in vivo. This line of cells possess features of both embryo cells, with normal diploid karyotype and developmental toti-or pluripotency, and that of normal cultured cells, proliferating indefinitely, tolerant of cryopreservation and genetic operations. Cattle as one of the most important domestic animal, its ES cells have a broad application prospect. It has turned to be an urgent problem to resolve the difficulties confronted in the establishment of ES cell line and to define the suitable condition for the growth of ES cells. In this research, different types and densities of feeder cells, three kinds of treatment of embryos and multiple classes of culture media have been explored to investigate the effects on culture of bovine ES cells. The expression of differential expressed mRNA and protein in different morphology and passages of bovine ES cell were tested in the method of DDRT-PCR and Western blotting, so that to provide the experimental basis for establishing bovine ES cells culture system.
Effects of different feeder layer cells on bESC-like cell growth
1. Effects of murine embryonic fibroblasts (MEF) or bovine embryonic fibroblasts (BEF) as feeder layer cells on bESC-like cell growth
BEF and MEF were used as feeder layers respectively for bESC-like cells culture. Formation of stem cells clones with morphological characteristics could be observed on both kinds of feeder cells. However, bESC-like cells on BEF layer could not grow as fast as that on MEF layer, and the colonies could not attach tightly. Disaggregation occurred till the fifth passage and all the clones disappeared. Whereas, ES cell colonies on MEFs could maintain undifferentiated state untill the10th passage.
2. Effects of feeder layer density on embryo attachment rates and ES cell colony forming rates
Three densities of MEF feeder cells were used to compare the effects on embryo attachment and ES cell colony formation. The results showed that the best derivation and maintenance of bESC-like cells could be acquired at a feeder cell density of1.25×105cells/ml, with efficient attachment and the highest primary colony forming rate.
Effects of embryo treatment and inoculation density on bESC-like cell isolation and culture
1. Effects of embryo treatment on bESC-like cell isolation and culture
The ICM used for bESC-like cells isolation were acquired from zona pellucida free blastocysts treated by pronase, naturally hatched blastocysts and mechanical isolated ICM. The result showed that the most efficient adherence was obtained in naturally hatched blastocysts, indicating this method to be beneficial for isolation and culture of bESC-like cells.
2. Effect of inoculation density on bESC-like cell isolation and culture
Naturally hatched blastocysts were inoculated at a density of1-4embryos/well in24-well plates, the highest primary colony forming rates (76%) were acquired at the density of2embryos/well.
Effects of culture medium formula on bESC-like cells culture
To confirm the effects of serum and IGF1on bESC-like cells culture, three different kinds of culture media were investigated, with a MEF feeder cells density of1.25×105/ml and inoculation density of2embryos/well in24-well plate. The results showed that there was no significant difference on bESC-like cells culture between normal serum and stem cell-specific serum supplemented in the culture media. The bESC-like cells cultured in media with10ng/ml IGF1possessed the most vigorous clone colony growth and the largest formed clone. However, the cells showed a distinct differentiation at the fifth passage, suggesting that IGF1facilitated bESC-like cells growth, but was not in favor of maintenance of stem cells pluripotency.
Verification of bESC-like cell
Immunofluorescence assay of OCT-4, SSEA-1, SSEA-4and TRA-1-61and RT-PCT assay of Nanog, Oct-4and Sox-2has been taken in bESC-like cells cultured in DMEM/F12media with15%FBS,0.1mM β-mercaptoethanol,0.1mM non-essential amino acids, penicillin/streptomycin,10ng/ml LIF and10ng/ml bFGF, with a feeder cells density of1.25×105/ml and inoculation density of2embryos/well in24-well plate. These analysis all showed positive results. The alkaline phosphatase assay of bESC-like cells in different shapes and passages also showed positive results. These results confirmed that the cultured and collected bESC-like cells shared the features of stem cells.
mRNA DDR-PCR assay and identification of bESC-like cells of different shapes
1. mRNA expression detection of differential expression genes
In this research, differential expression genes of bESC-like cells in sheet, vesicular and lump shapes have been screened by DDRT-PCR assay, expression quantity was analyzed by Q-PCR. Six DNA fragment has been acquired and confirmed to be homologous to RPL9、 LOC100850994、AMP、RPL31、Erbb2ip and CLIP1, respectively after sequencing and comparative analysis with the GenBank database. The results of real time PCR detection and SPSS statistical analysis of the six differential genes showed that RPL9expression quantity in lump and vesicular shaped bESC-like cells were0.67(P<0.01) and1.46(P<0.01) times as that in sheet shaped cells, LOC100850994expression quantity in lump and vesicular shaped bESC-like cells were0.33(P<0.01) and2.73(P<0.01) times as that in sheet shaped cells, RPL31expression quantity in lump and vesicular shaped bESC-like cells were0.71(P<0.05) and1.58(P<0.01) times as that in sheet shaped cells,, respectively. There were no significant difference for AMP, Erbb2IP and CLIP1expression quantity between sheet shaped cells and lump shaped cells, whereas their expression quantity in vesicular cells were2.11(P<0.05),1.43(P<0.05) and1.61(P<0.05) times as that in sheet shaped cells respectively.
2. Protein expression detection of differential expression genes
Protein expression of differential expression genes were detected by Western blotting, with sheet shaped bESC-like cells as calibration and a-tubulin as internal reference. The results showed that sheet shaped cells possessed the highest protein expression level of RPL9, lump shaped cells possessing the lowest level (P＜0.05). RPL31expression was almost same (P>0.05) between sheet and vesicular shaped cells but descended significantly in lump shaped cells (P＜0.05). There was no significant difference for AMP and CLIP1protein expression in the three shaped cells(P>0.05).
mRNA DDR-PCR assay and identification of bESC-like cells of different passages
1. Detection of differential genes expression
Differential expression genes in bESC-like cells of the first, fifth and tenth passage were screened by DDRT-PCR assay, and the expression quantity was analyzed by Q-PCR. Seven DNA fragments had been acquired and confirmed to be homologous to IK、TKDP1、BZW、 RPL31、PRL9、RP42and IGBP1respectively after sequencing and comparative analysis with the GenBank database. The results of real time PCR detection and SPSS statistical analysis showed that the expression level of IK, TKDP1, BZW, PRL9, RP42and IGBP1in the cells of the tenth passage descend significantly (P<0.05) compared with that of the first passage, and there was no significant difference for RPL31expression among them (P>0.05).
2. Protein expression detection of differential expression genes
Protein expression of differential genes was detected by Western blotting, with sheet shaped bESC-like cells as calibration and a-tubulin as internal reference. The results suggested that there was a consistently significant higher expression level of all the detected genes in the cells of the first passage than that of the tenth (P＜0.05).
引文
[1]Miyoshi K, Taquchi Y, Sendai Y, et al. Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes [J]. Biol Reprod,2000,62(6):1640-1646.
[2]Thomson J A, Itskovitz-Eldor J, Shapiro S S, et al. Embryonic stem cell lines derived from human blastocysts [J].Science,1998,282(5391):1145-147.
[3]Xu C, Inokuma MS, Denham J. Feeder-free growth of undifferentiated human embryonic stem cells [J].Nature,2001,19(10):971-974.
[4]Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos [J]. Nature,1981,292 (5819):154-156.
[5]李松,窦忠英,苗增民,等.牛ES细胞分离克隆及定向分化研究[J].医学研究杂志,2007,36(2)：79-80.
[6]李碧春,陈国宏,吴信生,等.牛体外受精胚胎衍生干细胞能力影响因素研究[J].畜牧兽医学报,2006,37(9)：928-932.
[7]Rexroad CE, Powell AM. Culture of blastomeres from in vitro—matured, fertilized, and cultured bovine embryos [J]. Mol Reprod Dev.1997,48:238-245.
[8]Pampai R, Minami N, Yamada M, et al. Asuitable passaging Technique for the establishment of bovine embryonic stem(ES)like cell lines[J]. Theriogenology,1998, 49(1):241-245.
[9]Talbot NC, Powell AM, Rexroad CE. In vitro pluripotency of epiblasts derived from bovine blastocysts[J]. Mol Reprod Dev,1995,42(1):35-52.
[10]Mitalipova M, Beyhan I, First W. Pluripotency of bovine embryonic stem cells derived from precompacting embryos[J]. Cloning,2001,3(2):59-67
[11]Liang P., Pardee A.B.,. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992,257:967-971.
[12]Pasentsis K,Falara V,Pateraki I,et al.Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display[J].J Exp Bot,2007,58(8):2203-16.
[13]Ikematsu K,Tsuda R,Nakasono I.Gene response of mouse skin to pressure injury in the neck region[J].Leg Med(Tokyo),2006,8(2):128-31.
[14]Pathak RU,Kanungo MS.Subtractive differential display:a modified differential display technique for isolating differentially expressed genes[J].Mol Biol Rep,2007,34(l):41-6.
[15]Zhang J,Turley RB,Stewart JM.Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display [J].Plant Cell Rep,2008,27(3):553-61.
[1]Evans MJ. Kaufman MH. Estabfishment in culture of pluripotential cells from mouse embryos[J]. Nature 1981:292(9):154-156
[2]李碧春,陈国宏.牛体外受精胚胎衍生干细胞能力影响因素的研究[D].扬州大学硕士研究生毕业论文,2005
[3]Cherny RA. Stokes TM, Merei J. et al. Strategies for the isolation and characterization of bovine embryonic stem cells[J]. Reprod Fert Dev 1994:6(5): 569-575
[4]Stice SL, Strelchenko NS, Keefer CL, et al. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer[J]. Biol Reprod 1996; 54(1):100-110
[5]Cibelli JB. Stice SL, Kane JJ. et al. Production of germline Chimeric bovine fetues from transgenic embryonic stem cells[J]. Theriogenology 1997:47(7):241
[6]Parnpai R. Minami N, Yamada M, et al. Asuitable passaging Technique for the establishment of bovine embryonic stem(ES) like cell lines[J]. Theriogenology 1998; 49(1):241-245
[7]Saito D, Strelchenko N, Niemann H. Bovine embryonic stem cell-like cultured over several passages[J]. Roux' S Arch Dev Biol,1992,201(3):134-140.
[8]Sim M, First NL. Production of calves by transfer of nuclei from cultured inner cell mass cells[J]. Proc Natl Acad Sci U S A.1994,91(13):6143—614
[9]White KL, Polejaeva IA, Bunch TD, et al. Effect of non-serum supplemented media of establishment and maintaince of bovine embryonic stem-like cells[J]. Theriogenology,1995,43(1):350
[10]Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells [J]. Dev Biol,1987,121(1):1-9.
[11]Li Wang, Enkui Duan, Li-ying Sung, et al. Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos [J]. Biology of reproduction, 2005,73(1):149-55.
[12]Seizo Hamano, Yoshie Watanabe, Sadahiro Azuma, et al. Establishment of embryonic stem (ES) cell-like cell lines derived from bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro [J]. Journal of reproduction and development,1998,44(3):297-303.
[13]M. Munoz, A. Rodriguez, C. De Frutos, et al. Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines [J]. Theriogenology,2008, 69(9):1159-1164.
[14]Strojek RM, Reed MA, Hoover J L, et al. A method for cultivation morphological undifferentiated embryonic stem cells from porcine blastocysts [J]. Theriogenology,1990,33:901-903.
[15]Vitezslav Bryja, Sonia Bonilla, Ernest Arenas. Derivation of mouse embryonic stem cells [J]. Nature Publishing Group,2006, 1(4):2082-2087.
[16]Marsha Roach, Li Wang, Xiangzhong Yang, et al. Bovine embryonic stem cells[J]. Methods in enzymology,2006,418:21-37.
[17]杨美玲.转过表达和低表达胰岛素样生长因子I基因牛成纤维细胞系的制备[D].内蒙古大学.2009.
[18]Schieke AS, Kind AJ, Ritchie WA. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts[J].Science,1997,278: 2130-2133.
[19]Baquisi A, Behboodi E, Melican DT. Production of goats by somatic cell nuclear transfer [J]. Nat Biotechnol,1999,17:456-461.
[20]Cibelli JB, Stice SL, Golueke PJ. Cloned transgenic calves produced from nonquiescent fetal fibroblasts[J].Science,1998,280:1256-1258.
[21]Marsha Roach, Li Wang, Xiangzhong Yang, et al. Bovine embryonic stem cells[J]. Methods in enzymology,2006,418:21-37.
[22]薛庆善.体外培养的原理与技术[M].北京,科学出版社.2001,958-961.
[23]屠迪,崔燕,余四九.牦牛胎儿成纤维细胞的分离培养[J].中国兽医科学.2006,36(07)：573-577.
[24]Masssgul.Stimulalion by insulin-like growth factor is required for cellular transformation by type transforming growth factor [J].Bio.chem.1985,260: 4551-4554.
[25]Nlchols.J, Edward P,et al. Establishment of germ-line-competent embryonic stem(ES)cells Using differentiation inhibiting activity [J]. Development Biology,1990,110:1341-1348.
[26]Smith Aostin, Lerlifer Nichols, et al. Differentiation inhibiting Activity (DLA/LIF) and mouse Development[J]. Development Biology,1992,151:339-351.
[27]张青丽,段因奎,曾国庆.白血病抑制因子及其在胚胎发育和胚泡着床中的生理功能[J].生理科学进展,1997,28(3)：240-242.
[28]Aitstin G. Smith. Diffentiation inhibiting activity (DLA/LIF) and mouse development[J].Dev.Biol.1992,151:339-351.
[29]Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells[J].Proteomics,2002,2(9):1187-1203.
[30]Strojek RM, Reed MA, Hoover JL, et al. A method for cultivating morphologically undifferentiated embryonic stem cells from porine blastocysts[J]. Theriogennology,1990,33(4):901-910.
[31]Rexroad CE Jr, Powell AM.. Culture of blastomeres from in vitro-matured, fertilized, and cultured bovine embryos[J]. Mol Reprod Dev1997,48:238-245.
[32]Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium.[J].Exp Cell Res,1999,247(1):241-248.
[33]Thomson J A, Kaalishman J, Golos T G, et al. Isolation of a primate embryonic stem cell line. [J].Proc Natl Acad Sci USA.1995,92(17):7844-7848.
[34]Li M, Ma W, Hou Y, et al. Improved isolation and culture of embryonic stem cells from Chinese miniature pig[J].Reprod Dev 2004.50:237-244.
[35]安立龙,杨奇,窦忠英等.某些因素对牛和小鼠类胚胎干细胞分离与培养的影响[J].中国兽医学报,2002,22(2)：187-190.
[36]Li M, Li YH, Hou Y, et al. Isolation and culture of pluripotent cells from in vitro produced porcine embryos [J].Cambridge University Press,2004,12(1):43-48.
[37]Thomson J A, Itskovitz-Eldor J, Shapiro S S, et al. Embryonic stem cell lines derived from human blastocysts [J].Science,1998,282(5391):1145-147.
[38]Munoz M, Diez C, Caamano JN, Jouneau A, Hue I, Gomez E. Embryonic stem cells in cattle[J]. Reprod Domest Anim 2008.4:32-37.
[39]Li Wang, Enkui Duan, Li-ying Sung, et al. Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos[J]. BIOLOGY OF REPRODUCTIO 2005:73,149-155.
[40]Robert ES, Morayma R, Lisa K, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells[J]. J Clin Invest, 2002,109:1291-1302.
[41]Talbot N C, Powell A M, Rexroad C E, In vitro pluripotency of epiblasts derived from bovine blastocysts [J] Mol Reprod Dev,1995,42:35-521.
[42]Cao S, Wang F, Chen Z, et al. Isolation and Culture of Primary Bovine Embryonic Stem Cell Colonies by a Novel Method[J]. JExp Zool A Ecol Genet Physiol 2009.311:368-76.
[43]Gj(?)rret JO, Maddox-Hyttel P. Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures[J]. Reprod Fertil Dev 2005.17:113-124.
[44]赵晶,董雅娟,柏学进等.饲养层细胞的接种密度对分离培养胚胎干细胞的影响[J].中国兽医学报,2009,29(4)：494-501.
[1]Turnpenny L, Spalluto CM, Perrett RM. Evaluating human embryonic germ cells: concord and conflict as pluripotent Stem cells [J].Stem Cells,2006,24:212-220.
[2]裴雪涛主编.干细胞生物学[M].北京：科学出版社,2003.7.
[3]Li Wang, Enkui Duan, Li-ying Sung, et al. Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos [J]. BIOLOGY OF REPRODUCTION,2005,73:149-155.
[4]Evans MJ, Kaufman MH. Establishment in culture of pluripotential cell from mouse embryos [J]. Nature,1981,293:154-156.
[5]Thomson JA, Itskovitz EJ, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts [J].Science,1998,282:1145-1147.
[6]Cao S, Wang F, Chen Z, et al. Isolation and Culture of Primary Bovine Embryonic Stem Cell Colonies by a Novel Method[J]. JExp Zool A Ecol Genet Physiol 2009.311:368-76.
[7]Gj(?)rret JO, Maddox-Hyttel P. Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures[J]. Reprod Fertil Dev 2005.17:113-124.
[8]Munoz M, Diez C, Caamano JN, et al. Embryonic stem cells in cattle[J]. Reprod Domest Anim 2008.4:32-37.
[9]Roach M,Wang L,Yang X,et al. Bovine embryonic stem cells[J]. Methods enzymol 2006.418:21-37.
[10]Saito S, Liu B, Yokoyama K. Animal embryonic stem (ES) cells:self-renewal, pluripotency, transgenesis and nuclear transfer[J]. Hum Cell 2004.17:107-115
[11]Sim M, First NL. Production of calves by transfer of nuclei from cultured inner cell mass cells[J]. Proc Natl Acad Sci USA 1994.91:6143-6614.
[12]Liang P., Pardee A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992,257:967-971.
[13]Ikematsu K,Tsuda R,Nakasono I.Gene response of mouse skin to pressure injury in the neck region[J].Leg Med(Tokyo),2006,8(2):128-31.
[14]Pathak RU,Kanungo MS.Subtractive differential display:a modified differential display technique for isolating differentially expressed genes[J].Mol Biol Rep,2007,34(1):41-6.
[15]Zhang J,Turley RB,Stewart JM.Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display [J].Plant Cell Rep,2008,27(3):553-61.
[16]Pasentsis K,Falara V,Pateraki I,et al.Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display[J].J Exp Bot,2007,58(8):2203-16.
[17]王梁燕,洪奇华,张耀洲.实时定量PCR技术及其应用[J].细胞生物学杂志,2004,26(1)：62-67.
[18]尹学钧DD-PCR技术及其在遗传毒理学的应用前景[J].国外医学：卫生学分册,1999,26(3)：156-160.
[19]Wool I.G., Extraribosomal functions of ribosomal proteins[J].Trends. Biochem. Sci.,1996,21:164-165.
[20]Walleczek J, Redl B, Stoffler-Meilicke Mat et al. G. Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques[J]. J. Biol. Chem.1989,264:4231-4237.
[21]Brimacombe R, Gornicki P, Greuer B, et al. The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques [J]. Biochim. Biophys. Acta 1990,1050:8-13.
[22]Nag B, Akella SS, Cann PA, et al. Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50S ribosomal subunit[J]. J. Biol. Chem.1991,266: 22129-22135.
[23]Agafonov DE, Kolb VA and Spirin AS. Proteins on ribosome surface: measurements of protein exposure by hot tritium bombardment technique [J]. Proc. Natl. Acad. Sci. U. S. A.1997,94:12892-12897.
[24]Hoffman DW, Davies C, Gerchman SE, et al. Crystal structure of prokaryotic ribosomal protein L9:a bi-lobed RNA-binding protein[J]. EMBO J.1994,13: 205-212.
[25]Lillemoen J, Cameron CS, Hoffman DW. The stability and dynamics of ribosomal protein L9:investigations of a molecular strut by amide proton exchange and circular dichroism[J]. J. Mol. Biol.1997,268:482-493.
[26]Fabijanski S, Pellegrini M. Identification of proteins at the peptidyl-tRNA binding site of rat liver ribosomes[J]. Mol. Gen. Genet.,1981,184:5551-5556.
[27]Durand S, Abadie P, Angeletti S, et.al. Identification of multiple differentially expressed messenger RNA in normal and pathological trophoblast [J]. Placenta,2003, 24:209-218.
[28]Wool I G, Chan Y L, Gluck A. Structure and evolution of mammalian ribosomal proteins[J]. Biochem Cell Biol.,1995,73:933-947.
[29]Hardie DG. AMP-activated/SNF1 protein kinases:conserved guardians of cellular energy[J]. Nat Rev Mol Cell Biol 2007.8(10):774-785.
[30]Fulco M, Cen Y, Zhao P, et.al. Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt[J]. Dev Cell 2008.14(5):661-673.
[31]Habinowski SA, Witters LA. The effects of AICAR on adipocyte differentiation of 3T3-L1 cells[J].Biochem Biophys Res Commun 2001.286(5):852-856.
[32]Kanazawa I, Yamaguchi T, Yano S, et.al. Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression[J]. Biochem Biophys Res Commun 2008,375(3):414-419.
[33]Lohmann J, Schickle H, Bosch T C. REN display, a rapid and efficient method for nonradioactive differential display and mRNA isolation [J]. Biotechniques, 1995,18(2):200-202.
[34]Allan G J, Flint D J, Patel K. Insulin like growth factor axis during embryonic development[J]. Reproduction,2001,122:31—39.
[35]李希水牛卵母细胞体外成熟前后基因差异表达的研究[D].南宁：广西大学,硕士学位论文.2008.
[36]Borg J. P., Marchetto S., Le Bivic A., et.al. ERBIN: A basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor [J]. Nature Cell Biology, 2000,2:407-414.
[37]Li D., Guo N. Research advances in mechenism of CIITA regulating MHC-II express. Chinese Journal of Cell and Molecular Immunology [J].2008,24 (1):97-99.
[38]Willers J., Haffner A., Zepter K. et.al. The interferon inhibiting cytokine IK is overexpressed in cutaneous T cell lymphoma derived tumor cells that fail to upregulate major histocompatibility complex class II upon interferon-gamma stimulation [J]. Journal of Investigative Dermatology,2001,116(6):874-9.
[39]van Wijk I., Griffioen S., Tjoa M. L. et.al. HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy[J]. American Journal of Obstetrics and Gynecology,2001,184(5):991-7.
[40]Mitra P, Vaughan PS, Stein JL, et.al.Purification and functional analysis of a novel leucine-zipper/nucleotide-fold protein, BZAP45, stimulating cell cycle regulated histone H4 gene transcription[J]. Biochemistry 2001, (40) 10693-10699.
[41]Li S, Chai Z, Li Y, et.al. BZW1, a novel proliferation regulator that promotes growth of salivary muocepodermoid carcinoma[J].Cancer Lett.2009 Oct 18;284(1):86-94.
[42]Onda M, Inui S, Maeda K, et.al.Expression and Chromosomal Localization of the Human a4/IGBP1 Gene, the Structure of Which Is Closely Related to the Yeast TAP42 Protein of the Rapamycin-Sensitive Signal Transduction Pathway[J].GENOMICS 199746,373-378
[43]Godfrey Grech, Montserrat Blazquez-Domingo, Andrea Kolbus, et.al. Hartmut Beug and Marieke von Lindern.Igbpl is part of a positive feedback loop in stem cell factor-dependent, selective mRNAtranslation initiation inhibiting erythroid differentiation[J]. blood 2008 112:2750-2760.
[44]Minami N, Tsukamoto S. Role of oocyte-specific genes in the development of mammalian embryos [J]. Reprod. Med. Biol.2006,5(3):175—182.
[1]Evans M. J, Kaufman M. H. Establishment in culture of pluripotential cells from mouse embryos[J]. Nature,1981,292(9):154-156
[2]Helena R, Axelord. Embryonic stem cell lins derived from blastocysts by a simplified technique[J].Development biolingy,1989,101:225-228
[3]Iannaccone PM, Taborn GU, Garton RL, et al. Pluripotent embvryonic stem cells from the rat are capable of producing chimeras [J]. Devel Biol,1994,288-292
[4]McDonald JW, Becker D, Holekamp TF, et al.Repair of the injured spinal cord and the potential of embryonic stem cell transplantation [J]. J Neurotrauma,2004, 21:383-393.
[5]Thomson JA, Kalishman J, Golos TC et al. Isolation of a primate embryonic stem cell line [J]. Proc Natl Acad Sci USA,1995,92:7844-7848.
[6]Doetschman T, Williams P, Maeda N.Establishment of hamster blastocyst-derived embryonic stem (ES) cells[J]. Devel Biol,1988,127:224-227.
[7]Piedrahita J A, Anderson G B, BonDurant R H. Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines[J]. Therogenology, 1990,34:865-877.
[8]Saito S, Strelchenko N, Niemann H. Bovine embryonic stem cell-like lines culture over several passages [J]. Rouxs Archives of Developmental Biology,1992, 201:134-141.
[9]Piedrahita JA., Anderson GB., BonDurant RH. On the isolation of embryonic stem cells:Comparative behavior of marine, porcine and ovine embryos [J]. Theriogenology,1990,34(5):879-901.
[10]Graves KH, Randall W. Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos [J]. Mol Reprod Dev,1993, 36:424-433.
[11]赖良学,郑瑞珍,孙方臻等.胚胎于细胞分离培养的影响因素[J].解剖科学进展,1995,1(3)：203-208.
[12]Sukoyan MA, Golubitsa AN, Zhelezova AI, et al. Isolation and cultivation of blastocyst-derived stem cell lines from American Mind (Mustela vison) [J]. Mol Reprod Dev,1992,33:418-431.
[13]Robertson E J, Evans M J, Kaufman M H. X-chromosome instability in pluripotential stem cell lines derived from parthenogenetic embryos [J]. Journal of Embryology and Experimental Morphology,1982,74:297-309.
[14]Harald R, Eistetter. Pluripotent embryonal stem cell lines can be established from disaggregated mouse morulae Develop ment[J]. Growth and Differ,1981,31(3): 275-282
[15]Wood SA, Pascoe WS, Schmidt C, et al. Simple and Efficient Production of Embryonic Stem Cell-Embryo Chimeras by Coculture[J]. PNAS,1993,90:4582.
[16]Peli J, Schmoll F, Laurincili J, et al. Comparison of aggregation and injection techniques in producing chimeras with ES cells in mice [J]. Theriogenology,1996, 45(4):833-842.
[17]Robertson E, Bradley A, Kuehn M et al. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector [J]. Nature,1986,323: 445-448.
[18]Chung Y, Klimanskaya I and Bocker S. Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres [J]. Nature,2006,439(10):1038-1042.
[19]Babinet C, Cohen-Tannoudji M. Genome engineering via homologous recombination in mouse embryonic stem (ES) cells:an amazingly versatile tool for the study of mammalian biology [J].AnAcad Bras Cienc,2001,73:365—383.
[20]Tomes M. The use of embryonic stem cells for the genetic manipulation of the mouse[J]. Curr Top Dev Biol,1998,36:99—114.
[21]Gossler A, Doetschman T, Korn R,et al. Transgenesis by means of blastocyst-derived embryonic stem cell lines [J]. Proc Natl Acad Sci USA,1986,83: 9065-9069.
[22]Thompson S, ClarkeAR, PowAM et al. Germ line transmis-sion and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells [J]. Cell,1989,56:313-321.
[23]Zambrowicz BP, Friedrich GA, Buxton EC, et al. Disruption and sequence identification of 2000 genes in mouse embryonic stem cells [J]. Nature 1998,392: 608-611.
[24]Bettiol E, Sartiani L, Chicha L, et al. Fetal bovine serum is essential for cardiac differentiation of human embryonic stem cell [J]. J Mol Cell Cardiol.,2007,42: s88-s101 (Abstract).
[25]Okumura AJ, Peterson LF, Lo MC, et al. Expression of AML/Runx and ETO/MTG family members during hematopoietic differentiation of embryonic stem cells [J]. Exp. Hematol.,2007,35:978-988.
[26]Lee SH, Lumelsky N, Studer L et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells [J]. Nat Biotechnol,2000,18: 675-679.
[27]Smith, A.G., Heath, J.K., Donaldson, D.D., et al. Inhibition of pluripotential embryonic stem cell differentiation by puried polypeptides [J]. Nature,1988,336: 688-690.
[28]Niwa H, Burdon T, Chambers I, Smith AG. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3 [J]. Genes Dev.,1998,12:2048-2060.
[29]Matsuda, T., Nakamura, T., Nakao, K., et al. STAT3 acti-vation is suf?cient to maintain an undifferentiated state of mouse embryonic stem cells [J].EMBO J.,1999, 18:4261-4269.
[30]Ogawa K, Matsui H, Ohtsuka S. et al. A novel mechanism for regulating clonal propagation of mouse ES cells [J]. Genes to Cells,2004,9:471-477.
[31]Brook F. A., Gardner R. L. The origin and efficient derivation of ESC in the mouse [J]. Proc Natl Acad Sci USA,1997,94:5709-5712.
[32]Zwaka T. P., Thomson J. A. A germ cell origin of embryonic stem cells [J]. Development,2005,132:227-233.
[33]Hirofumi Suemori, Horio Nakatsuji. Establishment of embryos derived stem (ES) cell lines from mouse blastocyst:effect of the feeder cell layer[J]. Development growth and diff.29 (2):133-139
[34]Piedrahita J. A, Anderson G. B, Bondurant R. H. on the isolation of embryonic stem cell:Camparative Behavior of murine, porcine, ovine embryos[J]. Thereiogenology.1990,39(5):879-901
[35]Labos PA, Barlow DP, Hogan BL. Embryonic germ cell lines and their derivation from mouse primordial germ cells [J]. CIBA Foundation symposium,1994,182: 157-178.
[36]Niemann H, Strelchenko N. Isolation and maintenance of rabbit embryonic stem cell lines [J].Theriogenllogy,1994,41:265.
[37]Du F, Giles J R, Footerh, et al. Nuclear transfer of putative rabbit embryonic stem cell leads to normal blastocyst development [J]. Reproduction and Fertilization,1995, 104:219-223.
[38]Moens A, Chastant S, Chesne P. Differential reprogramming capabilities,of male and female rabbit fetal gonadal cells [J]. Proc Cong Eur Biol Organ,1995,6:98.
[39]Schoonuans L, Albright GM, Li J L, et al. Pluripotent rabbit embryonic stem cells are capable of forming overt coat color chimeras following injection into blasto-cysts[J]. Mol Reprod Dev,1996,45:439-443.
[40]Nakayama N, Fang I, Elliott G. Natural killer and B-Lymphoid potential in CD34 cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor[J].Blood,1998,91(7):2283-295.
[41]Lee C.K., Weaks R. L., Johnson GA. Effects of Protease Inhibitors and Antioxidants on In Vitro Survival of Porcine Primordial Germ Cells [J]. Biol Reprod, 2000,63:887
[42]Shi-ping Luh, Chi-huei Chiang.Acute lung injury/acute respiratory distress syndrome (ALI/ARDS):the mechanism, present strategies and future perspectives of therapies[J]. JOURNAL OF ZHEJIANG UNIVERSITY.2007,8(1):60-69
[43]Piedrahita J A, Anderson G B, Martin G R, et al. Isolation of embryonic stem cell-lide colonies from porcine embryos[J]. Theriogenology,1988,29:286.
[44]Strojek RM, Reed M A, Hoover J L, et al. A method for cultivating morphologically undifferentiated embryonic stem cells from procine blastocysts[J]. Theriogenology,1990,33:901-913.
[45]Piedrahita JA, Anderson GB, Bondurant RH.On the isolation of embryonic stem cells:Comparative behavior of murine, porcine and ovine embryos[J].Theriogenology. 1990;34(5):879-901.
[46]Wheeler MB. Development and validation of swine embryonic stem cell:a review Reprod Fertil[J]. Dev,1994,6:563-568.
[47]Evans MJ, Notarianni E, Lauries, et al. Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts[J]. Theriogenology,1990,33:125-128.
[48]Cibelli JB, Stice SL, Golueke PJ, et al. Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells [J]. Nature Biotech,1998,16: 642-646
[49]Booth PJ, Perreau C, Hochereau-de Reviers. TEC-1 characterisation of porcine embryonic cells from day 11 embryonic discs cultured in serum-free medium [J]. Theriogenology,1997,47(1):167.
[50]Shim H, Gutierrez-Adan A, Chen LR, et al. Isolation of pluripotent stem cell from cultured porcine primordial germ cells [J]. Biol Reprod,1997,57:1089-1095.
[51]Shim H and Anderson GB. In vitro survival and proliferation of porcine embryonic germ cells [J].Theriogenology,1998,49:521—528.
[52]Piedrahita J, Moore K, Oetama B, et al. Generation of transgenic porcine chimeras using primordial germ cell-derived colonies [J]. Biol Reprod,1998,58: 1321—1329.
[53]Bongso A, Fong CY, Ng SC et al. The growth of inner cell mass cells from human blastocysts (Abstract) [J]. Theriogenology,1993,41:161.
[54]Bongso A, Fong CY, Ng SC et al. Isolation and culture of inner cell mass cells from human blastocysts [J]. Hum Reprod,1994,9:2110-2117.
[55]Thomson JA, Marshall VS. Primate embryonic stem cells[J]. Curr Top Dev Biol 1998,38:133-165.
[56]Thomson JA, Kalishman J, Golos TQ et al. Isolation of a primate embryonic stem cell line[J]. Proc Natl Acad Sci U S A,1995,55:254-259.
[57]Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts:somati differentiation in vitro [J]. Nat Biotechnol,2000,18:399-404.
[58]Amit M&Itskovitz-Eldor J. Derivation and spontaneous differentiation of human embryonic stet cells [J]. J Anat,2002,200(3):225-232.
[59]何志旭,黄绍良,李予等.人胚胎干细胞系的初步建立[J].中华医学杂志,2002,82(19)：1314-1318
[60]Kehat I, Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocyteswith structural and functional properties of cardiomyocytes [J].J Clip Invest,2001,108,407-414.
[61]VogelG. Developmental biology:The hottest stem cells are also the toughest[J]. Science,2001,292(5516):429
[62]Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursor from human embryonic stem cells [J]. Nat Biotechnol,2001,19: 1129-1133.
[63]Mummery C, Ward-van Oostvaard D, Doevendans P et al. Differentiation of human embryonicstet cells to cardiomyocytes:role of coculture with visceral endoderm-like cells[J]. Circulation,2003, (10),2733-2734.
[64]Levenberg S, Golub JS, Amit M, et al. Endothelial cells derived from human embryonic stem cells[J]. Proc Natl Acad Sci USA,2002,99:4391—4396
[65]Assa S, Maor G, Amit M et al. Insulin production by human embryonic stem cells [J]. Diabete,2001,50:1691—1697.
[66]Clark AT, Bodnar MS, Fox M et al. Spontaneous differentiation of germ cells from human embryoni stem cells in vitro[J]. Hum Mol Genet,2004,13:723—727.
[67]Xu RH, Chen X, Li DS et al. BMP4 initiates human embryonic stem cell differentiation to trophobla[J]. Nat Biotechnol,2002,20:1261—1264.
[68]Saito D, Strelchenko N, Niemann H. Bovine embryonic stem cell, like cultured over several passages [J]. Roux'S Arch Dev Biol,1992,201(3):134-140.
[69]Sim M, First NL. Production of calves by transfer of nuclei from cultured inner cell mass cells[J]. Proc Natl Acad Sci US A.1994,91(13):6143-6147
[70]White KL, Polejaeva IA, Bunch TD, et al. Effect of non-serum supplemented media of establishment and maintaince of bovine embryonic stem-like cells[J]. Theriogenology,1995,43(1):350
[71]Ito. f Influence of stage and derivation of bovine embryos on formation of embryonic stem(ES)like colonies[J]. Reprod Dev,1996,42(5):126-133.
[72]Cibelli JB, Stice SL, Kane JJ, et al. Production of germline chimeric bovine fetuses from transgenic embryonic stem cells[J]. Theriogenology,1997,47(1):241.
[73]Talbot NC, Powell AM, Rexroad CE. In vitro pluripotency of epiblasts derived from bovine blastocysts[J]. Mol Reprod Dev,1995,42(1):35-52.
[74]Rexroad CE, Powell AM. Culture of blastomeres from in vitro—matured, fertilized, and cultured bovine embryos[J]. Mol Reprod Dev.1997; 48:238-245.
[75]Pampai R, Minami N, Yamada M, et al. Asuitable passaging Technique for the establishment of bovine embryonic stem(ES)like cell lines[J]. Theriogenology,1998, 49(1):241-245.
[76]Mitalipova M, Beyhan I, First W. Pluripotency of bovine embryonic stem cells derived from precompacting embryos[J]. Cloning,2001,3(2):59-67
[77]Saito S, Sawai K, Ugai H, Moriyasu S, et al. Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells[J]. Biochemical and Biophysical Research Communications,2003,309:104-113
[78]Gjorret JO, Maddox—Hyttel P. Attempts towards derivation and establishment of bovine embryonic stem cell·like cultures[J]. Reprod Fertil Dev 2005; 17:113-124.
[79]Maddox—Hyttle P'Alexopoulos NI, Vajta G et al. Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos[J]. Reproduction.2003; 125:607-623.
[80]Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization[J]. J Assist Reprod Gen,1994:11: 515-529.
[81]冯秀亮,高志敏,杨春荣等.由猪囊胚内细胞团分离胚胎干细胞的研究[J].西 北农林科技大学学报(自然科学版),2003,31(3)：5-11.
[82]窦忠英,徐小明,华进联.牛胚胎干细胞建系研究进展及存在问题[J].农业生物技术学报,2003,11(5)：439-443.
[83]Cibelli JB, Stice SL, Golueke PJ, et al. Transgenic bovine chimeric offspring produced from somatic cell·derived stem-like cells[J]. Nat Biotechnol,1998,16: 642-646.
[84]Smith AG. Embryo—derived stem cells:of mice and men[J]. Annu Rev Cell Dev Bi01.2001,17:435-462.
[85]Reubinoff BE, Pera MF,Fong CY et al. Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro[J]. Nat Biotechnal.2000,18(4):399-404.
[86]De Felici M. Scaldaferri ML. Lobascio M, et al. Experimental approaches to the study of primordial germ eell lineage and proliferation[J]. Hum Reprod Update 2004; 10(31:197.206
[87]Mayanagilto K, Takahashi J. Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells[J]. Reproduction 2003:125(4):519-526
[88]冯书堂,董晓,刘立新,等.猪原始生殖嵴细胞(pGCs)建系因素的研究[J].畜牧兽医学报,2004,35(5)：477-480
[89]李松,窦忠英,华进联等.影响牛胚胎干细胞分离克隆因素的研究[J].生物技术通报,2002,3：35-39
[90]Chunhui XLJ, Inokuma M S, Denham J, et al. Feeder-free growth of undifierentiated human embryonic stem cells[J]. Nature 2001; 19(10):971.974
[91]杨奇.牛胚胎干细胞的分离与克隆[D].西北农业大学1999届硕士学位研究生学位论文
[92]Wang L, Duan E, Sung LY,et al. Generation and characte rization of pluripotent stem cells from cloned bovine embryos[J]. Biol Reprod 2005:73(1):149-155
[93]Ledda S, Naitana S, Loi P,et al. Transplantation of primordial germ cells(PGCs)into enucleatcd oocytes in sheep [J]. Theriogenology 1996:45:288
[94]Campbell KH。McW hir J. Ritchie W A, et al. Sheep cloned by nuclear transfer from a cultured cell line[J]. Nature 1996; 380(6569):64-66
[95]徐小明,华进联,冯秀亮,等.牛原始生殖细胞的分离与培养[J].西北农林科技大学学报(自然科学版),2004,32(1)：77-80
[96]葛秀国,徐小明,华进联,等牛胚胎生殖细胞的分离与培养[J].畜牧兽医学报,2005,36(12)：275-280
[2]Thomson J A, Itskovitz-Eldor J, Shapiro S S, et al. Embryonic stem cell lines derived from human blastocysts [J].Science,1998,282(5391):1145-147.
[3]Xu C, Inokuma MS, Denham J. Feeder-free growth of undifferentiated human embryonic stem cells [J].Nature,2001,19(10):971-974.
[4]Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos [J]. Nature,1981,292 (5819):154-156.
[5]李松,窦忠英,苗增民,等.牛ES细胞分离克隆及定向分化研究[J].医学研究杂志,2007,36(2)：79-80.
[6]李碧春,陈国宏,吴信生,等.牛体外受精胚胎衍生干细胞能力影响因素研究[J].畜牧兽医学报,2006,37(9)：928-932.
[7]Rexroad CE, Powell AM. Culture of blastomeres from in vitro—matured, fertilized, and cultured bovine embryos [J]. Mol Reprod Dev.1997,48:238-245.
[8]Pampai R, Minami N, Yamada M, et al. Asuitable passaging Technique for the establishment of bovine embryonic stem(ES)like cell lines[J]. Theriogenology,1998, 49(1):241-245.
[9]Talbot NC, Powell AM, Rexroad CE. In vitro pluripotency of epiblasts derived from bovine blastocysts[J]. Mol Reprod Dev,1995,42(1):35-52.
[10]Mitalipova M, Beyhan I, First W. Pluripotency of bovine embryonic stem cells derived from precompacting embryos[J]. Cloning,2001,3(2):59-67
[11]Liang P., Pardee A.B.,. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992,257:967-971.
[12]Pasentsis K,Falara V,Pateraki I,et al.Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display[J].J Exp Bot,2007,58(8):2203-16.
[13]Ikematsu K,Tsuda R,Nakasono I.Gene response of mouse skin to pressure injury in the neck region[J].Leg Med(Tokyo),2006,8(2):128-31.
[14]Pathak RU,Kanungo MS.Subtractive differential display:a modified differential display technique for isolating differentially expressed genes[J].Mol Biol Rep,2007,34(l):41-6.
[15]Zhang J,Turley RB,Stewart JM.Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display [J].Plant Cell Rep,2008,27(3):553-61.
[1]Evans MJ. Kaufman MH. Estabfishment in culture of pluripotential cells from mouse embryos[J]. Nature 1981:292(9):154-156
[2]李碧春,陈国宏.牛体外受精胚胎衍生干细胞能力影响因素的研究[D].扬州大学硕士研究生毕业论文,2005
[3]Cherny RA. Stokes TM, Merei J. et al. Strategies for the isolation and characterization of bovine embryonic stem cells[J]. Reprod Fert Dev 1994:6(5): 569-575
[4]Stice SL, Strelchenko NS, Keefer CL, et al. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer[J]. Biol Reprod 1996; 54(1):100-110
[5]Cibelli JB. Stice SL, Kane JJ. et al. Production of germline Chimeric bovine fetues from transgenic embryonic stem cells[J]. Theriogenology 1997:47(7):241
[6]Parnpai R. Minami N, Yamada M, et al. Asuitable passaging Technique for the establishment of bovine embryonic stem(ES) like cell lines[J]. Theriogenology 1998; 49(1):241-245
[7]Saito D, Strelchenko N, Niemann H. Bovine embryonic stem cell-like cultured over several passages[J]. Roux' S Arch Dev Biol,1992,201(3):134-140.
[8]Sim M, First NL. Production of calves by transfer of nuclei from cultured inner cell mass cells[J]. Proc Natl Acad Sci U S A.1994,91(13):6143—614
[9]White KL, Polejaeva IA, Bunch TD, et al. Effect of non-serum supplemented media of establishment and maintaince of bovine embryonic stem-like cells[J]. Theriogenology,1995,43(1):350
[10]Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells [J]. Dev Biol,1987,121(1):1-9.
[11]Li Wang, Enkui Duan, Li-ying Sung, et al. Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos [J]. Biology of reproduction, 2005,73(1):149-55.
[12]Seizo Hamano, Yoshie Watanabe, Sadahiro Azuma, et al. Establishment of embryonic stem (ES) cell-like cell lines derived from bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro [J]. Journal of reproduction and development,1998,44(3):297-303.
[13]M. Munoz, A. Rodriguez, C. De Frutos, et al. Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines [J]. Theriogenology,2008, 69(9):1159-1164.
[14]Strojek RM, Reed MA, Hoover J L, et al. A method for cultivation morphological undifferentiated embryonic stem cells from porcine blastocysts [J]. Theriogenology,1990,33:901-903.
[15]Vitezslav Bryja, Sonia Bonilla, Ernest Arenas. Derivation of mouse embryonic stem cells [J]. Nature Publishing Group,2006, 1(4):2082-2087.
[16]Marsha Roach, Li Wang, Xiangzhong Yang, et al. Bovine embryonic stem cells[J]. Methods in enzymology,2006,418:21-37.
[17]杨美玲.转过表达和低表达胰岛素样生长因子I基因牛成纤维细胞系的制备[D].内蒙古大学.2009.
[18]Schieke AS, Kind AJ, Ritchie WA. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts[J].Science,1997,278: 2130-2133.
[19]Baquisi A, Behboodi E, Melican DT. Production of goats by somatic cell nuclear transfer [J]. Nat Biotechnol,1999,17:456-461.
[20]Cibelli JB, Stice SL, Golueke PJ. Cloned transgenic calves produced from nonquiescent fetal fibroblasts[J].Science,1998,280:1256-1258.
[21]Marsha Roach, Li Wang, Xiangzhong Yang, et al. Bovine embryonic stem cells[J]. Methods in enzymology,2006,418:21-37.
[22]薛庆善.体外培养的原理与技术[M].北京,科学出版社.2001,958-961.
[23]屠迪,崔燕,余四九.牦牛胎儿成纤维细胞的分离培养[J].中国兽医科学.2006,36(07)：573-577.
[24]Masssgul.Stimulalion by insulin-like growth factor is required for cellular transformation by type transforming growth factor [J].Bio.chem.1985,260: 4551-4554.
[25]Nlchols.J, Edward P,et al. Establishment of germ-line-competent embryonic stem(ES)cells Using differentiation inhibiting activity [J]. Development Biology,1990,110:1341-1348.
[26]Smith Aostin, Lerlifer Nichols, et al. Differentiation inhibiting Activity (DLA/LIF) and mouse Development[J]. Development Biology,1992,151:339-351.
[27]张青丽,段因奎,曾国庆.白血病抑制因子及其在胚胎发育和胚泡着床中的生理功能[J].生理科学进展,1997,28(3)：240-242.
[28]Aitstin G. Smith. Diffentiation inhibiting activity (DLA/LIF) and mouse development[J].Dev.Biol.1992,151:339-351.
[29]Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells[J].Proteomics,2002,2(9):1187-1203.
[30]Strojek RM, Reed MA, Hoover JL, et al. A method for cultivating morphologically undifferentiated embryonic stem cells from porine blastocysts[J]. Theriogennology,1990,33(4):901-910.
[31]Rexroad CE Jr, Powell AM.. Culture of blastomeres from in vitro-matured, fertilized, and cultured bovine embryos[J]. Mol Reprod Dev1997,48:238-245.
[32]Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium.[J].Exp Cell Res,1999,247(1):241-248.
[33]Thomson J A, Kaalishman J, Golos T G, et al. Isolation of a primate embryonic stem cell line. [J].Proc Natl Acad Sci USA.1995,92(17):7844-7848.
[34]Li M, Ma W, Hou Y, et al. Improved isolation and culture of embryonic stem cells from Chinese miniature pig[J].Reprod Dev 2004.50:237-244.
[35]安立龙,杨奇,窦忠英等.某些因素对牛和小鼠类胚胎干细胞分离与培养的影响[J].中国兽医学报,2002,22(2)：187-190.
[36]Li M, Li YH, Hou Y, et al. Isolation and culture of pluripotent cells from in vitro produced porcine embryos [J].Cambridge University Press,2004,12(1):43-48.
[37]Thomson J A, Itskovitz-Eldor J, Shapiro S S, et al. Embryonic stem cell lines derived from human blastocysts [J].Science,1998,282(5391):1145-147.
[38]Munoz M, Diez C, Caamano JN, Jouneau A, Hue I, Gomez E. Embryonic stem cells in cattle[J]. Reprod Domest Anim 2008.4:32-37.
[39]Li Wang, Enkui Duan, Li-ying Sung, et al. Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos[J]. BIOLOGY OF REPRODUCTIO 2005:73,149-155.
[40]Robert ES, Morayma R, Lisa K, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells[J]. J Clin Invest, 2002,109:1291-1302.
[41]Talbot N C, Powell A M, Rexroad C E, In vitro pluripotency of epiblasts derived from bovine blastocysts [J] Mol Reprod Dev,1995,42:35-521.
[42]Cao S, Wang F, Chen Z, et al. Isolation and Culture of Primary Bovine Embryonic Stem Cell Colonies by a Novel Method[J]. JExp Zool A Ecol Genet Physiol 2009.311:368-76.
[43]Gj(?)rret JO, Maddox-Hyttel P. Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures[J]. Reprod Fertil Dev 2005.17:113-124.
[44]赵晶,董雅娟,柏学进等.饲养层细胞的接种密度对分离培养胚胎干细胞的影响[J].中国兽医学报,2009,29(4)：494-501.
[1]Turnpenny L, Spalluto CM, Perrett RM. Evaluating human embryonic germ cells: concord and conflict as pluripotent Stem cells [J].Stem Cells,2006,24:212-220.
[2]裴雪涛主编.干细胞生物学[M].北京：科学出版社,2003.7.
[3]Li Wang, Enkui Duan, Li-ying Sung, et al. Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos [J]. BIOLOGY OF REPRODUCTION,2005,73:149-155.
[4]Evans MJ, Kaufman MH. Establishment in culture of pluripotential cell from mouse embryos [J]. Nature,1981,293:154-156.
[5]Thomson JA, Itskovitz EJ, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts [J].Science,1998,282:1145-1147.
[6]Cao S, Wang F, Chen Z, et al. Isolation and Culture of Primary Bovine Embryonic Stem Cell Colonies by a Novel Method[J]. JExp Zool A Ecol Genet Physiol 2009.311:368-76.
[7]Gj(?)rret JO, Maddox-Hyttel P. Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures[J]. Reprod Fertil Dev 2005.17:113-124.
[8]Munoz M, Diez C, Caamano JN, et al. Embryonic stem cells in cattle[J]. Reprod Domest Anim 2008.4:32-37.
[9]Roach M,Wang L,Yang X,et al. Bovine embryonic stem cells[J]. Methods enzymol 2006.418:21-37.
[10]Saito S, Liu B, Yokoyama K. Animal embryonic stem (ES) cells:self-renewal, pluripotency, transgenesis and nuclear transfer[J]. Hum Cell 2004.17:107-115
[11]Sim M, First NL. Production of calves by transfer of nuclei from cultured inner cell mass cells[J]. Proc Natl Acad Sci USA 1994.91:6143-6614.
[12]Liang P., Pardee A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992,257:967-971.
[13]Ikematsu K,Tsuda R,Nakasono I.Gene response of mouse skin to pressure injury in the neck region[J].Leg Med(Tokyo),2006,8(2):128-31.
[14]Pathak RU,Kanungo MS.Subtractive differential display:a modified differential display technique for isolating differentially expressed genes[J].Mol Biol Rep,2007,34(1):41-6.
[15]Zhang J,Turley RB,Stewart JM.Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display [J].Plant Cell Rep,2008,27(3):553-61.
[16]Pasentsis K,Falara V,Pateraki I,et al.Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display[J].J Exp Bot,2007,58(8):2203-16.
[17]王梁燕,洪奇华,张耀洲.实时定量PCR技术及其应用[J].细胞生物学杂志,2004,26(1)：62-67.
[18]尹学钧DD-PCR技术及其在遗传毒理学的应用前景[J].国外医学：卫生学分册,1999,26(3)：156-160.
[19]Wool I.G., Extraribosomal functions of ribosomal proteins[J].Trends. Biochem. Sci.,1996,21:164-165.
[20]Walleczek J, Redl B, Stoffler-Meilicke Mat et al. G. Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques[J]. J. Biol. Chem.1989,264:4231-4237.
[21]Brimacombe R, Gornicki P, Greuer B, et al. The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques [J]. Biochim. Biophys. Acta 1990,1050:8-13.
[22]Nag B, Akella SS, Cann PA, et al. Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50S ribosomal subunit[J]. J. Biol. Chem.1991,266: 22129-22135.
[23]Agafonov DE, Kolb VA and Spirin AS. Proteins on ribosome surface: measurements of protein exposure by hot tritium bombardment technique [J]. Proc. Natl. Acad. Sci. U. S. A.1997,94:12892-12897.
[24]Hoffman DW, Davies C, Gerchman SE, et al. Crystal structure of prokaryotic ribosomal protein L9:a bi-lobed RNA-binding protein[J]. EMBO J.1994,13: 205-212.
[25]Lillemoen J, Cameron CS, Hoffman DW. The stability and dynamics of ribosomal protein L9:investigations of a molecular strut by amide proton exchange and circular dichroism[J]. J. Mol. Biol.1997,268:482-493.
[26]Fabijanski S, Pellegrini M. Identification of proteins at the peptidyl-tRNA binding site of rat liver ribosomes[J]. Mol. Gen. Genet.,1981,184:5551-5556.
[27]Durand S, Abadie P, Angeletti S, et.al. Identification of multiple differentially expressed messenger RNA in normal and pathological trophoblast [J]. Placenta,2003, 24:209-218.
[28]Wool I G, Chan Y L, Gluck A. Structure and evolution of mammalian ribosomal proteins[J]. Biochem Cell Biol.,1995,73:933-947.
[29]Hardie DG. AMP-activated/SNF1 protein kinases:conserved guardians of cellular energy[J]. Nat Rev Mol Cell Biol 2007.8(10):774-785.
[30]Fulco M, Cen Y, Zhao P, et.al. Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt[J]. Dev Cell 2008.14(5):661-673.
[31]Habinowski SA, Witters LA. The effects of AICAR on adipocyte differentiation of 3T3-L1 cells[J].Biochem Biophys Res Commun 2001.286(5):852-856.
[32]Kanazawa I, Yamaguchi T, Yano S, et.al. Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression[J]. Biochem Biophys Res Commun 2008,375(3):414-419.
[33]Lohmann J, Schickle H, Bosch T C. REN display, a rapid and efficient method for nonradioactive differential display and mRNA isolation [J]. Biotechniques, 1995,18(2):200-202.
[34]Allan G J, Flint D J, Patel K. Insulin like growth factor axis during embryonic development[J]. Reproduction,2001,122:31—39.
[35]李希水牛卵母细胞体外成熟前后基因差异表达的研究[D].南宁：广西大学,硕士学位论文.2008.
[36]Borg J. P., Marchetto S., Le Bivic A., et.al. ERBIN: A basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor [J]. Nature Cell Biology, 2000,2:407-414.
[37]Li D., Guo N. Research advances in mechenism of CIITA regulating MHC-II express. Chinese Journal of Cell and Molecular Immunology [J].2008,24 (1):97-99.
[38]Willers J., Haffner A., Zepter K. et.al. The interferon inhibiting cytokine IK is overexpressed in cutaneous T cell lymphoma derived tumor cells that fail to upregulate major histocompatibility complex class II upon interferon-gamma stimulation [J]. Journal of Investigative Dermatology,2001,116(6):874-9.
[39]van Wijk I., Griffioen S., Tjoa M. L. et.al. HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy[J]. American Journal of Obstetrics and Gynecology,2001,184(5):991-7.
[40]Mitra P, Vaughan PS, Stein JL, et.al.Purification and functional analysis of a novel leucine-zipper/nucleotide-fold protein, BZAP45, stimulating cell cycle regulated histone H4 gene transcription[J]. Biochemistry 2001, (40) 10693-10699.
[41]Li S, Chai Z, Li Y, et.al. BZW1, a novel proliferation regulator that promotes growth of salivary muocepodermoid carcinoma[J].Cancer Lett.2009 Oct 18;284(1):86-94.
[42]Onda M, Inui S, Maeda K, et.al.Expression and Chromosomal Localization of the Human a4/IGBP1 Gene, the Structure of Which Is Closely Related to the Yeast TAP42 Protein of the Rapamycin-Sensitive Signal Transduction Pathway[J].GENOMICS 199746,373-378
[43]Godfrey Grech, Montserrat Blazquez-Domingo, Andrea Kolbus, et.al. Hartmut Beug and Marieke von Lindern.Igbpl is part of a positive feedback loop in stem cell factor-dependent, selective mRNAtranslation initiation inhibiting erythroid differentiation[J]. blood 2008 112:2750-2760.
[44]Minami N, Tsukamoto S. Role of oocyte-specific genes in the development of mammalian embryos [J]. Reprod. Med. Biol.2006,5(3):175—182.
[1]Evans M. J, Kaufman M. H. Establishment in culture of pluripotential cells from mouse embryos[J]. Nature,1981,292(9):154-156
[2]Helena R, Axelord. Embryonic stem cell lins derived from blastocysts by a simplified technique[J].Development biolingy,1989,101:225-228
[3]Iannaccone PM, Taborn GU, Garton RL, et al. Pluripotent embvryonic stem cells from the rat are capable of producing chimeras [J]. Devel Biol,1994,288-292
[4]McDonald JW, Becker D, Holekamp TF, et al.Repair of the injured spinal cord and the potential of embryonic stem cell transplantation [J]. J Neurotrauma,2004, 21:383-393.
[5]Thomson JA, Kalishman J, Golos TC et al. Isolation of a primate embryonic stem cell line [J]. Proc Natl Acad Sci USA,1995,92:7844-7848.
[6]Doetschman T, Williams P, Maeda N.Establishment of hamster blastocyst-derived embryonic stem (ES) cells[J]. Devel Biol,1988,127:224-227.
[7]Piedrahita J A, Anderson G B, BonDurant R H. Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines[J]. Therogenology, 1990,34:865-877.
[8]Saito S, Strelchenko N, Niemann H. Bovine embryonic stem cell-like lines culture over several passages [J]. Rouxs Archives of Developmental Biology,1992, 201:134-141.
[9]Piedrahita JA., Anderson GB., BonDurant RH. On the isolation of embryonic stem cells:Comparative behavior of marine, porcine and ovine embryos [J]. Theriogenology,1990,34(5):879-901.
[10]Graves KH, Randall W. Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos [J]. Mol Reprod Dev,1993, 36:424-433.
[11]赖良学,郑瑞珍,孙方臻等.胚胎于细胞分离培养的影响因素[J].解剖科学进展,1995,1(3)：203-208.
[12]Sukoyan MA, Golubitsa AN, Zhelezova AI, et al. Isolation and cultivation of blastocyst-derived stem cell lines from American Mind (Mustela vison) [J]. Mol Reprod Dev,1992,33:418-431.
[13]Robertson E J, Evans M J, Kaufman M H. X-chromosome instability in pluripotential stem cell lines derived from parthenogenetic embryos [J]. Journal of Embryology and Experimental Morphology,1982,74:297-309.
[14]Harald R, Eistetter. Pluripotent embryonal stem cell lines can be established from disaggregated mouse morulae Develop ment[J]. Growth and Differ,1981,31(3): 275-282
[15]Wood SA, Pascoe WS, Schmidt C, et al. Simple and Efficient Production of Embryonic Stem Cell-Embryo Chimeras by Coculture[J]. PNAS,1993,90:4582.
[16]Peli J, Schmoll F, Laurincili J, et al. Comparison of aggregation and injection techniques in producing chimeras with ES cells in mice [J]. Theriogenology,1996, 45(4):833-842.
[17]Robertson E, Bradley A, Kuehn M et al. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector [J]. Nature,1986,323: 445-448.
[18]Chung Y, Klimanskaya I and Bocker S. Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres [J]. Nature,2006,439(10):1038-1042.
[19]Babinet C, Cohen-Tannoudji M. Genome engineering via homologous recombination in mouse embryonic stem (ES) cells:an amazingly versatile tool for the study of mammalian biology [J].AnAcad Bras Cienc,2001,73:365—383.
[20]Tomes M. The use of embryonic stem cells for the genetic manipulation of the mouse[J]. Curr Top Dev Biol,1998,36:99—114.
[21]Gossler A, Doetschman T, Korn R,et al. Transgenesis by means of blastocyst-derived embryonic stem cell lines [J]. Proc Natl Acad Sci USA,1986,83: 9065-9069.
[22]Thompson S, ClarkeAR, PowAM et al. Germ line transmis-sion and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells [J]. Cell,1989,56:313-321.
[23]Zambrowicz BP, Friedrich GA, Buxton EC, et al. Disruption and sequence identification of 2000 genes in mouse embryonic stem cells [J]. Nature 1998,392: 608-611.
[24]Bettiol E, Sartiani L, Chicha L, et al. Fetal bovine serum is essential for cardiac differentiation of human embryonic stem cell [J]. J Mol Cell Cardiol.,2007,42: s88-s101 (Abstract).
[25]Okumura AJ, Peterson LF, Lo MC, et al. Expression of AML/Runx and ETO/MTG family members during hematopoietic differentiation of embryonic stem cells [J]. Exp. Hematol.,2007,35:978-988.
[26]Lee SH, Lumelsky N, Studer L et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells [J]. Nat Biotechnol,2000,18: 675-679.
[27]Smith, A.G., Heath, J.K., Donaldson, D.D., et al. Inhibition of pluripotential embryonic stem cell differentiation by puried polypeptides [J]. Nature,1988,336: 688-690.
[28]Niwa H, Burdon T, Chambers I, Smith AG. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3 [J]. Genes Dev.,1998,12:2048-2060.
[29]Matsuda, T., Nakamura, T., Nakao, K., et al. STAT3 acti-vation is suf?cient to maintain an undifferentiated state of mouse embryonic stem cells [J].EMBO J.,1999, 18:4261-4269.
[30]Ogawa K, Matsui H, Ohtsuka S. et al. A novel mechanism for regulating clonal propagation of mouse ES cells [J]. Genes to Cells,2004,9:471-477.
[31]Brook F. A., Gardner R. L. The origin and efficient derivation of ESC in the mouse [J]. Proc Natl Acad Sci USA,1997,94:5709-5712.
[32]Zwaka T. P., Thomson J. A. A germ cell origin of embryonic stem cells [J]. Development,2005,132:227-233.
[33]Hirofumi Suemori, Horio Nakatsuji. Establishment of embryos derived stem (ES) cell lines from mouse blastocyst:effect of the feeder cell layer[J]. Development growth and diff.29 (2):133-139
[34]Piedrahita J. A, Anderson G. B, Bondurant R. H. on the isolation of embryonic stem cell:Camparative Behavior of murine, porcine, ovine embryos[J]. Thereiogenology.1990,39(5):879-901
[35]Labos PA, Barlow DP, Hogan BL. Embryonic germ cell lines and their derivation from mouse primordial germ cells [J]. CIBA Foundation symposium,1994,182: 157-178.
[36]Niemann H, Strelchenko N. Isolation and maintenance of rabbit embryonic stem cell lines [J].Theriogenllogy,1994,41:265.
[37]Du F, Giles J R, Footerh, et al. Nuclear transfer of putative rabbit embryonic stem cell leads to normal blastocyst development [J]. Reproduction and Fertilization,1995, 104:219-223.
[38]Moens A, Chastant S, Chesne P. Differential reprogramming capabilities,of male and female rabbit fetal gonadal cells [J]. Proc Cong Eur Biol Organ,1995,6:98.
[39]Schoonuans L, Albright GM, Li J L, et al. Pluripotent rabbit embryonic stem cells are capable of forming overt coat color chimeras following injection into blasto-cysts[J]. Mol Reprod Dev,1996,45:439-443.
[40]Nakayama N, Fang I, Elliott G. Natural killer and B-Lymphoid potential in CD34 cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor[J].Blood,1998,91(7):2283-295.
[41]Lee C.K., Weaks R. L., Johnson GA. Effects of Protease Inhibitors and Antioxidants on In Vitro Survival of Porcine Primordial Germ Cells [J]. Biol Reprod, 2000,63:887
[42]Shi-ping Luh, Chi-huei Chiang.Acute lung injury/acute respiratory distress syndrome (ALI/ARDS):the mechanism, present strategies and future perspectives of therapies[J]. JOURNAL OF ZHEJIANG UNIVERSITY.2007,8(1):60-69
[43]Piedrahita J A, Anderson G B, Martin G R, et al. Isolation of embryonic stem cell-lide colonies from porcine embryos[J]. Theriogenology,1988,29:286.
[44]Strojek RM, Reed M A, Hoover J L, et al. A method for cultivating morphologically undifferentiated embryonic stem cells from procine blastocysts[J]. Theriogenology,1990,33:901-913.
[45]Piedrahita JA, Anderson GB, Bondurant RH.On the isolation of embryonic stem cells:Comparative behavior of murine, porcine and ovine embryos[J].Theriogenology. 1990;34(5):879-901.
[46]Wheeler MB. Development and validation of swine embryonic stem cell:a review Reprod Fertil[J]. Dev,1994,6:563-568.
[47]Evans MJ, Notarianni E, Lauries, et al. Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts[J]. Theriogenology,1990,33:125-128.
[48]Cibelli JB, Stice SL, Golueke PJ, et al. Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells [J]. Nature Biotech,1998,16: 642-646
[49]Booth PJ, Perreau C, Hochereau-de Reviers. TEC-1 characterisation of porcine embryonic cells from day 11 embryonic discs cultured in serum-free medium [J]. Theriogenology,1997,47(1):167.
[50]Shim H, Gutierrez-Adan A, Chen LR, et al. Isolation of pluripotent stem cell from cultured porcine primordial germ cells [J]. Biol Reprod,1997,57:1089-1095.
[51]Shim H and Anderson GB. In vitro survival and proliferation of porcine embryonic germ cells [J].Theriogenology,1998,49:521—528.
[52]Piedrahita J, Moore K, Oetama B, et al. Generation of transgenic porcine chimeras using primordial germ cell-derived colonies [J]. Biol Reprod,1998,58: 1321—1329.
[53]Bongso A, Fong CY, Ng SC et al. The growth of inner cell mass cells from human blastocysts (Abstract) [J]. Theriogenology,1993,41:161.
[54]Bongso A, Fong CY, Ng SC et al. Isolation and culture of inner cell mass cells from human blastocysts [J]. Hum Reprod,1994,9:2110-2117.
[55]Thomson JA, Marshall VS. Primate embryonic stem cells[J]. Curr Top Dev Biol 1998,38:133-165.
[56]Thomson JA, Kalishman J, Golos TQ et al. Isolation of a primate embryonic stem cell line[J]. Proc Natl Acad Sci U S A,1995,55:254-259.
[57]Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts:somati differentiation in vitro [J]. Nat Biotechnol,2000,18:399-404.
[58]Amit M&Itskovitz-Eldor J. Derivation and spontaneous differentiation of human embryonic stet cells [J]. J Anat,2002,200(3):225-232.
[59]何志旭,黄绍良,李予等.人胚胎干细胞系的初步建立[J].中华医学杂志,2002,82(19)：1314-1318
[60]Kehat I, Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocyteswith structural and functional properties of cardiomyocytes [J].J Clip Invest,2001,108,407-414.
[61]VogelG. Developmental biology:The hottest stem cells are also the toughest[J]. Science,2001,292(5516):429
[62]Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursor from human embryonic stem cells [J]. Nat Biotechnol,2001,19: 1129-1133.
[63]Mummery C, Ward-van Oostvaard D, Doevendans P et al. Differentiation of human embryonicstet cells to cardiomyocytes:role of coculture with visceral endoderm-like cells[J]. Circulation,2003, (10),2733-2734.
[64]Levenberg S, Golub JS, Amit M, et al. Endothelial cells derived from human embryonic stem cells[J]. Proc Natl Acad Sci USA,2002,99:4391—4396
[65]Assa S, Maor G, Amit M et al. Insulin production by human embryonic stem cells [J]. Diabete,2001,50:1691—1697.
[66]Clark AT, Bodnar MS, Fox M et al. Spontaneous differentiation of germ cells from human embryoni stem cells in vitro[J]. Hum Mol Genet,2004,13:723—727.
[67]Xu RH, Chen X, Li DS et al. BMP4 initiates human embryonic stem cell differentiation to trophobla[J]. Nat Biotechnol,2002,20:1261—1264.
[68]Saito D, Strelchenko N, Niemann H. Bovine embryonic stem cell, like cultured over several passages [J]. Roux'S Arch Dev Biol,1992,201(3):134-140.
[69]Sim M, First NL. Production of calves by transfer of nuclei from cultured inner cell mass cells[J]. Proc Natl Acad Sci US A.1994,91(13):6143-6147
[70]White KL, Polejaeva IA, Bunch TD, et al. Effect of non-serum supplemented media of establishment and maintaince of bovine embryonic stem-like cells[J]. Theriogenology,1995,43(1):350
[71]Ito. f Influence of stage and derivation of bovine embryos on formation of embryonic stem(ES)like colonies[J]. Reprod Dev,1996,42(5):126-133.
[72]Cibelli JB, Stice SL, Kane JJ, et al. Production of germline chimeric bovine fetuses from transgenic embryonic stem cells[J]. Theriogenology,1997,47(1):241.
[73]Talbot NC, Powell AM, Rexroad CE. In vitro pluripotency of epiblasts derived from bovine blastocysts[J]. Mol Reprod Dev,1995,42(1):35-52.
[74]Rexroad CE, Powell AM. Culture of blastomeres from in vitro—matured, fertilized, and cultured bovine embryos[J]. Mol Reprod Dev.1997; 48:238-245.
[75]Pampai R, Minami N, Yamada M, et al. Asuitable passaging Technique for the establishment of bovine embryonic stem(ES)like cell lines[J]. Theriogenology,1998, 49(1):241-245.
[76]Mitalipova M, Beyhan I, First W. Pluripotency of bovine embryonic stem cells derived from precompacting embryos[J]. Cloning,2001,3(2):59-67
[77]Saito S, Sawai K, Ugai H, Moriyasu S, et al. Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells[J]. Biochemical and Biophysical Research Communications,2003,309:104-113
[78]Gjorret JO, Maddox—Hyttel P. Attempts towards derivation and establishment of bovine embryonic stem cell·like cultures[J]. Reprod Fertil Dev 2005; 17:113-124.
[79]Maddox—Hyttle P'Alexopoulos NI, Vajta G et al. Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos[J]. Reproduction.2003; 125:607-623.
[80]Plante L, King WA. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization[J]. J Assist Reprod Gen,1994:11: 515-529.
[81]冯秀亮,高志敏,杨春荣等.由猪囊胚内细胞团分离胚胎干细胞的研究[J].西 北农林科技大学学报(自然科学版),2003,31(3)：5-11.
[82]窦忠英,徐小明,华进联.牛胚胎干细胞建系研究进展及存在问题[J].农业生物技术学报,2003,11(5)：439-443.
[83]Cibelli JB, Stice SL, Golueke PJ, et al. Transgenic bovine chimeric offspring produced from somatic cell·derived stem-like cells[J]. Nat Biotechnol,1998,16: 642-646.
[84]Smith AG. Embryo—derived stem cells:of mice and men[J]. Annu Rev Cell Dev Bi01.2001,17:435-462.
[85]Reubinoff BE, Pera MF,Fong CY et al. Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro[J]. Nat Biotechnal.2000,18(4):399-404.
[86]De Felici M. Scaldaferri ML. Lobascio M, et al. Experimental approaches to the study of primordial germ eell lineage and proliferation[J]. Hum Reprod Update 2004; 10(31:197.206
[87]Mayanagilto K, Takahashi J. Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells[J]. Reproduction 2003:125(4):519-526
[88]冯书堂,董晓,刘立新,等.猪原始生殖嵴细胞(pGCs)建系因素的研究[J].畜牧兽医学报,2004,35(5)：477-480
[89]李松,窦忠英,华进联等.影响牛胚胎干细胞分离克隆因素的研究[J].生物技术通报,2002,3：35-39
[90]Chunhui XLJ, Inokuma M S, Denham J, et al. Feeder-free growth of undifierentiated human embryonic stem cells[J]. Nature 2001; 19(10):971.974
[91]杨奇.牛胚胎干细胞的分离与克隆[D].西北农业大学1999届硕士学位研究生学位论文
[92]Wang L, Duan E, Sung LY,et al. Generation and characte rization of pluripotent stem cells from cloned bovine embryos[J]. Biol Reprod 2005:73(1):149-155
[93]Ledda S, Naitana S, Loi P,et al. Transplantation of primordial germ cells(PGCs)into enucleatcd oocytes in sheep [J]. Theriogenology 1996:45:288
[94]Campbell KH。McW hir J. Ritchie W A, et al. Sheep cloned by nuclear transfer from a cultured cell line[J]. Nature 1996; 380(6569):64-66
[95]徐小明,华进联,冯秀亮,等.牛原始生殖细胞的分离与培养[J].西北农林科技大学学报(自然科学版),2004,32(1)：77-80
[96]葛秀国,徐小明,华进联,等牛胚胎生殖细胞的分离与培养[J].畜牧兽医学报,2005,36(12)：275-280
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