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1.
小鼠囊胚的不同遗传背景对形成ES细胞集落的影响   总被引:8,自引:1,他引:8  
使用正常囊胚经过内细胞团增殖后的离散程序,比较了小鼠C57BL/6品系、129品系和C57BL/6与129杂交的囊胚在形成胚胎干细胞(ES细胞)集落上的差异。C57BL/6品系的正常囊胚经培养后只有17.4%的胚胎出现ES集落,细胞生长迅速但极易分化。129品系为41.0%,细胞生长比较缓慢。而杂交鼠胚易于出现ES细胞集落,高达75.0%,有利于ES细胞系的建立。文中讨论了在嵌合体工作中使用这种杂交鼠胚ES细胞的可能性。  相似文献   

2.
以昆明白小鼠成纤维细胞和胚胎干(ES)细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎.在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率(24.4%相对于56.9%,P〈0.05),1.8%的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率(89.6%)和囊胚发育率(18.8%)明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率(54.2%)和囊胚发育率(4.2%).  相似文献   

3.
小鼠体细胞核移植及ES细胞样集落分离   总被引:4,自引:0,他引:4  
利用小鼠皮肤成纤维细胞为核供体进行体细胞核移植并从重构胚中分离胚胎干细胞(ES)样集落,以便对体细胞核移植重构胚来源的ES细胞样集落进行研究.结果显示,小鼠皮肤成纤维细胞作为核供体,核移植重构胚激活率为60.48%(254/420),囊胚发育率为6.90%(29/420),6个囊胚中分离出ES细胞样集落,分离率为1.43%(6/420),3个ES细胞样集落能够稳定传代,至第5代时核型正常率分别为77.84%,75.18%,77.20%.分离出的ES细胞样集落具有岛屿状团状隆起结构,碱性磷酸酶染色呈阳性,体外可自发分化成上皮样或梭形细胞.实验证实小鼠唇部皮肤成纤维细胞能够支持体细胞核移植重构胚发育至囊胚,并能分离出可以稳定传代的ES细胞样集落.  相似文献   

4.
研究了昆明小鼠胚胎成纤维细胞的分离、培养和生长特征,建立了快速、稳定的优质饲养层细胞培养体系.从不同日龄的胎鼠均分离到胚胎成纤维细胞,但最佳分离时间为13.5~14.5d;三种分离原代胚胎成纤维细胞的方法中胰酶消化法效果最好,能在较短的周期内获得大量原代及传代细胞;MEF细胞形态以小梭形为主,呈漩涡状、火焰状生长;增殖速度较快,每1~2d可传一代,按1∶3比例常规传代;5代以内适宜制作饲养层用,5代以后细胞开始变形呈现典型的衰老特征.  相似文献   

5.
大鼠心肌条件培养基对形成小鼠ES细胞集落的影响   总被引:11,自引:0,他引:11  
报道一种新的从小鼠囊胚中培养和分离出ES细胞集落的方法。取出生2-3周龄大鼠的心肌组织制备单层培养物,收集6代以内的条件培养基。以RHCM培养C57BL/6J小鼠的囊胚,对照组以小鼠胚胎原代成纤维细胞为饲养层,并补加LIF因子。  相似文献   

6.
为了检验胚胎干的全能性,通过用1日龄卵巢移植后,经体外受精得到的囊胚的饲养建立了小鼠的ES系,并在较高的代数下(第41代)制出了5只毛色嵌合体小鼠,但是没有得到性腺嵌合的小鼠。研究结果证实,高代数的ES细胞仍然具有构建嵌合体的能力。  相似文献   

7.
小鼠胚胎成纤维细胞的分离培养   总被引:1,自引:0,他引:1  
目的探讨小鼠胚胎成纤维细胞(MEF)的分离与培养。方法取BALB/c小鼠胚胎分离成纤维细胞,利用体外培养体系,对MEF的生长形态进行观察,并对传代细胞培养液、胚胎胎龄、胰蛋白酶浓度进行筛选。结果 MEF在体外为贴壁生长型细胞,第三、四、五代细胞纯度较高且增殖最为旺盛;添加血清的M199和DMEM均能较好的满足原代细胞的生长,两种培养液中细胞增殖的速度无明显差异;11.5~16.5d胎龄的小鼠胎儿MEF分离效果最好;0.1%胰蛋白酶消化MEF时间以8~11min为宜。结论通过对MEF分离培养影响因素的筛选,为MEF的进一步研究奠定了基础。  相似文献   

8.
兔ES样细胞系的建立及其特性分析   总被引:5,自引:0,他引:5  
报道从237枚家兔胚胎中建成7个可连续传代的ES样细胞系。建系条件为,使用小鼠原始胚胎成纤维细胞(ME)作饲养层,以含10%胎年血清和10%兔血清的DMEM/F12为培养基,添加白血病抑制因子(LIF)或上皮生长因子(EGF),胚龄为90,96h。该细胞系的细胞。在许多方面类似于小鼠ES细胞,具干细胞的形态特征,呈集落型生长,可连续传代并保持其形态特征,具有一定的自发分化和诱导分化的能力,悬浮培养  相似文献   

9.
胚胎干细胞的体外扩增   总被引:2,自引:0,他引:2  
探讨了胚胎干细胞在体外大量扩增的方法。将胚胎干细胞复苏后,选择合适的培养基(含高糖和谷氨酰胺的DMEM,加入ψ=20%胎牛血清,10^5U/L青链霉素双抗,0.1mmol/L L—谷氨酰胺,10^3IU/L白血病抑制因子,0.1mmol/L β-巯基乙醇)进行培养。此外,使用早代胚胎于细胞;培养时使用不涂明胶的一次性培养瓶;掌握好消化、复苏和冻存时机并及时换液;按照严格步骤进行培养、传代、冻仔。对扩增后的ES细胞进行染色体和全能性检测。结果表明,在较短时问内(5d)可将胚胎干细胞扩增16倍以上。扩增后的ES细胞较好地保持了胚胎干细胞的全能性和核型。可见使用合适的培养基,按照严格步骤操作,短期内完全可以大量扩增胚胎干细胞。  相似文献   

10.
克服昆明小鼠早胚2-细胞阻滞的研究   总被引:3,自引:0,他引:3  
目的:评价两种培养液对昆明小鼠胚胎2-细胞阻滞的克服效果。方法:用添加15%FCS或BSA的M16和CZB培养液培养昆明小鼠2-细胞胚胎和体外受精卵,以观察这两种培养液对昆明小鼠早胚发育的影响及在克服昆明小鼠2-细胞阻滞中的作用,并通过CZB培养液中葡萄糖成份的增减,了解其作用及效应的时间。结果:添加FCS的M16和CZB培养注均能较好地支持2-细胞胚胎发育到囊胚(78.9%,72.7%),当进行  相似文献   

11.
Effects of the steel gene product on mouse primordial germ cells in culture.   总被引:21,自引:0,他引:21  
I Godin  R Deed  J Cooke  K Zsebo  M Dexter  C C Wylie 《Nature》1991,352(6338):807-809
Mutations at the steel (sl) and dominant white spotting (W) loci in the mouse affect primordial germ cells (PGC), melanoblasts and haemopoietic stem cells. The W gene encodes a cell-surface receptor of the tyrosine kinase family, the proto-oncogene c-kit. In situ analysis has shown c-kit messenger RNA expression in PGC in the early genital ridges. The Sl gene encodes the ligand for this receptor, a peptide growth factor, called here stem cell factor (SCF). SCF mRNA is expressed in many regions of the early mouse embryo, including the areas of migration of these cell types. It is important now to identify the role of the Sl-W interaction in the development of these migratory embryonic stem cell populations. Using an in vitro assay system, we show that SCF increases both the overall numbers and colony sizes of migratory PGC isolated from wild-type mouse embryos, and cultured on irradiated feeder layers of STO cells (a mouse embryonic fibroblast line). In the absence of feeder cells, SCF causes a large increase in the initial survival and apparent motility of PGC in culture. But labelling with bromodeoxyuridine shows that SCF is not, by itself, a mitogen for PGC. SCF does not exert a chemotropic effect on PGC in in vitro assays. These results suggest that SCF in vivo is an essential requirement for PGC survival. This demonstrates the control of the early germ-line population by a specific trophic factor.  相似文献   

12.
人胎儿骨髓间充质干细胞的分离及生物学鉴定   总被引:2,自引:1,他引:2  
通过原代细胞培养,从引产胎儿骨髓组织中分离干细胞,然后进行生物学鉴定,旨在体外建立培养胎儿骨髓干细胞的有效方法,为进一步研究干细胞奠定基础.本研究对四个月的引产胎儿骨髓组织进行原代细胞培养,采用贴壁筛选法,在含有15%胎牛血清的L-DMEM/IMDM(1:1)混和培养液中培养,7 d后细胞可长满瓶底.显微镜下观察,细胞形态均一,呈长梭形.传代后,在8代以内的细胞贴壁能力较强,生长速度较快.将其命名为BMMS-03.在第3代时,对培养的细胞进行了于细胞标志物的生物学鉴定,采用流式细胞仪对经免疫荧光染色的细胞进行检测.结果显示:97.2%的细胞呈CD105阳性反应,66.0%的细胞呈CD106阳性反应, 9.2%的细胞呈CD34阳性反应.阴性对照组阳性反应为0.5%.生物学鉴定的初步结果提示,从胎儿骨髓组织中分离培养成功的细胞为骨髓间充质干细胞,其细胞形态学特征、CD105 、CD106 和CD34-的检测结果均符合间充质干细胞的特征.本研究成功地建立了体外培养胎儿骨髓间充质干细胞的有效方法,所获得的间充质干细胞纯度较高,增殖较快,适用于干细胞生物学和组织工程学的研究.  相似文献   

13.
鸡胚胎干细胞的分离、培养与鉴定的初步研究   总被引:3,自引:0,他引:3  
采用饲养层培养法,以高糖DM EM为基础培养基,同时添加胎牛血清以及5 ng/m l SCF,l0 ng/m l bFGF和1 000 IU/m l mL IF等细胞因子与鸡胚浸出液等,对鸡的X期胚盘细胞进行离体培养,并利用形态学鉴定、AKP染色、体外分化实验等方法对所获得的细胞克隆进行鉴定。实验结果表明,本实验建立的培养体系培养出了具有多分化潜能的类胚胎干细胞。  相似文献   

14.
新生小鼠神经干细胞的分离、培养和鉴定   总被引:1,自引:0,他引:1  
目的:探讨新生小鼠神经干细胞(NSCs)分离、培养以及鉴定的方法。方法:分离新生昆明种小鼠(出生24h内)的大脑组织,经胰酶消化加机械吹打后,利用无血清培养基悬浮培养细胞,获得具有自我增殖能力的细胞克隆,并将细胞克隆贴壁培养测其分化能力。应用抗Ncstin、Musashi、SSEA-1、NSE免疫细胞化学方法及BrdU标记方法鉴定细胞克隆。结果:从新生昆明种小鼠大脑组织分离的细胞悬液,经悬浮培养,生成大量具有增殖能力的细胞,可形成神经球(neurosphercs),抗Nestin、Musashi、SSEA-1阳性,BrdU标记也呈阳性。细胞克隆贴壁培养后神经球分化为神经细胞,抗NSE阳性。结论:上述方法分离的细胞具有自我更新、自我复制及分化为神经细胞的能力,属于中枢神经系统干细胞。  相似文献   

15.
建立一种简单、有效体外分离和培养精原干细胞(Spermatogonia stem cell,SSCs)的方法。采用酶消化6—8d新生小鼠睾丸制备睾丸细胞悬液后进行培养,碱性磷酸酶(AKP)染色和间接免疫荧光对培养的细胞进行鉴定;结果显示:SSCs培养3~5天可观察到细胞克隆的产生,AKP染色强阳性;间接免疫荧光显示GCNF阴性,oct-4、c—kit均呈阳性反应。由此可知以DMEM+15%胎牛血清+白血病抑制因子(leukemia inhibitory factor,LIF)+L-谷氨酰胺等为培养基,成功建立了精原干细胞的体外培养体系。  相似文献   

16.
The nucleus of a somatic cell could be dedifferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16.1%, which was significantly higher than that of both the adult mouse fibroblast cells (0%-3.1%, P < 0.05) and fetus mouse fibroblast cells (2.1%-3.7%, P < 0.05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryos development normally than fibroblast cells.  相似文献   

17.
Pluripotency of spermatogonial stem cells from adult mouse testis   总被引:2,自引:0,他引:2  
Guan K  Nayernia K  Maier LS  Wagner S  Dressel R  Lee JH  Nolte J  Wolf F  Li M  Engel W  Hasenfuss G 《Nature》2006,440(7088):1199-1203
Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.  相似文献   

18.
To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application ,we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state,We Success-fully use human fibroblasts derved from aborted fetus and adult prepuces as feeder layer to maintain human embryonic stem cells growth ,During the passage and growth on this feeder layer,the human embryonic stem cells can keep their undifferentiated state.  相似文献   

19.
Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.  相似文献   

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