昆明小鼠胚胎干细胞建系的初步研究
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摘要
胚胎干细胞(embryonic stem cells,ESCs)是具有自我更新和高度分化潜能的细胞,能在体外保持未分化状态无限增殖传代。目前已建立了多种动物和人的ESCs系。昆明系小鼠是我国生物实验中应用最多的实验动物。故本试验以昆明小鼠为实验材料,探讨了小鼠ESCs(mouse ESCs,mESCs)建系的技术方法,为实验室下一步研究水牛ESCs建系提供一套可行的技术理论体系。
本试验包括两部分内容,第一部分为类mESCs分离方法的研究。主要探讨不同胚胎期(扩展囊胚和孵化囊胚)和传代方法对分离类mESCs效率的影响。第二部分为类mESCs鉴定的研究。包括碱性磷酸酶(AKP)染色、RT-PCR分析Oct4和Sox2表达、核型分析和分化能力鉴定。其中检测类mESCs分化能力又通过自发分化和bFGF诱导分化两方面进行。
结果显示:1)扩展囊胚分离类mESCs效率高于孵化囊胚(P＜0.05)。虽然在贴壁率和原代克隆形成率二者没有显著差异(P＞0.05),但在孵化囊胚组未获得类mESCs;2)从类mESCs生长形态和AKP染色上看,5代后类mESCs传代使用机械法或Trypsin/EDTA消化法,对维持类mESCs未分化状态没有差异,说明无论机械法还是Trypsin/EDTA消化法都适合对类mESCs进行传代;3)本实验获得的类mESCs传至25代未见形态上的分化,AKP染色呈强阳性,RT-PCR分析显示表达Oct4和Sox2,核型分析显示为正常核型40XY,正常核型率为70%,说明本试验分离的类mESCs具有ESCs的基本特征;4)类mESCs在去除饲养层和LIF、bFGF之后,经悬浮培养形成类胚体(EB),EB贴壁后在无LIF、bFGF下自发分化为多种类型细胞;在无LIF、血清以及含20 ng/mL bFGF下诱导分化成神经细胞;AKP染色显示,这两种方式分化的细胞均呈阴性;用Nestin检测诱导分化,呈阳性。说明类mESCs具有体外分化潜能。
综上所述,本试验成功分离出类mESCs,而且所分离的类mESCs符合ESCs的一般特征。这为本实验室下一步研究水牛ESCs建系打下了基础。
Embryonic stem cells(ESCs)can self-renew and proliferate indefinitely in vitro,which retain their capability to differentiate into all cell types.By now, various kinds of animals and human ESC lines had been established. KUNMING(KM)mouse is used most-widely for biological experiment in our country.Therefore in this experiment the KM mouse was chosen to explore the isolation methods of mouse ESCs(mESCs)line to provide feasible methods and theories for Buffalo ESCs isolation.
The study included two parts.The first part focused on the effects of embryonic stages and passage methods on putative mESCs isolation.The second part focused on the detection of putative mESCs.The alkaline phosphatase(AKP)staining,RT-PCR analysis of Oct4 and Sox2,karyotype analysis and differentiation capability assays were used to testify the charaterastics of putative mESCs.The differentiation capability assays were carried out by two approachs.One was spontaneous and the other was inducing putative mESCs to differentiate into neural cells.
The results showed that:1)The isolation efficiencies of the expanded blastocysts was higher than that of the hatched blastocysts(P＜0.05),although there were not significant differences in the attachment and the primary colonies formation rates between the expanded and hatched blastocysts(P＞0.05), putative mESCs were not successfully obtained from the hatched blastocysts;2) Judging from the putative mESCs morphology and AKP staining,there wasn't significant difference by the mechanical and trypsinized passage methods after passage five,which revealed that both of the passage methods were suitable to passage putative mESCs;3)putative mESCs remained undifferentiated culturing to passage 25 in vitro,showed the high activity of AKP,expressed Oct4 and Sox2,and had a normal karyotype 40XY,which indicated that the isolated putative mESCs had the general feature of putative mESCs;4)During suspension culture in the absence of feeder layers,LIF and bFGF,putative mESCs formed embryoid bodies(EB).When the EB were cultured in the absence of LIF and bFGF,which was called spontaneous differentiation,they were able to differentiate into various cell types.When the EB were cultured in the absence of LIF and serum with additional 20 ng/mL bFGF,which was called induced differentiation,they differentiated into neural cells.The AKP staining showed both the differentiated cells of two approachs were negative and the immunofluorescence assay showed the cells derived from induced differentiation expressed Nestin,which demonstrated that putative mESCs had the ability to differentiate into somatic cells in vitro.
In conclusion,putative mESCs were successfully isolated from the expanded blastocytes in this study,and were pluripotent,which might lay the foundations for further study of Buffalo ESCs in our lab.
本试验包括两部分内容,第一部分为类mESCs分离方法的研究。主要探讨不同胚胎期(扩展囊胚和孵化囊胚)和传代方法对分离类mESCs效率的影响。第二部分为类mESCs鉴定的研究。包括碱性磷酸酶(AKP)染色、RT-PCR分析Oct4和Sox2表达、核型分析和分化能力鉴定。其中检测类mESCs分化能力又通过自发分化和bFGF诱导分化两方面进行。
结果显示:1)扩展囊胚分离类mESCs效率高于孵化囊胚(P＜0.05)。虽然在贴壁率和原代克隆形成率二者没有显著差异(P＞0.05),但在孵化囊胚组未获得类mESCs;2)从类mESCs生长形态和AKP染色上看,5代后类mESCs传代使用机械法或Trypsin/EDTA消化法,对维持类mESCs未分化状态没有差异,说明无论机械法还是Trypsin/EDTA消化法都适合对类mESCs进行传代;3)本实验获得的类mESCs传至25代未见形态上的分化,AKP染色呈强阳性,RT-PCR分析显示表达Oct4和Sox2,核型分析显示为正常核型40XY,正常核型率为70%,说明本试验分离的类mESCs具有ESCs的基本特征;4)类mESCs在去除饲养层和LIF、bFGF之后,经悬浮培养形成类胚体(EB),EB贴壁后在无LIF、bFGF下自发分化为多种类型细胞;在无LIF、血清以及含20 ng/mL bFGF下诱导分化成神经细胞;AKP染色显示,这两种方式分化的细胞均呈阴性;用Nestin检测诱导分化,呈阳性。说明类mESCs具有体外分化潜能。
综上所述,本试验成功分离出类mESCs,而且所分离的类mESCs符合ESCs的一般特征。这为本实验室下一步研究水牛ESCs建系打下了基础。
Embryonic stem cells(ESCs)can self-renew and proliferate indefinitely in vitro,which retain their capability to differentiate into all cell types.By now, various kinds of animals and human ESC lines had been established. KUNMING(KM)mouse is used most-widely for biological experiment in our country.Therefore in this experiment the KM mouse was chosen to explore the isolation methods of mouse ESCs(mESCs)line to provide feasible methods and theories for Buffalo ESCs isolation.
The study included two parts.The first part focused on the effects of embryonic stages and passage methods on putative mESCs isolation.The second part focused on the detection of putative mESCs.The alkaline phosphatase(AKP)staining,RT-PCR analysis of Oct4 and Sox2,karyotype analysis and differentiation capability assays were used to testify the charaterastics of putative mESCs.The differentiation capability assays were carried out by two approachs.One was spontaneous and the other was inducing putative mESCs to differentiate into neural cells.
The results showed that:1)The isolation efficiencies of the expanded blastocysts was higher than that of the hatched blastocysts(P＜0.05),although there were not significant differences in the attachment and the primary colonies formation rates between the expanded and hatched blastocysts(P＞0.05), putative mESCs were not successfully obtained from the hatched blastocysts;2) Judging from the putative mESCs morphology and AKP staining,there wasn't significant difference by the mechanical and trypsinized passage methods after passage five,which revealed that both of the passage methods were suitable to passage putative mESCs;3)putative mESCs remained undifferentiated culturing to passage 25 in vitro,showed the high activity of AKP,expressed Oct4 and Sox2,and had a normal karyotype 40XY,which indicated that the isolated putative mESCs had the general feature of putative mESCs;4)During suspension culture in the absence of feeder layers,LIF and bFGF,putative mESCs formed embryoid bodies(EB).When the EB were cultured in the absence of LIF and bFGF,which was called spontaneous differentiation,they were able to differentiate into various cell types.When the EB were cultured in the absence of LIF and serum with additional 20 ng/mL bFGF,which was called induced differentiation,they differentiated into neural cells.The AKP staining showed both the differentiated cells of two approachs were negative and the immunofluorescence assay showed the cells derived from induced differentiation expressed Nestin,which demonstrated that putative mESCs had the ability to differentiate into somatic cells in vitro.
In conclusion,putative mESCs were successfully isolated from the expanded blastocytes in this study,and were pluripotent,which might lay the foundations for further study of Buffalo ESCs in our lab.
引文
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