影响小鼠胚胎干细胞和牛胚胎生殖细胞分离克隆的因素
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摘要
本研究以BALB／c小鼠为材料，建立免疫外科分离克隆小鼠ES细胞方法；并以牛胎儿为材料，从牛原始生殖细胞中分离克隆出牛EG细胞。对影响小鼠ES细胞、牛EG细胞分离与克隆的影响因素进行了探讨，为进一步建立小鼠ES及牛EG细胞系奠定了基础。实验主要结果如下：
1．用免疫外科法从45枚BALB／c小鼠囊胚中分离得到20枚除去滋养层细胞的ICM，接种在MEF饲养层上，使用DMEM(高糖)+15％FBS+0.1mMβ-巯基乙醇+0.01mM非必需氨基酸+1000 IU／ml LIF+100 IU／mL青霉素+100 IU／ml链霉素培养液，17枚形成典型的ICM集落，有一枚胚胎传至第10代。用于全胚培养的BALB／c小鼠胚胎共102枚，使用与免疫外科相同的培养方法，66枚形成典型的ICM集落，其中一枚胚胎传至第8代。
2．收集妊娠29～100d牛胎儿46例，使用DMEM(高糖)+15％NBS(或FBS)+0.1mMβ-巯基乙醇+2mM谷氨酰胺斗0.01mM非必需氨基酸牛100 IU／mL青霉素+100 IU／ml链霉素培养液从生殖嵴(腺)或类似物原始生殖细胞中分离克隆牛EG细胞，25例出现EG细胞集落，最高一例传至6代。胎龄为29～45d牛胎儿最适合牛EG细胞分离与克隆，胎龄大于70 d，很难得到牛EG细胞。
3．从45d牛胎儿中分离培养出牛胎儿成纤维细胞，传至28代。发现α-MEM是一种很好牛胎儿成纤维细胞分离培养的培养基。
4．小鼠胚胎贴壁率及ICM集落形成率在MEF、STO及BEF三种饲养层上无显著差异(P＞0.05)；MEF、STO、BEF及HEF饲养层均能促进牛EG细胞体外增殖，但STO效果最好，MEF及BEF次之，HEF效果最差。
5．DMEM培养基对牛PGCs的分离培养及传代明显好于F12；15％NBS在小鼠胚胎贴壁率及ICM集落形成率上明显好于10％NBS(P＜0.05)；15％FBS较15％NBS有利于小鼠ES细胞(P＜0.05)、牛EG细胞的分离与克隆。
6．添加LIF(10ng／ml)有利于小鼠ES细胞(P＜0.05)、牛EG细胞的分离与克隆；LIF、SCF及bFGF(添加浓度均为10 ng／ml)三种因子联合作用能明显促进牛EG细胞分离克隆。
7．BALB／c小鼠ES细胞对胰酶很敏感，0.05％胰酶+0.008％EDTA是较好的ES细胞消化液，对细胞综合损伤力小，且传代后ES细胞集落形成能力也较高(P＜0.05)。
分离得到的小鼠ES细胞、牛EG细胞，经形态学观察，AKP(或PAS)染色，体
外分化实验，核型分析证明其具有胚胎干细胞的诸多特性。
The major object of this paper is to isolate and culture embryonic stem (ES) cells from BALB/c strain mouse using a method of immunosurgery and to isolate and culture bovine embryonic germ (EG) cells derived from primordial germ cells (PGCs) as well as to demonstrate pluripotent of mouse ES and bovine EG cells from various aspects. Some factors influencing the efficiency of the isolation and culture mouse ES cells and bovine EG cells have been discussed. Our work may be useful to further establish mouse ES cell and bovine EG cell lines. The results obtained were as follows:
1. 20 pure ICMs were obtained from 45 embryos of BALB/c strain mouse using immunosurgical method. ICMs were plated on mitomicin-inactivated MEF feeders and cultured in a humidified environment of 5% CO2 in air, 37 癈. ES-like colonies were observed from 17 of 20 pure ICMs and one ES-like cell line had maintained undifferentiation for 10 passages. In addition, ES-like colonies were also obtained from 66 of 102 embryos of BALB/c strain mouse using a method of intact embryo culture. Both methods used mouse ES culture medium composed of DMEM (high glucose) containing 15% FBS, 0.1 mM p-mercaptoethanol, 0.01 mM non-essential amino acid, 10 ng/ml LIF, 100 lU/ml penicillin, and 100 IU/ml streptomycin.
2. Primordial germ cells were isolated and cultured from gonads, genital ridges or urogenital ridges of 46 fetuses with the age of 29-100 days of gestation. Bovine EG cell culture medium contains DMEM (high glucose), 15% NBS (or FBS), 0.1 mM P-mercaptoethanol, 0.01 mM non-essential amino acid, 2 mM glutamine, 100 lU/ml penicillin, and 100 lU/ml streptomycin. One EG cell line had maintained an undifferentiated state for 6 passages. 29-45 days of gestation embryos are optimum for the isolation and culture of bovine EG cells. It is difficult to isolate and culture bovine EG cells if the age of fetuses is more than 70 days of gestation.
3. Bovine fetal fibroblasts were isolated from a 45-day-old fetus, cultured in vitro, and passaged 28 passages. We find that a -MEM is a good cell culture medium for bovine fetal fibroblasts.
4. MEF, STO and BEF feeders all can sustain mouse ES cells, there are no distinct difference in the rate of embryo attaching and ICM colonies formig (P>0.05). MEF, STO, BEF and HEF feeders all can promote growth of bovine EG cells in vitro, but STO is the best, HEF is the last and MEF, BEF is in the middle.
5. DMEM medium is better than F12 medium in the isolation and culture of bovine PGCs and the formation rate of EG cells colonies. 15% NBS is better than 10% NBS in the rate of embryo attanching and ICM colonies forming in mouse (P<0.05). 15% FBS is more beneficial to isolate and culture mouse ES cells and bovine EG cells than 15% NBS (P<0.05).
6. Supplement with LIF (10 ng/ml) is advantage to the isolation and culture of mouse ES cells (PO.05) and bovine EG cells. Moreover, the influence was distinct for bovine EG cells if LIF, SCF and bFGF (each of them is 10 ng/ml) are all in presence.
7. BALB/c strain mouse ES cells is susceptive to trypsin. 0.05% trypsin-0.008% EDTA is a good dispersed liquid for mouse ES cells, which is relative gentle to ES cells and is better for forming new ES colonies (P<0.05).
The mouse ES cells and bovine EG cells which are pluipotential cells have been indentified by colony morphology, AKP (or PAS) staining, in vitro differentiation and karyotyping.
1．用免疫外科法从45枚BALB／c小鼠囊胚中分离得到20枚除去滋养层细胞的ICM，接种在MEF饲养层上，使用DMEM(高糖)+15％FBS+0.1mMβ-巯基乙醇+0.01mM非必需氨基酸+1000 IU／ml LIF+100 IU／mL青霉素+100 IU／ml链霉素培养液，17枚形成典型的ICM集落，有一枚胚胎传至第10代。用于全胚培养的BALB／c小鼠胚胎共102枚，使用与免疫外科相同的培养方法，66枚形成典型的ICM集落，其中一枚胚胎传至第8代。
2．收集妊娠29～100d牛胎儿46例，使用DMEM(高糖)+15％NBS(或FBS)+0.1mMβ-巯基乙醇+2mM谷氨酰胺斗0.01mM非必需氨基酸牛100 IU／mL青霉素+100 IU／ml链霉素培养液从生殖嵴(腺)或类似物原始生殖细胞中分离克隆牛EG细胞，25例出现EG细胞集落，最高一例传至6代。胎龄为29～45d牛胎儿最适合牛EG细胞分离与克隆，胎龄大于70 d，很难得到牛EG细胞。
3．从45d牛胎儿中分离培养出牛胎儿成纤维细胞，传至28代。发现α-MEM是一种很好牛胎儿成纤维细胞分离培养的培养基。
4．小鼠胚胎贴壁率及ICM集落形成率在MEF、STO及BEF三种饲养层上无显著差异(P＞0.05)；MEF、STO、BEF及HEF饲养层均能促进牛EG细胞体外增殖，但STO效果最好，MEF及BEF次之，HEF效果最差。
5．DMEM培养基对牛PGCs的分离培养及传代明显好于F12；15％NBS在小鼠胚胎贴壁率及ICM集落形成率上明显好于10％NBS(P＜0.05)；15％FBS较15％NBS有利于小鼠ES细胞(P＜0.05)、牛EG细胞的分离与克隆。
6．添加LIF(10ng／ml)有利于小鼠ES细胞(P＜0.05)、牛EG细胞的分离与克隆；LIF、SCF及bFGF(添加浓度均为10 ng／ml)三种因子联合作用能明显促进牛EG细胞分离克隆。
7．BALB／c小鼠ES细胞对胰酶很敏感，0.05％胰酶+0.008％EDTA是较好的ES细胞消化液，对细胞综合损伤力小，且传代后ES细胞集落形成能力也较高(P＜0.05)。
分离得到的小鼠ES细胞、牛EG细胞，经形态学观察，AKP(或PAS)染色，体
外分化实验，核型分析证明其具有胚胎干细胞的诸多特性。
The major object of this paper is to isolate and culture embryonic stem (ES) cells from BALB/c strain mouse using a method of immunosurgery and to isolate and culture bovine embryonic germ (EG) cells derived from primordial germ cells (PGCs) as well as to demonstrate pluripotent of mouse ES and bovine EG cells from various aspects. Some factors influencing the efficiency of the isolation and culture mouse ES cells and bovine EG cells have been discussed. Our work may be useful to further establish mouse ES cell and bovine EG cell lines. The results obtained were as follows:
1. 20 pure ICMs were obtained from 45 embryos of BALB/c strain mouse using immunosurgical method. ICMs were plated on mitomicin-inactivated MEF feeders and cultured in a humidified environment of 5% CO2 in air, 37 癈. ES-like colonies were observed from 17 of 20 pure ICMs and one ES-like cell line had maintained undifferentiation for 10 passages. In addition, ES-like colonies were also obtained from 66 of 102 embryos of BALB/c strain mouse using a method of intact embryo culture. Both methods used mouse ES culture medium composed of DMEM (high glucose) containing 15% FBS, 0.1 mM p-mercaptoethanol, 0.01 mM non-essential amino acid, 10 ng/ml LIF, 100 lU/ml penicillin, and 100 IU/ml streptomycin.
2. Primordial germ cells were isolated and cultured from gonads, genital ridges or urogenital ridges of 46 fetuses with the age of 29-100 days of gestation. Bovine EG cell culture medium contains DMEM (high glucose), 15% NBS (or FBS), 0.1 mM P-mercaptoethanol, 0.01 mM non-essential amino acid, 2 mM glutamine, 100 lU/ml penicillin, and 100 lU/ml streptomycin. One EG cell line had maintained an undifferentiated state for 6 passages. 29-45 days of gestation embryos are optimum for the isolation and culture of bovine EG cells. It is difficult to isolate and culture bovine EG cells if the age of fetuses is more than 70 days of gestation.
3. Bovine fetal fibroblasts were isolated from a 45-day-old fetus, cultured in vitro, and passaged 28 passages. We find that a -MEM is a good cell culture medium for bovine fetal fibroblasts.
4. MEF, STO and BEF feeders all can sustain mouse ES cells, there are no distinct difference in the rate of embryo attaching and ICM colonies formig (P>0.05). MEF, STO, BEF and HEF feeders all can promote growth of bovine EG cells in vitro, but STO is the best, HEF is the last and MEF, BEF is in the middle.
5. DMEM medium is better than F12 medium in the isolation and culture of bovine PGCs and the formation rate of EG cells colonies. 15% NBS is better than 10% NBS in the rate of embryo attanching and ICM colonies forming in mouse (P<0.05). 15% FBS is more beneficial to isolate and culture mouse ES cells and bovine EG cells than 15% NBS (P<0.05).
6. Supplement with LIF (10 ng/ml) is advantage to the isolation and culture of mouse ES cells (PO.05) and bovine EG cells. Moreover, the influence was distinct for bovine EG cells if LIF, SCF and bFGF (each of them is 10 ng/ml) are all in presence.
7. BALB/c strain mouse ES cells is susceptive to trypsin. 0.05% trypsin-0.008% EDTA is a good dispersed liquid for mouse ES cells, which is relative gentle to ES cells and is better for forming new ES colonies (P<0.05).
The mouse ES cells and bovine EG cells which are pluipotential cells have been indentified by colony morphology, AKP (or PAS) staining, in vitro differentiation and karyotyping.
引文
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