兔类胚胎干细胞体外培养体系的优化与建立
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摘要
本研究以兔为材料,系统地研究了不同培养条件和传代方法对兔类ES、EG细胞分离培养的影响,为完善兔类ES、EG细胞建系的技术路线提供资料。实验主要结果如下:
1试验比较不同饲养层和不同培养液对ES细胞分离培养的影响。选用DMEM+15%FBS+0.1mMβ-巯基乙醇+0.1mM MEM+100IU/ml青霉素+100IU/ml链霉素+1000IU/ml LIF+10mg/mL胰岛素作为体外分离培养家兔ES细胞的培养液,胚胎能较快贴壁、增殖,兔类ES细胞最高能够传至5代而不分化。无饲养层培养与在MEF、REF饲养层中培养的家兔胚胎贴壁率,ICM增殖率之间有极显著差异(P<0.05),几乎不能传代。MEF作为饲养层,胚胎的贴壁率为67.4%,最高传代至第5代;以REF为饲养层胚胎的贴壁率为63.2%,最高传至第4代。
2本试验对ICM和类ES细胞集落采用三种不同的传代方法进行传代,结果发现,连续消化法相对一次消化法ES细胞克隆的比率大为提高,连同饲养层一起消化产生ES克隆的比率太低,ES细胞只传1代,不适合用作ES细胞的早期分离、传代。
3 14～18d胎龄的兔胎儿均能分离得到PGCs原代集落,但是15、16d的胎儿分离得到的集落数要比14、17、18d胎儿得到的集落要多,类EG细胞最高传了9代,适合克隆兔类EG细胞。SD大鼠心肌条件培养基能够较好地促进兔PGCs的分离培养,与添加LIF组差异不显著(P>0.05),但明显好于未添加LIF组,可以用于兔PGCs的分离培养。
4将取得的兔PGCs分别培养在MEF﹑REF饲养层上或与同源兔胎儿生殖脊成纤维细胞(HEFgr)﹑兔睾丸支持细胞(RSC)共培养。结果表明,采用共培养的模式分离得到的集落明显多于传统的饲养层模式。与RSCs共培养得到的EG细胞集落数最多,保持未分化的时间最长,并传了8代,与HEFgr共培养得到的EG细胞集落数与RSCs相比差异不明显,但最高只传了6代。培养在MEF上的兔类EG细胞也能较好的保持未分化状态,传了4代。培养在REF上的兔类EG集落数目较少,出现分化时间较早,只传了3代。因此可以用与RSCs或HEFgr共培养的方法分离培养兔PGCs。
5试验比较了不同传代方法对兔类EG细胞传代的影响,结果发现手工传代和连同饲养层细胞一起消化传代法均获得了较高的P1集落数,两者差异不显著,但手工传代EG集落能够传至第9代,而连同饲养层细胞一起消化只能将EG细胞传至第4代,不适合作为兔类EG细胞传代的方法。
6分离得到的兔ES、EG细胞,经形态学观察、AKP染色、体外分化能力检测实验等,证明其具有胚胎干细胞的诸多特性。
This paper is to systematically study the effect of isolating and cloning Embryonic stem cells (ESC) and embryonic germ cells( EGC) in the different culture conditions and the passage way. It might provide datas to perfect route to establish rabbit ES、EG cell line. The results obtained were as follows:
1 The research Compared the Effect of Different culture medium and Feeder Layers on isolating and cloning of ES cells. With the culture medium of DMEM, 15%FBS, 0.1mMβ-mercaptoethanol, 0.01mM non-essential amino acid, 100IU/ml penicillin, 100IU/ml streptomycin, 1000IU/ml LIF and 10mg/mL insulin, the Rabbit Embryo can Attach and proliferate very fast. ES cell-like line clone in this culture medium had maintained an undifferentiated state for 5 passages. Compare with the feeder layer of MEF and the REF, the rates of embryo attaching, ICM propagating for the embryo cultivated on free-feeding layer culture system were significant differences(P<0.05), and have no passage. With the feeder layer of MEF and the REF, The rate of attaching of embryos is 67.4% and 63.2%, and they had maintained an undifferentiated state for 5 passages.
2 The method of Continuous digestive for ES cells is better than The single digestive method, it improve the rate of forming ESC clones. Digestion with the feeder layer has the lowest rate of ES clones, ES cell had maintained an undifferentiated state for only 1 passages, not suitable for ES cells separation and passage in the early time.
3 PGCs colonies can be isolated from 14-18 days of gestation embryos of rabbit, but the PGCs colonies isolate from 15, 16 days Fetus are more than 14, 17 and 18 days’; and One EG cell line in this two days maintained undifferentiation for 9 passages, suitable for cloning rabbits EG-like cells. Rat fetus cardiomyocyte cells condition medium is as good as EG cell medium with LIF, both are better than the medium contained no cytokine, it showed that rat fetus cardiomyocyte cell conditioned medium is fit for the isolation and culture of rabbit PGCs.
4 Let the PGCs culture on mouse embryonic fibroblasts(MEF), rabbit embryonic fibroblasts ( REF) and co-cultured with the homogenous embryonic fibroblast from genital ridges(HEFgr)or co-cultured with Rabbit Sertoli cells(RSCs). The Result is that the manner of co-cultured was better than cultured depended on mitotically inactive feed layer. PGCs co-cultured with RSCs gained the more EG than other ways. They remained undifferentiated state for the longest period and cultured for 8 passages. There was not significant difference in PGCs co-cultured with the HEFgr from genital ridges and co-cultured with RSCs, but PGCs were just cultured for 6 passages. PGCs culture on MEF can also remained undifferentiated and cultured for 4 passages. PGCs culture on REF gained fewer EGs, they remained undifferentiated state were shorter and just cultured for 3 passages. So the manner of co-cultured with RSCs and co-cultured with the HEFgr can be used to isolation and cultivation rabbit PGCs.
5 Comparing the effect of different methods on the subculture of Rabbit EG like cells, the method of Passage by hand as well as the methods of digestion together with freeder layer can obtain the more F1 clones, they are no obvious difference. But the method of Passage by hand in EG clones can cultured for 9 passages, The methods of digestion together with freeder layer just support the EG like cell for 4 passages, so it is not a good passaging methods for rabbit EG like cell.
6 When isolated rabbit ESC and EGC were examined by observing morphology, AKP stain and in vitro differentiation capacity, it have a series of characters of Embryonic Stem Cells.
1试验比较不同饲养层和不同培养液对ES细胞分离培养的影响。选用DMEM+15%FBS+0.1mMβ-巯基乙醇+0.1mM MEM+100IU/ml青霉素+100IU/ml链霉素+1000IU/ml LIF+10mg/mL胰岛素作为体外分离培养家兔ES细胞的培养液,胚胎能较快贴壁、增殖,兔类ES细胞最高能够传至5代而不分化。无饲养层培养与在MEF、REF饲养层中培养的家兔胚胎贴壁率,ICM增殖率之间有极显著差异(P<0.05),几乎不能传代。MEF作为饲养层,胚胎的贴壁率为67.4%,最高传代至第5代;以REF为饲养层胚胎的贴壁率为63.2%,最高传至第4代。
2本试验对ICM和类ES细胞集落采用三种不同的传代方法进行传代,结果发现,连续消化法相对一次消化法ES细胞克隆的比率大为提高,连同饲养层一起消化产生ES克隆的比率太低,ES细胞只传1代,不适合用作ES细胞的早期分离、传代。
3 14～18d胎龄的兔胎儿均能分离得到PGCs原代集落,但是15、16d的胎儿分离得到的集落数要比14、17、18d胎儿得到的集落要多,类EG细胞最高传了9代,适合克隆兔类EG细胞。SD大鼠心肌条件培养基能够较好地促进兔PGCs的分离培养,与添加LIF组差异不显著(P>0.05),但明显好于未添加LIF组,可以用于兔PGCs的分离培养。
4将取得的兔PGCs分别培养在MEF﹑REF饲养层上或与同源兔胎儿生殖脊成纤维细胞(HEFgr)﹑兔睾丸支持细胞(RSC)共培养。结果表明,采用共培养的模式分离得到的集落明显多于传统的饲养层模式。与RSCs共培养得到的EG细胞集落数最多,保持未分化的时间最长,并传了8代,与HEFgr共培养得到的EG细胞集落数与RSCs相比差异不明显,但最高只传了6代。培养在MEF上的兔类EG细胞也能较好的保持未分化状态,传了4代。培养在REF上的兔类EG集落数目较少,出现分化时间较早,只传了3代。因此可以用与RSCs或HEFgr共培养的方法分离培养兔PGCs。
5试验比较了不同传代方法对兔类EG细胞传代的影响,结果发现手工传代和连同饲养层细胞一起消化传代法均获得了较高的P1集落数,两者差异不显著,但手工传代EG集落能够传至第9代,而连同饲养层细胞一起消化只能将EG细胞传至第4代,不适合作为兔类EG细胞传代的方法。
6分离得到的兔ES、EG细胞,经形态学观察、AKP染色、体外分化能力检测实验等,证明其具有胚胎干细胞的诸多特性。
This paper is to systematically study the effect of isolating and cloning Embryonic stem cells (ESC) and embryonic germ cells( EGC) in the different culture conditions and the passage way. It might provide datas to perfect route to establish rabbit ES、EG cell line. The results obtained were as follows:
1 The research Compared the Effect of Different culture medium and Feeder Layers on isolating and cloning of ES cells. With the culture medium of DMEM, 15%FBS, 0.1mMβ-mercaptoethanol, 0.01mM non-essential amino acid, 100IU/ml penicillin, 100IU/ml streptomycin, 1000IU/ml LIF and 10mg/mL insulin, the Rabbit Embryo can Attach and proliferate very fast. ES cell-like line clone in this culture medium had maintained an undifferentiated state for 5 passages. Compare with the feeder layer of MEF and the REF, the rates of embryo attaching, ICM propagating for the embryo cultivated on free-feeding layer culture system were significant differences(P<0.05), and have no passage. With the feeder layer of MEF and the REF, The rate of attaching of embryos is 67.4% and 63.2%, and they had maintained an undifferentiated state for 5 passages.
2 The method of Continuous digestive for ES cells is better than The single digestive method, it improve the rate of forming ESC clones. Digestion with the feeder layer has the lowest rate of ES clones, ES cell had maintained an undifferentiated state for only 1 passages, not suitable for ES cells separation and passage in the early time.
3 PGCs colonies can be isolated from 14-18 days of gestation embryos of rabbit, but the PGCs colonies isolate from 15, 16 days Fetus are more than 14, 17 and 18 days’; and One EG cell line in this two days maintained undifferentiation for 9 passages, suitable for cloning rabbits EG-like cells. Rat fetus cardiomyocyte cells condition medium is as good as EG cell medium with LIF, both are better than the medium contained no cytokine, it showed that rat fetus cardiomyocyte cell conditioned medium is fit for the isolation and culture of rabbit PGCs.
4 Let the PGCs culture on mouse embryonic fibroblasts(MEF), rabbit embryonic fibroblasts ( REF) and co-cultured with the homogenous embryonic fibroblast from genital ridges(HEFgr)or co-cultured with Rabbit Sertoli cells(RSCs). The Result is that the manner of co-cultured was better than cultured depended on mitotically inactive feed layer. PGCs co-cultured with RSCs gained the more EG than other ways. They remained undifferentiated state for the longest period and cultured for 8 passages. There was not significant difference in PGCs co-cultured with the HEFgr from genital ridges and co-cultured with RSCs, but PGCs were just cultured for 6 passages. PGCs culture on MEF can also remained undifferentiated and cultured for 4 passages. PGCs culture on REF gained fewer EGs, they remained undifferentiated state were shorter and just cultured for 3 passages. So the manner of co-cultured with RSCs and co-cultured with the HEFgr can be used to isolation and cultivation rabbit PGCs.
5 Comparing the effect of different methods on the subculture of Rabbit EG like cells, the method of Passage by hand as well as the methods of digestion together with freeder layer can obtain the more F1 clones, they are no obvious difference. But the method of Passage by hand in EG clones can cultured for 9 passages, The methods of digestion together with freeder layer just support the EG like cell for 4 passages, so it is not a good passaging methods for rabbit EG like cell.
6 When isolated rabbit ESC and EGC were examined by observing morphology, AKP stain and in vitro differentiation capacity, it have a series of characters of Embryonic Stem Cells.
引文
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