胚胎干细胞培养与鉴定的方法研究
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摘要
目的培养胚胎干细胞R1系及CGR8系并对其生物学特性进行检测。为探讨胚胎干细胞替代牙源性上皮细胞的研究奠定基础。方法采用GIBCO公司生产的浓度为15胎牛血清培养液,形成的ES细胞样集落,继续培养后观察集落的生长状态,并通过碱性磷酸酶染色,在体外形成拟胚体,移植入裸鼠皮下4周后取材,组织学观察进行鉴定。结果 ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物作用下,未分化的ES细胞显微镜下为棕褐色,分化的不着色。悬滴法培养ES细胞两天后生成拟胚体,它是胚胎干细胞在体外一定条件下自发形成的类似早期胚胎的球体结构,这种结构与胚胎发育过程中的卵圆柱期结构十分相似。体内研究表明,裸鼠皮下移植4周后,可形成含有三胚层组织的畸胎瘤,这种畸胎瘤细胞核型仍为二倍体,无恶性病变。结论胚胎干细胞具有高度的复制能力及多向分化的潜能,为后续实验奠定了基础。
Objective To culture ESCs R1 cell line and CGR8 cell line and confirm the identity of mESCs and to explore the possibility of mESCs for odontogenic differentiation. Methods MESCs were cultured in the absence of feeder cells for 4 days. The culture medium was Dulbecco's modified Eagle medium supplemented with 15% heat-inactivated fetal bovine serum(FBS; Gibco), Alkaline-phosphatase(AP) staining was performed according to the manufacturer's recommendations(Millipore) and EB formation was achieved. For teratoma formation, approximately 2 ×10~6 ESCs were injected into the subcutaneous pockets of 6-week-old nude mice. At 4 weeks after ESC injection,xenografted masses were observed and dissected. Results ES cells have the typical morphological characteristics: the nest-like colony with clear edge and smooth surface; the boundaries between cells is not clear and the single cell volume is small. Next we evaluated the AP activity of mESCs; enzyme assay results revealed high AP activity. In vitro methods involving EB formation are simple and widely used because this method created suitable conditions for the differentiation into the cells of all 3 germ layers. Teratomas were formed 4 weeks after injection in vivo.HE staining revealed that the teratomas displayed the derivatives of all 3 embryonic germlayers. Conclusion ESCs possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells,suggesting the potential of using ESCs as an alternative candidate cell source in tooth regeneration research.
Objective To culture ESCs R1 cell line and CGR8 cell line and confirm the identity of mESCs and to explore the possibility of mESCs for odontogenic differentiation. Methods MESCs were cultured in the absence of feeder cells for 4 days. The culture medium was Dulbecco's modified Eagle medium supplemented with 15% heat-inactivated fetal bovine serum(FBS; Gibco), Alkaline-phosphatase(AP) staining was performed according to the manufacturer's recommendations(Millipore) and EB formation was achieved. For teratoma formation, approximately 2 ×10~6 ESCs were injected into the subcutaneous pockets of 6-week-old nude mice. At 4 weeks after ESC injection,xenografted masses were observed and dissected. Results ES cells have the typical morphological characteristics: the nest-like colony with clear edge and smooth surface; the boundaries between cells is not clear and the single cell volume is small. Next we evaluated the AP activity of mESCs; enzyme assay results revealed high AP activity. In vitro methods involving EB formation are simple and widely used because this method created suitable conditions for the differentiation into the cells of all 3 germ layers. Teratomas were formed 4 weeks after injection in vivo.HE staining revealed that the teratomas displayed the derivatives of all 3 embryonic germlayers. Conclusion ESCs possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells,suggesting the potential of using ESCs as an alternative candidate cell source in tooth regeneration research.
引文
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2 Chai Y,Slavkin HC.Prospects for tooth regeneration in the 21st century:a perspective.Microsc Res Tech,2003,60(5):469-479.
3 Hu B,Unda F,Bopp-Kuchler S,et al.Bone marrow cells can give rise to ameloblast-like cells.J Dent Res,2006,85(5):416-421.
4 Petrovic V,Stefanovic V.Dental tissue-new source for stem cells.Scientific World Journal,2009,9:1167-1177.
5 Bourne S,Polak JM,Hughes SP,et al.Osteogenic differentiation of mouse embryonic stem cells:differential gene expression analysis by c DNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting.Tissue Eng,2004,10(N5-6):796-806.
6 Thomson JA,Itskovitz-Eldor J,Shapiro SS,et al.Embryonic stem cell lines derived from human blastocyst Science,1998,282(N5391):1145-1147.
7 Heng BC,Cao T,Stanton LW,et al.Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro.J Bone Miner Res,2004,19(9):1379-1394.
8 Deutsch G,Jung T,Zheng M,et al.A bipotential precursor population for pancreas and liver within the embryonic endoderm.Development,2001,128(6):871-881.
9 Duncan SA.Mechanisms controlling early development of the liver.Mech.Dev,2003,120(1):19-33.
10 Young CS,Terada S,Vacanti JP,et al.Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.J Dent Res,2002,81(10):695-700.
11 Nakao K,Morita R,Saji Y,et al.The development of a bioengineered organ germ method.Nat Methods,2007,4(3):227-230.
12 Tucker A,Sharpe P.The cutting-edge of mammalian development;how the embryo makes teeth.Nat Rev Genet,2004,5(7):499-508.