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背景:人胚胎干细胞是一种全能型细胞,可以分化为3个胚层的组织,目前国内对其无饲养层生长的研究较少。成纤维细胞生长因子是维持胚胎干细胞不分化状态的重要因子。目的:探讨长期培养过程中不同质量浓度成纤维细胞生长因子对人胚胎干细胞未分化状态和全能性维持的影响。方法:两株人胚胎干细胞在鼠胚胎成纤维细胞条件培养基中培养3代,分别转移到含100,160,250μg/L成纤维细胞生长因子的鼠胚胎成纤维细胞条件培养基中培养8代。从培养皿中移出胚胎干细胞,用IV胶原酶消化聚集成团的细胞,观察细胞分化状态和全能性情况。收集传8代后的胚胎干细胞,种植于SCID小鼠体内。对所得细胞做形态学评估,并进行碱性磷酸酶染色、表面标记免疫组化检测、RT-PCR检测OCT-4的表达、体内致瘤情况。结果与结论:在含160,250μg/L成纤维细胞生长因子的鼠胚胎成纤维细胞条件培养基中,两株人胚胎干细胞可以保持原有性状,即细胞克隆呈圆形,核质比较高,中间大片区域为未分化细胞,周围为分化细胞;呈碱性磷酸酶强阳性表达;表达OCT-4转录因子蛋白;细胞表面标志SSEA-4,TRA-1-60,TRA-1-81均呈阳性表达;聚集成团的胚胎干细胞培养10d后形成拟胚体;种植于SCID小鼠体内可得含3个胚层组织的畸胎瘤。含100μg/L成纤维细胞生长因子的鼠胚胎成纤维细胞条件培养基不足以维持人胚胎干细胞的长期增殖,4代以后大部分细胞分化死亡。提示成纤维细胞生长因子质量浓度达160μg/L以上时,可以单独支持人胚胎干细胞的体外稳定增殖,且不影响细胞分化状态和全能性。
BACKGROUND: Human embryonic stem cells are pluripotent cells that can differentiate into three germ layers. At present, there are few researches on feeder-free growth in China. Fibroblast growth factor is an important factor to maintain the undifferentiated state of embryonic stem cells. OBJECTIVE: To investigate the effects of different concentrations of fibroblast growth factor on the maintenance of undifferentiated human embryonic stem cells in long-term culture. Methods: Two human embryonic stem cells were cultured in conditioned medium of mouse embryonic fibroblasts for three generations and transferred to conditioned medium of mouse embryonic fibroblasts containing 100,160,250μg / L of fibroblast growth factor respectively for 8 generations. Embryonic stem cells were removed from the petri dish and aggregated into clusters of cells by IV collagenase to observe the state of cell differentiation and pluripotency. Eight passages of embryonic stem cells were collected and seeded in SCID mice. Morphological evaluation of the resulting cells, and alkaline phosphatase staining, surface labeling immunohistochemical detection, OCT-4 expression by RT-PCR, in vivo tumorigenicity. RESULTS AND CONCLUSION: In human embryonic fibroblast conditioned medium containing 160, 250μg / L fibroblast growth factor, the two human embryonic stem cells can retain the original traits, that is, the cell clone is round with relatively high nuclei, Large area of undifferentiated cells, surrounded by differentiated cells; strongly alkaline phosphatase expression; expression of OCT-4 transcription factor protein; cell surface markers SSEA-4, TRA-1-60, TRA-1-81 were positive The embryonic stem cells aggregated into clusters formed embryoid body after 10 days, and teratomas with 3 germ layers in SCID mice were obtained. Mouse embryonic fibroblast conditioned medium containing 100 μg / L of fibroblast growth factor was not sufficient to maintain the long-term proliferation of human embryonic stem cells, and most of the cells died after 4 passages. The results suggest that when the concentration of fibroblast growth factor is more than 160μg / L, it can support the stable proliferation of human embryonic stem cells in vitro without affecting the cell differentiation state and the pluripotency.