不同功能状态滋养层对人胚胎生殖嵴干细胞生长作用的实验研究

目的筛选并建立高效胚胎干细胞滋养层体外培养的最佳条件,利于人胚胎生殖嵴原始生殖细胞（PGC）的增殖。方法分离培养孕14.5 d（E14.5 d）和新生0 d（P0）昆明小鼠原代胚胎成纤维细胞（MEF）并传代制备滋养层,以60Coγ射线照射后继续培养并接种人PGC。采用活细胞拒染法、免疫细胞化学染色,对比观察滋养层的生长活性和照射后分泌功能;采用碱性磷酸酶（AKP）染色技术鉴定人PGCs的生长状态。结果 （1）E14.5 d和P0来源的第3～5代MEF生长旺盛。P0来源第6代与E14.5 d来源同代MEF相比深染变形细胞增达32%,经60Coγ照射后衰老细胞明显增多（P〈0.05）。（2）E14.5 d来源第3～6代和P0来源第3～4代MEF经60Coγ照射后培养5 d时,Ⅰ型胶原蛋白免疫阳性细胞数目较多,染色较深;但P0来源第5代其阳性细胞数目明显减少,染色变浅。（3）人PGCs接种于E14.5 d第3代制备的滋养层3～4 d后见典型的ESC小克隆团,细胞呈圆形;AKP染色呈深蓝色团;而接种于P0来源第6代MEF制备的滋养层5～6 d,可见ESC克隆松散、变大,周围边界不清,细胞紊乱。结论以60Coγ射线照射小鼠E14.5 d来源第3～6代MEF是制备滋养层的最佳来源,可维持人PGCs增殖。

Effects of different functional status of mouse embryonic fibroblast feeder layers on growth of embryonic stem cells from human genital ridge

Objective To screen the optimized conditions of feeder layers for the culture of human embryonic stem cells in vitro and to establish a high-performance method that is conducive to the proliferation of primordial germ cells（PGCs） from human genital ridge.Methods Kunming mouse embryonic fibroblasts（MEFs） isolated from the fetus at 14.5 d of gestation（E14.5 d） and newborns of 0 d（P0） were cultured and passed in vitro as feeder layers.After radiated by 60Co γ-ray,they were cultured continuously and vaccinated with human PGC.The growth activity and secretary function of MEF feeder layers before and after irradiation were observed comparatively by trypan blue staining and immunocytochemistry,respectively.The growth state of human PGCs was determined by alkaline phosphatase（AKP）.Results（1） Generation 3-5 MEFs from E14.5 d and P0 grew vigorously.The trachychromatic deformed cells of generation 6 from P0 increased by 32% compared with those from E14.5 d.Moreover,senescent cells from P0 significantly increased after 60Co γ-ray radiation compared with those from E 14.5 d（P0.05）.（2） Generation 3-6 MEFs from E14.5 d and generation 3-4 MEFs from P0 were relatively trachychromatic and more cells with the positive staining of typeⅠcollagen protein were seen on day 5 of culture after 60Co γ-ray radiation.Nevertheless,generation 5 MEFs from P0 showed paler staining and those with the positive staining of typeⅠcollagen protein decreased significantly.（3） Typical small clone groups of ESC with deep blue staining of AKP and round cells were seen after human PGCs were seeded in feeder layers prepared by generation 3 MEFs for 3-4 d.However,loose and bigger ESC clones with obscure boundary and disordered cells were seen after human PGCs were seeded in feeder layers prepared by generation 6 MEFs for 5-6 d.Conclusion Generation 3-6 MEFs from E14.5 d of mice receiving 60Co γ-ray radiation are the best sources for the preparation of feeder layers.They can maintain the proliferation of human PGCs.