人胚骨髓基质细胞饲养层对人胚胎生殖细胞生长的作用

Effect of Human Embryonic Primary Bone Marrow Stromal Cell Feeder Layer on Culture of Human Embryonic Stem Cells

目的:研究人胚原代骨髓基质细胞(BMSC)饲养层对人胚胎生殖细胞(hEGC)生长的支持作用。方法:以非意愿妊娠流产胎儿股骨为材料制备BMSC饲养层;同时取胎儿组织分离培养人成纤维细胞,以成纤维细胞培养液上清制备条件培养液(CM)。另取13.5d胎鼠制备原代小鼠胎儿成纤维细胞(PMEF)饲养层。分组培养人原始生殖细胞:A组(PMEF饲养层)、B组(BMSC饲养层)、C组(CM)、D组(BMSC饲养层+CM),观察不同条件下hEGC生长增殖行为。结果:D组与A组均有利于支持人胚胎生殖细胞的体外生长,并保持其不分化的状态和多分化的潜能,两组比较无明显差异。单用B组可支持人胚胎生殖细胞的体外生长,但效率低于上述两组。单用条件培养液不能获得贴壁生殖细胞克隆。结论:BMSC饲养层+CM可支持hEGC的体外培养,且无异源性蛋白的污染,更有利于临床应用。

Objective: To study the effect of human embryonic primary bone marrow stromal cell (BMSC) feeder layer on culture of human embryonic germ cells (hEGC), replace primary mouse empryofibroblast (PMEF) feeder layer. Methods: Human fetus bone marrow prepared from thighbones was cultured to establish BMSC feeder layer. At the same time, fibroblast cells were separated from the same fetus, culture medium were used as conditioned medium(CM). Some 13.5-day-old mouse embryos were used to establish PMEF feeder layer. Four groups, i.e. PMEF feeder layer, BMSC feeder layer, CM, BMSC feeder layer+CM, were established. The growth characters of embryonic germ cells in different conditions were analyzed. Results: The results showed human embryonic primary BMSC feeder layer, under the collaboration of CM, play the same role with PMEF in proliferation and maintaining undifferentiation state of hEGC in vitro. Without the help of CM, the proliferate efficiencydecreased. CM itself can't keep hEGC to proliferate. Conclusion: Human embryonic primary bone marrow stromal cell feeder layer, under the collaboration of CM, can support hEGC expansion without the infection of the heterogeneous protein, therefore it is adapted to clinical applications.