基因重组白细胞介素3刺激胎肝巨核细胞祖细胞生长的特性

IN VITRO GROWTH CHARACTERISTICS OF rIL3 STIMULATED MEGAKARYOCYTIC PROGENTOR CELLS (CFU-MK) OF FETAL LIVER

本文采用血浆凝块、甲基纤维素半固体和液体培养体系结合流式细胞仪巨核细胞DNA双荧光分析，观察了4～5个月胎肝和成人骨髓CFU－MK在体外的增殖和分化特点。结果表明，无论以基因重组白细胞介素3（Interleukin－3，rIL3）或再障狗血清（Meg－CSA）作为刺激因子，股肝CFU－MK集落均较大（大于50个细胞／集落），而成人骨髓CFU－MK集落均较小（小于50个细胞／集落）。在以rIL3（2ng／ml）为刺激因子时，胎肝巨核细胞祖细胞（CFU－MK）集落产率（36.40±16．60）高于成人骨髓（10．05±2．81）（P＜0．01）。与此相反，胎肝CFU－MK源巨核细胞DNA倍性分布以2N和4N为主，成人骨髓则以8N和4N为主，前者的平均倍性值（5．46±0．86）明显低于后者（10．13±1．30）（P＜0．01）。同样，在以Meg－CSA为刺激因子时，也获得了类似的结果，提示胎肝CFU－MK具有很强的增殖能力和较低的分化（倍体化）能力。另外，胎肝CFU－MK集落产率在rIL3浓度为0．5～2ng／ml时逐渐上升，而在2～8ng／ml则持续下降。但在相同浓度范围内，rIL3对成人骨髓CFU－MK和胎肝CFU－GM的作用均无这种双向效应。Meg－CSA（5％～25％）对股肝、成人骨髓CFU－MK和胎肝CFU－GM的作用也呈明显的剂量效应曲线。而且，胎肝CFU－MK源巨核细胞DNA倍性?

In this study, plasma clot and methycellulose semi-solid and liquid culture technigues were employed to observe the in nitro growth characteristics of proliferation and differetiation of 4~5 months fetal liver and adult bone marrow CFU-MKs. Fetal liver MK colonies in plasma clot in the presence of rIL3 or Meg-CSA were larger (usually with >50 cells/colony) than that of the adult (usually with <50 cells/colony). The number (36. 40±16. 60/1×105 cells) of fetal liver Mk colonies in presence of 2 ng/ml rIL3 was larger than that (10. 05±2.81 /1 × 105 cells) of human adult bone marrow MK colonies (P<0. 01). In contrast, the major DNA ploidy classes of megakaryocytes derived from fetal liver CFU-MK were those of 2N and 4N cells and the major DNA ploidy classes of megakaryocytes derived from human adult bone marrow were those of 8N and 4N cells. The mean (5. 45±0. 86) of the ploidy of the former was lower than that (10. 13±1. 30) of the latter (P<0. 01 ). The same results were obtained with the presence of 10% Meg-CSA. These present results indicated that CFU-MK in fetal liver has a high ability of proliferation and low capacity of differentiation (polyploidization). Interestingly, the number of fetal liver MK colonies increased in the range of rIL3 concentration from 0. 5 ng/ml to 2 ng/ml. At higher rIL3 concentration (2~8 ng/ml), the colony growth showed a steady decrease from the maximum value instead of an increase. However, in the same range of rIL3 concentraion, the numbers of adult bone marrow MK colonies numbers and fetal liver CFU-GM colonies increased steadily and finally reached to a plateau. Furthermore, both fetal liver and adult bone marrow MK colonies showed dose-dependent response in the range of MegCSA concentration from 5% to 25%. In addition, there was no difference on DNA ploidy distributions of megakaryocytes derived from fetal liver CFU-MK between rIL3 and Meg-CSA as growth factors. Moreover, the DNA ploidy distribution of fetal liver derived megakaryocytes stimulited by rIL3 could not be changed by addition of rIL6 (100u/ml). In summary, the above data suggest that CFU-MK in the fetal liver undergoes some intrinsic cellular modification in order to suit the need of ontogensis.