摘要
背景：目前细胞因子支持的脐血体外扩增是安全性较高、最有可能运用于临床的方法之一。目的：比较白血病抑制因子、白细胞介素6及脂多糖对脐血干／祖细胞体外增殖的影响。设计、时间及地点：细胞学体外对照实验，于2003-08／2004—05在广东医学院附属医院中心实验室完成。材料：10例正常脐血标本取自广东医学院附属医院妇产科及湛江市妇幼保健院妇产科，自血病抑制因子为达科为试剂公司产品，白细胞介素6为晶美试剂公司产品，脂多糖由广东医学院附属医院李延平教授自制。方法：密度梯度法体外分离培养脐血单个核细胞，应用磁分选器和单克隆抗体免疫磁珠（阳性）分选法提取脐血CD34＋细胞。建立液体培养体系，为含体积分数为40％的胎牛血清、20g／L牛血清白蛋白、20μg／L粒巨集落刺激因子、20ug／L干细胞因子、20μg／L自细胞介素3的RPMI1640培养液。取2×10^8L^-1脐血CD34＋细胞，加入配制的液体培养体系50μL，设立8组：第1组仅加入RPMI1640作为对照，第2-4组分别单独加入脂多糖、白血病抑制因子、白细胞介素6，第5—7组分别加入脂多糖、白血病抑制因子、白细胞介素6的两两混合液，第8组加入3种刺激因子的混合液，上述各组脂多糖、白血病抑制因子、白细胞介素6的终浓度分别为10u，mL，10μg／L，10mg／L。主要观察指标：MTT法测定不同刺激因子在液体培养体系中对脐血CD34＋细胞增殖的影响。结果：培养3d后与对照组比较，单用白血病抑制因子对脐血CD34＋细胞具有促增殖作用（F=3．33，P〈0．01），单用脂多糖或白细胞介素6的效果均不显著（F=-2．14，183，P〉0．05）；刺激因子两两联用及3种刺激因子联用时，对脐血CD34＋细胞均具有促增殖作用（F=3．54-4．06，P均〈0．01），且3种刺激因子联用时的促增殖效果尤为显著。结论：在特定的液体培养体系中，单独使用白血病抑制因子可明显促进脐血干，祖细胞体外增殖，联合情况下白细胞介素6、脂多糖与白血病抑制因子具有促增殖协同作用。
BACKGROUND： Cord blood in vitro amplification supported by cytokines may be more safe and easier to be used in clinic.
OBJECTIVE： To compare the effects of leukaemia inhibitory factor, interleukin-6 and lipopolysacchadde on in vitro proliferation of cord blood hematopoietic stem/progenitor ceils.
DESlNG, TIME AND SETTING： The cytological in vitro controlled study was performed at the Central Laboratory, Hospital Affiliated to Guangdong Medical College from August 2003 to May 2004.
MATERIALS： Ten normal cord blood samples were obtained from the Department of Gynaecology and Obstetrics, Hospital Affiliated to Guangdong Medical College, and Department of Gynaecology and Obstetrics, Zhanjiang Health Center for Women and Children. Leukaemia inhibitor factor （Dake, China）, interleukin-6 （Jingmei, China）, and lipopolysaccharide （made by Professor Li Yan-ping） were used for this study.
METHODS： Cord blood mononuclear ceils were in vitro isolated by the density gradient method. Cord blood CD34＋ cells were extracted by the magnetic sorting machine and monoclonal antibody immunomagnetic beads （positive） sorting. Liquid culture system was created, which was RPMI 1640 medium containing 40% fetal bovine serum, 20 g/L bovine serum albumin, 20 μg/L granulocyte-macrophage colony-stimulating factor, 20μg/L stem cell factor, and 20μg/L interleukin-3. Purified CD34＋ cells （2×10^8/L） were incubated in 50μL above-mentioned liquid medium. There were 8 groups. Cells in the group 1 were treated with RPMI 1640 medium as controls. Cells in the groups 2-4 were respectively treated with lipopolysaccharide, leukaemia inhibitory factor, and interleukin-6. Cells in the groups 5 7 were subjected to pairwise mixed liquor of lipopolysaccharide, ieukaemia inhibitory factor, and interleukin-6. Cells in the group 8 were given the mixed liquor of lipopolysaccharide, leukaemia inhibitory factor and interleukin-6. The final concentrations of lipopolysaccharide, teukaemia inhibitory factor and interleukin-6 were respectively 10 U/mL, 10 μ g/L and 10 mg/L.
MAIN OUTCOME MEASURES： MTT assay was used to detect effects of various stimulating factors on cord blood CD34＋ cell proliferation in liquid medium.
RESULTS： Compared with controls, at 3 days leukaemia inhibitory factor alone could promote proliferation of cord blood CD34＋ cells （F=3.33, P 〈 0.01 ）, but the effects of lipopotysaccharide or interleukin-6 alone were not significant （F=2.14, 1.83, P〈0.05）. Peirwise combination of stimulating factors and combination of three stimulating factors could accelerate proliferation of cord blood CD34＋ cells （F=3.54-4.06, P 〈 0.01 ）. The effects of combination of three stimulating factors were the most significant.
CONCLUSION： Under a special liquid medium, leukaemia inhibitory factor alone significantly enhances proliferation of cord blood hematopoietic stem/progenitor cells in vitro. Under combination conditions, interleukin-6, lipopolysaccharide and leukaemia inhibitory factor have synergistic effects on promoting proliferation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第23期4471-4474,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
