联合应用粒细胞集落刺激因子和干细胞因子促进骨髓单个核细胞增殖和动员的实验研究

Experimental Study on the Effects of Granulocyte Colony-stimulating Factor and Stem Cell Factor on Proliferation and Mobilization of Bone Marrow Mononuclear Cells

目的探讨联合应用粒细胞集落刺激因子(G-CSF)和干细胞因子(SCF)对小鼠骨髓单个核细胞(BM-MNCs)增殖和动员的影响。方法将昆明种小鼠随机分为4组(n=10):①G-CSF组,皮下注射重组鼠粒细胞集落刺激因子(rmG-CSF)100μg/(kg.d),连续使用5 d;②SCF组,皮下注射重组鼠干细胞因子(rmSCF)25μg/(kg.d),连续使用5d;③G-CSF/SCF组,皮下注射rmG-CSF 100μg/(kg.d)和rmSCF 25μg/(kg.d),连续使用5 d;④空白对照组,皮下注射等剂量的生理盐水。于最后一次注射后6 h取小鼠骨髓,分离培养MNCs,在倒置相差显微镜下观察MNCs的生长情况,用苏木精-伊红常规染色和细胞荧光免疫化学法染色观察MNCs形态。计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪检测MNCs细胞周期及表面抗原特征。结果G-CSF/SCF组和G-CSF组,CFU-F形成能力显著增强(均P<0.05);并且G-CSF/SCF组的CFU-F计数大于G-CSF组(P<0.05)。而SCF组CFU-F计数并没有显著地增加(P>0.05)。MNCs培养形成的CFU-F,其细胞表面抗原呈CD13+、CD29+、CD90+、CD105+,而不表达CD44。G-CSF/SCF组的MNCs,其G0/G1期细胞百分率较对照组低(P<0.05),而细胞增殖指数(PI)增高(P<0.05),SCF组对MNCs细胞周期的作用与对照组相比差异没有统计学意义(P>0.05)。结论G-CSF/SCF可有效地诱导BM-MNCs增殖和动员,且其作用强于单独应用G-CSF。而单独应用小剂量SCF对促进BM-MNCs增殖和动员的作用不明显。

Objective To investigate the effects of granulocyte colony-stimulating factor（G-CSF） combined with stem cell factor（SCF） on proliferation and mobilization of bone marrow mononuclear cells（MNCs） in mice.Methods Kunming mice were randomly divided into 4 groups（n=10 each）.The mice in the G-CSF group were subjected to subcutaneous injections of rmG-CSF at a dose of 100 μg/kg per day for 5 days;those in the SCF group to subcutaneous injections of rm-SCF at a dose of 25 μg/kg per day for 5 days;those in the G-CSF/SCF group to subcutaneous injections of G-CSF at a dose of 100 μg/kg per day and SCF at a dose of 25 μg/kg per day for 5 days;and those in the control group to the same volume of normal saline for 5 days.The bone marrow was obtained from the mice at the 6th h after the last injection.The MNCs were separated and cultured,and observed under an inverted phase contrast microscope.The morphology of the MNCs was observed by HE staining and immunocytochemical staining.The colony forming unit-fibroblast（CFU-F） was counted.The cell cycle of the MNCs and the surface antigens of the CFU-F were analyzed by flow cytometry.Results The G-CSF/SCF and G-CSF had an obvious effect on the expansion capability of CFU-F（P〈0.05）,and the number of CFU-F in the G-CSF/SCF group was more than that in the G-CSF group（P〈0.05）,but the number of CFU-F in SCF group was not significantly increased（P〉0.05）.Flow cytometric detection of MNCs surface markers showed that CFU-F was positive for CD13＋,CD29＋,CD90＋,CD105＋,but didn＇t express CD44.After mobilization with G-CSF/SCF,the percentage of BM-MNCs in G0/G1 phases was decreased as compared with the control group（P〈0.05）,while PI was increased（P〈0.05）.The influence of SCF on the cell cycle of MNCs was not different from that in control group（P〉0.05）.Conclusion This study confirmed that G-CSF/SCF effectively induced BM-MNCs proliferation and mobilizetion,and the effect of the G-CSF/SCF was greater than the G-CSF alone.There is no obvious effect on proliferation and mobilization of MNCs by using low dose of SCF alone.