补肾生血药干预不同时间对环磷酰胺所致骨髓抑制小鼠细胞周期与细胞增殖的影响

目的观察补肾生血药干预不同时间对环磷酰胺诱发骨髓抑制小鼠骨髓细胞周期及细胞增殖的影响。方法52只雄性BALB/c小鼠随机分为空白对照组、模型对照组、阳性对照组、补肾生血药低剂量组、补肾生血药高剂量组。先预防用药3 d,补肾生血药低剂量组、高剂量组灌胃不同剂量补肾生血药,其余组灌胃蒸馏水。第4~6天造模,除空白对照组腹腔注射生理盐水外,其余组均腹腔注射环磷酰胺。造模同时进行干预,阳性对照组皮下注射重组人粒细胞刺激因子,其余组给药同预防用药,分别持续到造模后第10天、第12天、第14天。收集小鼠骨髓细胞,流式细胞仪检测10 d、12 d、14 d骨髓细胞周期,12 d、14 d骨髓细胞CD34+率;造血祖细胞培养法观察10 d、14 d红细胞集落形成单位(CFU-E)、爆式红细胞集落形成单位(BFU-E)、粒细胞-巨噬细胞集落形成单位(CFU-GM)数量。结果细胞周期分析,10 d时造模各组明显阻滞在G₀/G₁期、S期、G₂/M期(P<0.01),补肾生血药低剂量组则明显阻滞在G₀/G₁期、G₂/M期(P<0.05,P<0.01);12 d时细胞周期阻滞解除,各组间比较差异无统计学意义(P>0.05);14 d时补肾生血药高剂量组明显由G₀/G₁期进入到G₂/M期(P<0.05)。CD34+率,12 d时各组间比较差异无统计学意义(P>0.05),14 d时补肾生血药高剂量组明显升高(P<0.01)。集落计数,10 d时补肾生血药高剂量组BFU-E数量明显增加(P<0.01);14 d时补肾生血药高剂量组CFU-E、BFU-E数量明显增加(P<0.01),补肾生血药低剂量组CFU-E、BFU-E、CFU-GM数量明显增加(P<0.001,P<0.01)。结论对于环磷酰胺所致骨髓抑制小鼠,应用补肾生血药干预14 d时能发挥较明显的促骨髓细胞增殖作用,表现为促进骨髓细胞进入增殖周期,刺激红系、粒-巨系造血祖细胞增殖,提升造血干/祖细胞比例。

Effects of Bushen Shengxue Intervention at Different Times on Cell Cycle and Cell Proliferation in Mice with Bone Marrow Suppression Induced by Cyclophosphamide

Objective To clarify the effects of Bushen Shengxue intervention at different times on cell cycle and cell proliferation in mice with bone marrow suppression induced by cyclophosphamide.Methods 52 male BALB/c mice were randomly divided into 5 groups:blank control group,model control group,positive control group,high-dose and low-dose Bushen Shengxue intervention group.Prevention medication for 3d,high-dose and low-dose Bushen Shengxue intervention group were given different doses of Bushen Shengxue intervention,the other groups were given distilled water.4-6d except for blank control group,mice in other groups were injected with cyclophosphamide(100mg/kg.d).Intervention during and after modeling,positive control group were given recombinant human granulocyte colony-stimulating factor(30μg/kg.d),the other groups were the same as the preventive medication.It was continued until the 10th day,12th day and 14th day after modeling.Bone marrow cells were collected,the cell cycle at 10 d,12 d,14 d and the CD34+rate at 12 d,14 d were detected by flow cytometry.CFU-E,BFU-E,and CFU-GM were observed at 10 d,14 d by hematopoietic progenitor cell culture.Results Cell cycle analysis,at 10 d the model groups were significantly blocked in G₀/G₁,S and G₂/M phases(P<0.01),and the Bushen Shengxue intervention low-dose group was significantly blocked in G₀/G₁and G₂/M phases(P<0.05,P<0.01);The cell cycle arrest was lifted at 12 d,and there was no significant difference betweenthe groups(P>0.05);At 14 d the Bushen Shengxue intervention high-dose group significantly changed from G₀/G₁to G₂/M(P<0.05).The CD34+rate was not significantly different between the groups at 12 d(P>0.05),and the Bushen Shengxue intervention high-dose group was significantly increased at 14 d(P<0.01).Colony counting,BFU-E increased significantly in the high-dose Bushen Shengxue intervention group at 10 d(P<0.01);CFU-E and BFU-E increased significantly in the high-dose Bushen Shengxue intervention group(P<0.01),and CFU-E,BFU-E,CFU-GM were significantly increased in the low-dose Bushen Shengxue intervention group(P<0.001,P<0.01)at 14 d.Conclusion For cyclophosphamide-induced bone marrow suppression mice,application of Bushen Shengxue intervention on 14 d can play a more obvious role in promoting bone marrow cell proliferation.It appears to promote bone marrow cells into the proliferation cycle,stimulate the proliferation of erythroid,granulosa-megaloblastic progenitor cells,and increase the ratio of hematopoietic stem/progenitor cells.