健脾补血方改善环磷酰胺诱导的小鼠骨髓及免疫抑制的机制研究

Mechanism of Jianpi Buxue Decoction on Myelosuppression and Immunosuppression Induced by Cyclophosphamide in Mice

目的观察健脾补血方对环磷酰胺诱导的小鼠外周血细胞变化、血浆因子浓度及造血干细胞表型的影响,同时观察其对外周血单个核细胞(PBMC)和胸腺中CD3和CD8a表达的影响,探讨健脾补血方对小鼠骨髓及免疫抑制的改善作用。方法将C57BL/6小鼠正常饲养7 d,分为正常对照组、模型组、阳性药物组和高(2.5 g·mL-1)、中(1.25 g·mL-1)、低(0.62 g·mL-1)剂量中药组。模型组、阳性药物组与中药组腹腔注射环磷酰胺,25 mg·kg-1·d-1,给药10 d。阳性药物组皮下注射粒-巨噬细胞集落刺激因子(GM-CSF),5μg·kg-1·d-1,中药组给予中药汤剂灌胃,给药12 d,分别取外周血及骨髓细胞,检测外周血细胞、血浆因子浓度及造血干细胞表型的变化,外周血单个核细胞和胸腺采用流式细胞术和蛋白质印迹(Western Blot)检测CD3和CD8a的表达。结果与正常对照组比较,模型组的白细胞数量、血浆中GM-CSF、促血小板生成素(TPO)、促红细胞生成素(EPO)的浓度、骨髓细胞中造血干细胞表型Sca1和C-kit、外周血单个核细胞和胸腺组织中CD3和CD8a明显减少(P<0.01,P<0.05)。与模型组比较,阳性药物组和中药组均能显著提高白细胞数量及血浆因子浓度(P<0.01);中药组可升高Scal和C-kit表达(P <0.01)、外周血单个核细胞和胸腺组织中CD3和CD8a的表达(P<0.01,P<0.05)。结论健脾补血方对环磷酰胺诱导小鼠的外周血细胞、血浆中造血因子浓度及骨髓细胞中造血干细胞表面分子的表达均有一定的调节作用,并且能上调外周血单个核细胞和胸腺中CD3和CD8a表达,提示健脾补血方对环磷酰胺诱导的骨髓及免疫抑制具有改善作用。

Objective To observe the effect of Jianpi Buxue decoction on the changes of peripheral blood cells,plasma factors and the phenotype of bone marrow cells induced by cyclophosphamide in mice,and to observe the influence on CD3 and CD8 a expression in the peripheral blood mononuclear cells(PBMC)and thymus,to explore the effect of Jianpi Buxue decoction on bone marrow and immunity improvement.Methods C57 BL/6 mice were fed for 7 days.They were divided into normal control group,model group,positive drug group and high,medium and low dose Chinese medicine groups.Mice in the model group,the positive drug group and the traditional Chinese medicine groups were intraperitoneal injected with cyclophosphamide for 10 days;the positive drug group was subcutaneously injected with GM-CSF,and the drugs were given 12 days.The peripheral blood and bone marrow cells were taken to detect the amount of peripheral blood cells,concentrations of plasma factors and the surface molecules of hematopoietic cells.Flow cytometry and Western Blot were used to detect the expression of CD3 and CD8 a in peripheral blood mononuclear cells and thymus.Results There were significant differences in blood cells,plasma factor concentration,surface markers of bone marrow cells,expression of CD3 and CD8 a.Compared with the normal control group,the number of white blood cells,concentration of GM-CSF,TPO and EPO in the plasma,Sca1,C-kit,expression of CD3 and CD8 a in the model group was significantly reduced(P<0.01,P0.05).Compared with the model group,the number of white blood cells was significantly increased in the positive drug group and the Chinese medicine groups(P<0.01).The concentration of GM-CSF,TPO and EPO in the plasma of the positive drug group and the Chinese medicine groups were significantly improved(P<0.01).The expression of CD3 and CD8 a in the peripheral blood mononuclear cells and thymus tissues of the Chinese medicine group increased significantly(P<0.01,P<0.05).Conclusion The Jianpi Buxue decoction can regulate the concentration of hematopoiesis factor in peripheral blood cells,plasma and the expression of hematopoietic stem cell surface markers in bone marrow cells,and upregulate the expression of CD3 and CD8 a in peripheral blood mononuclear cells and thymus,suggesting that the prescription can improve myelosuppression and immunosuppression induced by cyclophosphamide.