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目的探讨当归多糖(ASP)与阿糖胞苷(Ara-C)联合注射对移植性人白血病模型小鼠骨髓单个核细胞(BMMCs)的影响,并探讨其调控白血病细胞衰老的可能机制。方法每只小鼠尾静脉移植2×107个K562细胞,建立移植性人白血病NOD/SCID小鼠模型,模型建立后随机分为模型组、ASP组、Ara-C组和ASP+Ara-C组,移植K562细胞第31天开始分别ip ASP(200 mg/kg)、Ara-C(2.5 mg/kg)和ASP(200 mg/kg)+Ara-C(2.5 mg/kg)治疗,共14 d,模型组注射等量生理盐水。药物注射完成第2天眼球取血检测外周血白细胞总数与分类计数,取股骨测定每只股骨BMMCs细胞数;CCK8测定BMMCs增殖能力,流式细胞术分析BMMCs增殖周期,混合集落(CFU-Mix)培养检测BMMCs形成集落能力;衰老β半乳糖苷酶(SA-β-Gal)染色观察衰老细胞百分率;Western blotting检测衰老相关蛋白P16、Rb、CDK4及Cyclin D1表达。结果与模型组比较,无论是ASP、Ara-C单独注射或ASP+Ara-C联合用药均能有效降低白血病模型小鼠外周血白细胞总数,降低中性粒细胞百分率,提高淋巴细胞百分率;降低股骨BMMCs细胞数,明显抑制BMMCs增殖,降低CFU-Mix产率,提高G1期细胞比例,减少S期细胞比例;显著提高SA-β-Gal染色阳性细胞百分率;上调P16、Rb表达,下调CDK4、Cyclin D1表达,且联合用药组效果更为明显。结论 ASP与Ara-C可能通过调节衰老相关蛋白P16、Rb、CDK4及Cyclin D1表达,进而促进移植小鼠白血病细胞衰老,为临床治疗白血病提供了新思路。
Objective To investigate the effect of combination of Angelica sinensis polysaccharide (ASP) and cytarabine (Ara-C) on bone marrow mononuclear cells (BMMCs) of mice with human leukemia and to explore its possible mechanism of regulating leukemia cell senescence. Methods 2 × 107 K562 cells were transplanted into the caudal vein of each mouse to establish a mouse model of NOD / SCID of human leukemia. The models were randomly divided into model group, ASP group, Ara-C group and ASP + Ara-C group The K562 cells were treated with ip ASP (200 mg / kg), Ara-C (2.5 mg / kg) and ASP (200 mg / kg) The model group was injected with normal saline. The total number of leukocytes in peripheral blood and the number of the differentiated cells were measured by blood sampling on the second day after drug injection. The number of BMMCs in each femur was determined by femur. The proliferation of BMMCs was measured by CCK8. The proliferation cycle, CFU-Mix, The colony formation ability of BMMCs was detected by flow cytometry. The percentage of senescent cells was observed by aging β-galactosidase (SA-β-Gal) staining and the expressions of P16, Rb, CDK4 and Cyclin D1 were detected by Western blotting. Results Compared with the model group, both ASP and Ara-C alone or combined with ASP + Ara-C could effectively reduce the leukocyte count, decrease the percentage of neutrophils and increase the percentage of lymphocytes in leukemia mice, The number of BMMCs significantly inhibited the proliferation of BMMCs, decreased the production of CFU-Mix, increased the percentage of cells in G1 phase and decreased the percentage of cells in S phase, significantly increased the percentage of SA-β-Gal-positive cells, up-regulated the expression of P16 and Rb, D1 expression, and combined treatment group effect is more obvious. Conclusion ASP and Ara-C may promote the senescence of leukemic cells in mice by regulating the expression of aging-related proteins P16, Rb, CDK4 and Cyclin D1, providing a new idea for the clinical treatment of leukemia.