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CyclinD1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、CyclinD1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CyclinD1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 explore the effects and the possible mechanism of combined injection of Angelica sinensis polysaccharide (ASP) and cytarabine (Ara-C) on the bone marrow mononuclear cells (BMMCs) of the transplanted human leukemia mouse model. Methods K562 cells (2×10⁷)were transplanted into the tail vein of mice to establish the transplanted human leukemia NOD/SCID mouse model. Then the leukemia mice were randomly divided into model, ASP, Ara-C, and ASP+Ara-C groups. After the 30 d transplantation, the mice were ip injected with ASP (200 mg/kg/d), Ara-C (2.5 mg/kg/d), and ASP (200 mg/kg/d)+Ara-C (2.5 mg/kg/d), respectively for 14 d, and the mice in the model group were injected with saline (equal volume and time). The next day after the treatment, the eyeball blood was collected to detect the amount and classification of white blood cells (WBC). The femurs were taken to count BMMCs of each femur. The proliferation of BMMCs was detected by CCK-8; The distribution of cell cycles was analyzed by flow cytometry (FCM); The capability of colony forming was examined by CFU-Mix cultivation; The ratio of the SA-β-Gal staining positive BMMCs was counted; The aging related proteins of P16, Rb, CDK4, and CyclinD1 were detected by Western blotting. Results Compared with the model group, ASP or Ara-C injected alone and their combined injection can obviously reduce the amount of the peripheral blood WBC, the percentage of neutrophiles, and the number of femur BMMCs; effectively inhibit the proliferation of BMMCs, CFU-Mix forming, and the ratio of S stages; markedly raise the percentage of lymphocytes, ratio of G₁ stages, and the percentage of SA-β-Gal positive cells; down-regulate the expression of the aging related proteins of CDK4 and CyclinD1; and up-regulate the expression of P16 and Rb protein. The effects of ASP+Ara-C group were much better than those in the other groups. Conclusion The aging mechanism of BMMCs for the transplanted human leukemia mice induced by ASP and Ara-C may be ascribed to the regulation of the expression of the aging related proteins of P16, Rb, CDK4, and CyclinD1. Our research provides a new idea to treat leukemia in clinic.

国家自然科学基金资助项目(81173398)