Damage of chemotherapy agents to bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.50.008

Abstract

Abstract:

BACKGROUND: Many chemotherapy drugs can produce adverse reactions, such as bone marrow suppression, when they kill tumor cells. It cannot only damage hematopoietic cells, but also damage bone marrow mesenchymal stem cells. To understand the role of various chemotherapy agents on bone marrow mesenchymal stem cells is conductive to rational choice of chemotherapy drugs as well as achievement of high-efficiency, low-toxicity, clinical efficacy.
OBJECTIVE: To investigate the changes in proliferation, differentiation and hematopoiesis support ability of bone marrow mesenchymal stem cells after exposure to chemotherapy agents.
METHODS: Mesenchymal stem cells were isolated from normal adult bone marrow and cultured in media with different chemotherapy agents (cyclophosphamide, busulfan, cytarabine, daunorubicin, vincristine, etoposide, methotrexate, dexamethasone). After exposure to chemotherapeutic agents, the changes of cell proliferation,
apoptosis and recovery ability were detected. In vitro drug-treated bone marrow mesenchymal stem cells differentiated into adipocytes and osteocytes, identified by oil red O and Von Kossa staining. RT-PCR method was used to detect the expression of hematopoietic cytokines in bone marrow mesenchymal stem cells after drug treatment. Colony formation assay was used to detect the hematopoiesis support ability of bone marrow mesenchymal stem cells after drug treatment.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells were resistant to three agents: cyclophosphamide, busulfan and methotrexate, and other agents could induce apoptosis of bone marrow mesenchymal stem cells and reduce the proliferation of bone marrow mesenchymal stem cells. However, bone marrow mesenchymal stem cells recovered after withdrawal of vincristine and dexamethasone. Bone marrow mesenchymal stem cells, after exposure to chemotherapy agents, still had the ability of multi-directional differentiation, which could differentiate into adipocytes and osteoblasts in vitro. Moreover, chemotherapy agent-treated bone marrow mesenchymal stem cells could express hematopoietic cytokines (stem cell factor, monocyte colony stimulating factor, interleukin-6 and granulocyte colony-stimulating factor) and possessed hematopoietic supportive ability. But, after exposure to daunorubicin, vincristine, etoposide, and cytarabine, bone marrow mesenchymal stem cells showed limited proliferation capacity and impaired haematopoiesis support ability. These findings demonstrate that chemotherapy agents commonly used clinically (daunorubicin, vincristine, etoposide, and cytarabine) can impact the proliferation and hematopoiesis supportabilities of bone marrow mesenchymal stem cells, but these agents cannot affect the multi-directional differentiation ability and expression of hematopoietic cytokines of bone marrow mesenchymal stem cells. Moreover, several chemotherapy agents have no effects on biological characteristics of bone marrow mesenchymal stem cells, such as cyclophosphamide, busulfan and methotrexate.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, drug therapy, combination, transplantation conditioning

CLC Number:

R392.4

Wang Jin-huan, Wang Zhen-ling, Wang Xiao-fang, Zhao Zhi-gang. Damage of chemotherapy agents to bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(50): 8080-8087.

Figures/Tables 3

2.1 化疗药物对骨髓间充质干细胞增殖的影响为了检测化疗药物对骨髓间充质干细胞的影响，不同剂量(常规剂量组，大剂量组)、不同类型化疗药物(环磷酰胺、阿糖胞苷、甲氨蝶呤、足叶乙甙、地塞米松、长春新碱、柔红霉素、马利兰)加入骨髓间充质干细胞中，处理3 d后检测骨髓间充质干细胞的增殖情况。发现任何浓度的环磷酰胺、甲氨蝶呤、马利兰和常规剂量的地塞米松不影响骨髓间充质干细胞的增殖；而常规剂量和大剂量长春新碱、足叶乙甙、柔红霉素、阿糖胞苷和大剂量地塞米松均不同程度抑制骨髓间充质干细胞的增殖，见图1。 2.2 化疗药物作用后骨髓间充质干细胞的恢复如图2所示，柔红霉素、足叶乙甙和阿糖胞苷持续性抑制骨髓间充质干细胞的增殖。当柔红霉素、足叶乙甙和阿糖胞苷撤除后，继续培养9 d，骨髓间充质干细胞仍处于增殖受抑状态。而长春新碱、地塞米松对骨髓间充质干细胞的增殖抑制作用随着药物的撤除有所恢复，但是不能恢复到正常。环磷酰胺、甲氨蝶呤和马利兰作用后骨髓间充质干细胞的早期(3 d)和晚期(12 d)的增殖能力并不发生改变。 2.3 化疗药物诱导骨髓间充质干细胞凋亡 5种检测的化疗药物(阿糖胞苷、柔红霉素、长春新碱、足叶乙甙、地塞米松)均可以诱导骨髓间充质干细胞凋亡。不同类型、不同剂量的化疗药物诱导骨髓间充质干细胞凋亡的程度存在差异。大剂量组化疗药物对骨髓间充质干细胞的凋亡作用要明显强于常规剂量组(P < 0.05)，不同类型化疗药物诱导骨髓间充质干细胞凋亡的作用存在差异，其中大剂量组柔红霉素诱导细胞凋亡的能力最强，而地塞米松诱导凋亡的作用最弱；此外，不同类型、不同剂量化疗药物对骨髓间充质干细胞的凋亡率与处理时间成正相关。随着处理时间的延长，细胞凋亡率上升，见图3。 2.4 化疗药物处理后骨髓间充质干细胞的分化能力化疗药物处理后和未处理骨髓间充质干细胞都能在体外向脂肪和成骨诱导分化。成脂肪诱导1周后看见大部分细胞胞体变大，胞浆出现大量脂肪滴，油红O染色证实为脂肪滴，RT-PCR证实诱导后细胞表达脂肪形成特异性基因脂蛋白脂酶。成骨诱导3周后细胞呈现多层生长的结节状，Von kossa 染色证实为钙盐沉积，RT-PCR证实上述被诱导的细胞表达骨形成特异性基因Osteocalcin(图4)。化疗药物处理前后的骨髓间充质干细胞具有相似的体外分化能力。 2.5 造血相关因子的表达应用RT-PCR方法检测化疗药物处理前后骨髓间充质干细胞中造血相关因子的表达，结果显示干细胞因子(SCF)，单核细胞集落刺激因子(M-CSF)，白细胞介素6(IL-6)和粒细胞集落刺激因子(G-CSF)在化疗药物处理前后的骨髓间充质干细胞中稳定表达，但是没有检测到粒、单核细胞集落刺激因子，白细胞介素3(IL-3)和血小板生长因子(TPO)的表达(图5)。2.6 体外支持造血能力为了明确化疗药物是否影响骨髓间充质干细胞支持造血的能力，将不同类型、不同剂量处理后的骨髓间充质干细胞照射后，将骨髓单个核细胞接种其中，在长期骨髓培养基中培养4周后检测CFU生成情况。各种化疗药物均不同程度影响骨髓间充质干细胞支持造血的能力，见图6。"

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