细胞毒药物对小鼠肺组织和骨组织间充质干细胞增殖和分化的影响

Effects of cytotoxic agents on proliferation and differentiation of mesenchymal stem cells from mouse lung and compact bone

背景:肿瘤患者接受高剂量化疗时,细胞毒药物不仅对肿瘤细胞产生杀伤作用,对正常组织细胞甚至组织干细胞也产生一定损伤。目的:探讨细胞毒药物体外对小鼠肺组织间充质干细胞和骨组织间充质干细胞增殖及分化能力的影响。设计、时间及地点:细胞学体外对照观察,于2007-05/2008-08在解放军军事医学科学院基础医学研究所细胞生物学室和解放军北京军区总医院肿瘤科完成。材料:清洁级3～5周龄C57BL/6小鼠3只,由解放军军事医学科学院实验动物中心提供。方法:从胶原酶消化的小鼠肺组织和骨片中分离培养肺组织间充质干细胞和骨组织间充质干细胞,并在含体积分数为10%胎牛血清的α-MEM培养液中培养、传代、扩增。采用MTT法测定马利兰、阿霉素、环磷酰胺、足叶乙甙、甲氨蝶呤、长春新碱、顺铂对两种间充质干细胞生长增殖的影响,参照文献设置药物浓度范围,并设立空白对照,仅加入含体积分数为10%胎牛血清的α-MEM培养液。药物作用后,分别诱导两种间充质干细胞向脂肪和成骨细胞分化。主要观察指标:不同药物作用后,肺组织间充质干细胞和骨组织间充质干细胞的生长抑制、增殖情况及分化能力。结果:①在治疗剂量范围内,肺组织间充质干细胞和骨组织间充质干细胞对环磷酰胺、马利兰和甲氨蝶呤耐受,而对长春新碱、足叶乙甙、顺铂、阿霉素中度敏感。肺组织间充质干细胞和骨组织间充质干细胞对环磷酰胺、马利兰的敏感性相似,但小鼠肺组织间充质干细胞对甲氨蝶呤、长春新碱、足叶乙甙、阿霉素的敏感性均低于骨组织间充质干细胞,对顺铂的敏感性高于骨组织间充质干细胞。②与空白对照比较,环磷酰胺、马利兰和甲氨蝶呤作用后对两种间充质干细胞的增殖能力影响较小。③在环磷酰胺、马利兰、甲氨蝶呤、长春新碱和足叶乙甙等药物作用24h内,两种间充质干细胞仍能被诱导分化为成骨细胞和脂肪细胞。药物作用72h后,两种间充质干细胞向脂肪和成骨细胞的分化能力减弱甚至消失。环磷酰胺、马利兰和甲氨蝶呤对肺组织间充质干细胞和骨组织间充质干细胞的分化功能影响相对较小。结论:细胞毒药物对小鼠肺组织间充质干细胞和骨组织间充质干细胞具有毒性作用,但不同组织器官来源的间充质干细胞对细胞毒药物的敏感性存在一定差异;药物作用后可影响两种间充质干细胞的增殖和分化能力。

BACKGROUND： During high-dose chemotherapy, cytotoxic agents have killing effects on tumor cells, and can damage normal cells, even stem cells, in tumor patients. OBJECTIVE： To study the influence of cytotoxic agents on proliferation and differentiation of mesenchymal stern cells （MSCs） derived from mice lung and compact bone. DESIGN, TIME AND SETTING： In vitro controlled cytological study was performed at the Room of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA, and the Department of Oncology, General Hospital of Beijing Military Area Command of Chinese PLA from May 2007 to August 2008. MATERIALS： A total of 3 clean C57BL/6 mice aged 3-5 weeks were obtained from the Experimental Animal Center of Academy of Military Medical Sciences of Chinese PLA. METHODS： Adherent cells from digested lung and compact bone were cultured and expanded in 10% fetal bovine serum/α-MEM These cells were identified as mesenchymal stem cells, and then examined in vitro using MTT assay to find out their sensitivity to cytotoxic agents, comprising myleran, addamycin, cyclophosphamide, etoposide, methotrexate, vincristine and cis-diamminedichloroplatinum. Drug concentration range was set according to a reference. Cells in the blank control group were administered α-MEM containing 10% fetal bovine serum. After exposure to cytotoxic agents, the cells were induced to differentiate into adipocytes and osteoblasts. MAIN OUTCOME MEASURES： Growth inhibition, proliferation and differentiation of lung and compact bone-derived MSCs were measured following treatment of various drugs. RESULTS： Lung-and compact bone-derived MSCs were resistant to busulphan, cyclophosphamide and methotrexate and mildly sensitive to vincristine, etoposide, cis-diamminedichloroplatinum and adriamycin. The sensitivity to cyclophosphamide and myleran was similar between lung and compact bone-derived MSCs. However, the sensitivity to methotrexate, vincristine, etoposide and adriamycin was less in lung-derived MSCs compared with the compact bone-derived MSCs in mice. The sensitivity to cis-diamminedichloroplatinum was greater in lung-derived MSCs compared with the compact bone-derived MSCs in mice. Compared with blank control group, treatment of cyclophosphamide, myleran and methotrexate had small effects on proliferation of lung-and compact bone-derived MSCs. Within 24 hours of treatment of cyclophosphamide, myleran, methotrexate, vincristine and etoposide, lung- and compact bone-derived MSCs also could differentiate into adipocytes and osteoblasts. Following 72 hours of treatment, the differential ability of lung-and compact bone-derived MSCs weakened, even disappeared. Cyclophosphamide, myleran and methotrexate had small effects on the differential ability of lung- and compact bone-derived MSCs. CONCLUSION： Cytotoxic agents could damage the MSCs in lung and compact bone. MSC from lung and compact bone had different sensitivity to the same cytotoxic agent. The cytotoxic agents influence the proliferation and differentiation of MSCs from lung and compact bone.