阿米福汀在非霍奇金淋巴瘤患者骨髓间充质干细胞体外化疗损伤中的保护作用

Role of amifostine against the effect of etoposide on non-Hodgkin lymphoma bone marrow derived mesenchymal stem cells:an in vitro experiment

目的研究阿米福汀(WR-2721)在保护非霍奇金淋巴瘤(NHL)来源的骨髓间充质干细胞(MSC)免受化疗药物依托泊甙(VP-16)体外损伤中的作用,为临床细胞移植治疗提供依据。方法获取 NHL 患者的骨髓 MSC,应用极限稀释法获取单克隆来源的 MSC,并在低血清培养液中培养和扩增,后按处理因素分为空白对照组(A)、WR-2721组(B)、WR-2721+VP-16组(c)和 VP-16组(D),分别计数细胞绘制生长曲线,流式细胞仪检测其免疫表型及凋亡,甲基纤维素半固体培养、MSC作为滋养层检测其支持造血能力,油红 O 染色和 Von Kossa 染色检测 MSC 向脂肪和骨的分化。结果各组因素对 MSC 免疫表型无影响。(B)组 MSC 的增殖、凋亡和支持造血能力较空白对照组差异无统计学意义(均 P>0.05);(c)组 MSC 的增殖和凋亡较(D)组差异有统计学意义(均 P<0.05),其支持造血能力均明显高于(D)组(P<0.05)。各组 MSC 均能被诱导分化为骨和脂肪。结论 WR-2721能显著降低 MSC 遭受化疗药物 VP-16的体外损伤,其对 NHL 骨髓来源 MSC 体外的增殖、凋亡、免疫表型、造血支持能力和分化能力无明显影响。

Objective To study the protective role of amifostine （ WR-2721 ） in the mesenchymal stem cells （MSC） derived from non-Hodgkin lymphoma （NHL） bone marrow treated with etoposide （VP-16）. Methods MSC were obtained from non-Hodgkin lymphoma bone marrow, cultured in expanded medium, and then divided into 4 groups： Group A, without treatment by either WR-2721 or VP-16 and used as control group, Group B, treated with WR-2721, Group C, treated with WR-2721 ＋ VP-16, and Group D, treated with VP-16 alone. Inverted microscopy was used to observe the growth of the MSC. Flow cytometry was used to observe the apoptosis and immunophenotypes of the MSC. Mononuclear growth and apoptosis of the MSC Mononuclear bone marrow cells were obtained from 5 healthy volunteers and added into the MSC of the 4 groups, 4 weeks later methylcellulose progenitor assay and then inverted microscopy were used to measure the numbers of colony so as to detect the hematopoiesis. MSC of different groups were put into osteocyte-inducing and adipocyte-inducing media respectively, and 2 weeks later Von Kossa staining and oilred O staining were used to identify the differentiation into bone and fat. Results The NHL derived MSC of the 4 groups all showed a typical fibroblast-like morphology and were all positive in CD29, CD44, and CD105, while negative in CDllb, CD31, CD34, CD45, and HLA-DR. The apoptotic rate of Group D was 31.2% ＋ 4.3 %, significantly higher than those of the other 3 groups （ all P 〈 0.05 ）, and the apoptotic rate of Group C was significantly higher than those of Groups A and B （ both P 〈 0. 05 ）, however, still significantly lower than that of Group D （ P 〈 0.05 ）. The ability to support hematopoiesis of Group B was not significantly different from that of Group A, and Groups C and D showed a lower ability to support hematopoiesis in comparison with Groups A and B, However, the ability of Group C was still significantly higher than that of Group D （ P 〈 0. 05 ）. Under suitable conditions, all the MSC differentiated into osteocytes or adipocytes. Conclusion Amifostine alone has no effects on proliferation, apoptosis, immuophenotype or ability of hematopoiesis support of the MSC in vitro; however, it can protect NHL patient-derived MSC from injury hy VP-16 in vitro.