环磷酰胺对正常小鼠骨髓造血细胞的影响及其作用机制

摘要

本研究检测化疗药物环磷酰胺(CTX)对正常小鼠骨髓造血细胞的影响及其可能的作用机制,尤其是在骨髓造血细胞自我更新、定向分化中的作用.首先建立CTX处理的小鼠模型(一次性腹腔注射CTX 200 mg/kg),通过定期检测小鼠外周血及骨髓细胞中造血干细胞(HSC,LKS+)比例,确定小鼠骨髓及外周血细胞数量完全恢复的时间；应用竞争性移植、非竞争性移植、多色流式细胞仪分析等实验,研究CTX对骨髓造血细胞自我更新、定向分化等功能的影响；通过检测连续移植模型小鼠骨髓细胞中p16Ink4a mRNA的相对表达水平及SA-β-半乳糖苷酶(gal)的染色情况,探讨CTX对骨髓造血细胞衰老的影响.结果表明:尽管CTX处理后小鼠骨髓造血细胞数量能够完全恢复正常,但是却导致造血细胞功能的长期损伤,降低骨髓造血细胞的重建能力；并且在连续移植模型中,移植了CTX处理组小鼠骨髓造血细胞的受体小鼠出现骨髓细胞p16Ink4a mRNA高表达和SA-β-gal蓝染.结论:CTX诱发的骨髓造血细胞衰老可能在其骨髓长期损伤中发挥重要的作用.%This study was purposed to investigate the effect of chemotherapeutic drug cyclophosphamide (CTX) on normal murine bone marrow hematopoietic cells, especially on the self-renewal, proliferation and differentiation of bone marrow hematopoietic cells, and possible mechanisms. The CTX-treated mouse model was established by CTX 200 mg/ kg, ip. The exact time of complete recovery of hematopoiesis was determined by monitoring the recovery level of differential blood counts and the proportion of LKS+ cells in bone marrow cells. The function of bone marrow hematopoietic cells such as self-renewal, proliferation and differentiation were assessed by non-competitive and competitive bone marrow transplantation. The potential effect of CTX on senescence of bone marrow hematopoietic cells was analyzed by detecting p16Ink4a mRNA relative expression and SA-β-galactosidase (gal) staining. The results showed that the CTX could induce long-term but latent damage to bone marrow hematopoietic cell function and lead to the decrease in competency of bone marrow hematopoietic cells to reconstitute while seemingly permitting a complete recovery. Furthermore, the serial-transplantation model showed that these mice received transplantation of bone marrow hematopoietic cells from CTX-treated mice exhibited a high expression of p16Ink4a mRNA and SA-β-gal staining. It is concluded that CTX-induced bone marrow cellular senescence may play an important role in CTX-induced long-term injury to bone marrow hematopoietic cells.