Xu Yanjun, Yi Tiantian, Xu Xiaoxiao, Pei Fuyu, He Yuelin, Wu Xuedong
Department of Pediatrics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Department of Pediatrics, Foshan Women and Children's Hospital Affiliated to Southern Medical University, Foshan 528000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Jan 30;40(1):110-117. doi: 10.12122/j.issn.1673-4254.2020.01.18.
To explore the effect of cyclophosphamide on hematopoietic stem cells (HSCs) in mice with iron overload.
Mouse models of iron overload were established in 30 male C57BL/6 mice by intraperitoneal injections of iron dextran at low (0.25 g/kg), moderate (0.5 g/kg), and high (1 g/kg) doses (n=10), with another 10 PBS-treated mice as the control group. The changes in body weight, liver, spleen and bone marrow of the mice were recorded, and serum level of ferritin was detected. The mice receiving a moderate dose of iron dextran were further divided into 8 groups for observation at different time points (D1, D2, D3, D4, D5, D6, D7, and D14 groups) and were given intraperitoneal injection of 50 mg/kg cyclophosphamide (Cy) for 2 consecutive days. Peripheral blood cells, bone marrow mononuclear cells (BMMNCs), and the frequencies of different HSCs (HPCs, HSCs, LT-HSCs) in the BMMNCs were monitored. The cell cycle distribution in the HSCs, level of reactive oxygen species and the microenvironment of the HSCs were analyzed using flow cytometry.
Compared with the control mice, the mice with iron overload showed obvious weight loss with significantly increased serum ferritin level, enlargement of the liver and spleen, and iron deposition in the organs ( < 0.05). No significant changes were noted in the peripheral blood of the mice with iron overload. The cyclophosphamide-treated mice exhibited significantly decreased number of WBCs and lymphocyte ratio at days 1 to 4 ( < 0.05). The numbers of BMMNCs and HPCs in mice with iron overload did not show significant changes as compared with those in the control mice, but the numbers of HSCs and LTHSCs decreased significantly in the mice with iron overload ( < 0.05). In cyclophosphamide-treated mice, the number of HSCs increased since day 1 and reached the peak level on day 3 ( < 0.05). Compared with those in the control group, the HSCs did not exhibit significant changes in cell cycle distribution in mice with iron overload, but the proportion of G0/G1 cells decreased significantly in cyclophosphamide group since day 1 and reached the lowest level on day 3 ( < 0.05).
Iron deposition in the bone marrow causes long- term damages of the HSCs in the bone marrow but does not induce obvious changes in the peripheral blood. In mice with iron overload, intraperitoneal injection of 50 mg/kg cyclophosphamide for two days promotes cell cycle changes of the resting HSCs to mobilize the HSCs, and this effect is the most obvious on day 4.
探讨环磷酰胺对铁过载小鼠造血干细胞（HSCs）的影响。
将30只雄性C57BL/6小鼠通过腹腔注射低（0.25 g/kg）、中（0.5 g/kg）、高（1 g/kg）剂量的右旋糖酐铁建立铁过载小鼠模型（n = 10），另10只经PBS处理的小鼠作为对照组。记录小鼠体重、肝脏、脾脏和骨髓的变化，并检测血清铁蛋白水平。将接受中剂量右旋糖酐铁的小鼠进一步分为8组，在不同时间点（D1、D2、D3、D4、D5、D6、D7和D14组）进行观察，并连续2天腹腔注射50 mg/kg环磷酰胺（Cy）。监测外周血细胞、骨髓单个核细胞（BMMNCs）以及BMMNCs中不同造血干细胞（HPCs、HSCs、LT-HSCs）的频率。采用流式细胞术分析造血干细胞的细胞周期分布、活性氧水平及造血干细胞微环境。
与对照小鼠相比，铁过载小鼠体重明显减轻，血清铁蛋白水平显著升高，肝脏和脾脏肿大，器官中有铁沉积（P < 0.05）。铁过载小鼠外周血无明显变化。环磷酰胺处理的小鼠在第1至4天白细胞数量和淋巴细胞比例显著降低（P < 0.05）。与对照小鼠相比，铁过载小鼠的BMMNCs和HPCs数量无明显变化，但铁过载小鼠的HSCs和LT-HSCs数量显著减少（P < 0.05）。在环磷酰胺处理的小鼠中，造血干细胞数量自第1天开始增加，并在第3天达到峰值水平（P < 0.05）。与对照组相比，铁过载小鼠造血干细胞的细胞周期分布无明显变化，但环磷酰胺组自第1天起G0/G1期细胞比例显著降低，并在第3天达到最低水平（P < 0.05）。
骨髓中的铁沉积会对骨髓中的造血干细胞造成长期损害，但不会引起外周血的明显变化。在铁过载小鼠中，腹腔注射50 mg/kg环磷酰胺连续两天可促进静止造血干细胞的细胞周期变化，从而动员造血干细胞，且该作用在第4天最为明显。
