HIF-1α介导铁过载对骨髓红系造血功能损伤的研究
发布时间：2019-03-12 20:30
【摘要】：目的研究铁过载状态下缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)在骨髓照射损伤铁过载小鼠骨髓细胞内的表达情况及其对骨髓红系造血功能的影响及机制研究。方法1.将20只C57BL/6小鼠随机分成4组,除Ctrl组小鼠外,后3组小鼠均接受4Gy 137Csγ射线照射,照射量率为1.0Gy/min,IO组和RAPA组分别给予腹腔注射右旋糖酐铁25mg/ml(0.2ml/只),Ctrl组和IR组给予腹腔注射生理盐水(0.2ml/只),注射4周,IR组和RAPA组铁累积剂量50mg。RAPA组另予灌胃雷帕霉素4mg/kg体重,每周1次,累计4周,两个月后颈椎脱臼处死取材;2.流式细胞仪检测骨髓单个核细胞可变铁池(labile iron pool,LIP)及骨髓细胞甩片行普鲁士蓝染色验证铁过载模型。3.半固体甲基纤维素的方法检测造血干祖细胞红系造血集落形成能力:CFU-E(clony-forming units-erythroid,红系造血集落形成单位)、BFU-E(burst clony-forming units-erythroid,爆式红系造血集落形成);流式检测骨髓原始红细胞:早期红细胞(Ter119+CD71+)、晚期红细胞(Ter119+CD71-)的数量和比例;4.实时荧光定量PCR检测骨髓细胞HIF-1α及HIF-1α信号通路分子(PI3K/AKT/m TOR)m RNA的表达水平;ELISA检测血清EPO的表达水平。结果IR组、IO组的HIF-1α较Ctrl组表达升高,分别是Ctrl组的(1.41±0.09)倍、(2.12±0.17)倍,IO组较IR组表达升高(P均0.05)。IR组、IO组早期红细胞比例较Ctrl组降低,差异无统计学意义(P0.05);IR组、IO组的晚期红细胞比例较Ctrl组降低,IO组较IR组晚期红细胞比例降低,差异具有统计学意义(P均0.05)。IR组的BFU-E和CFU-E数目较Ctrl组明显减低,IO组的BFU-E和CFU-E数目较IR组明显降低(P均0.05)。IR组和IO组HIF-1α相关信号通路分子(PI3K/AKT/m TOR)较Ctrl组表达升高(P0.05)。应用m TOR抑制剂RAPA使HIF-1α表达明显降低,其相关信号分子表达也明显降低,差异有统计学意义(P0.05);BFU-E明显升高,差异有统计学意义(P0.05),CFU-E数目升高,但无统计学差异(P0.05);早期红细胞和晚期红细胞的比例略有升高,无统计学差异(P0.05)。结论1.铁过载可损伤骨髓的红系造血功能。通过构建铁过载动物模型发现,铁过载可以影响铁过载小鼠的原始细胞红系造血集落(CFU-E、BFU-E)形成能力、影响早期原始红细胞和晚期原始红细胞的比例,尤以对晚期红细胞的影响为显著,照射更加重了这种损伤。2.铁过载小鼠骨髓单个核细胞HIF-1α表达水平明显升高,提示HIF-1α与铁过载致骨髓红系造血功能损伤有关。研究HIF-1α及其相关信号转导通路发现,铁过载使HIF-1α相关通路信号转导分子PI3K、AKT、m TOR表达均明显升高。3.铁过载对骨髓红系造血功能的损伤是可逆的。通过应用m TOR抑制剂雷帕霉素后,抑制细胞内HIF-1α的表达,可部分恢复铁过载对骨髓红系造血功能的损伤作用。这提示了铁过载对骨髓红系造血功能的损伤有望通过抑制HIF-1α来缓解。
[Abstract]:Aim to study the expression of hypoxia inducible factor (hypoxia inducible factor-1 伪 (HIF-1 伪) in bone marrow cells of irradiated mice with iron overload and its effect on erythroid hematopoiesis and its mechanism. Method 1. Twenty C57BL/6 mice were randomly divided into 4 groups. All the mice in the latter 3 groups were exposed to 4Gy 137Cs 纬-ray with the dose rate of 1.0 Gy / min. The mice in IO group and RAPA group were given intraperitoneal injection of iron dextran 25mg/ml (0.2ml/ only), with the exception of the latter three groups receiving 4Gy 137Cs 纬-ray irradiation at a dose of 1.0 Gy 路min ~ (- 1). The rats in Ctrl group and IR group were given intraperitoneal injection of saline (0.2ml/) for 4 weeks. The cumulative dose of iron in IR group and RAPA group was added to 50mg.RAPA group, and the body weight of rapamycin was given once a week for 4 weeks. Two months later, the cervical vertebrae were dislocated and the samples were taken. 2. Flow cytometry for detection of variable iron pool (labile iron pool,LIP in bone marrow mononuclear cells and Prussian blue staining for bone marrow cells flick to verify iron overload model. 3. Hemopoietic colony forming ability of hemopoietic stem progenitor cells was detected by semi-solid methylcellulose method: CFU-E (clony-forming units-erythroid, erythroid colony forming unit), BFU-E (burst clony-forming units-erythroid, burst erythroid hematopoietic colony formation); Flow cytometry was used to detect the number and proportion of early erythrocytes (Ter119 CD71) and late erythrocytes (Ter119 CD71-) in bone marrow. Real-time fluorescence quantitative PCR was used to detect the expression of HIF-1 伪 and HIF-1 伪 signal pathway molecules (PI3K/AKT/m TOR) m RNA) in bone marrow cells and ELISA to detect the expression level of serum EPO. Results the expression of HIF-1 伪 in IR group and IO group was (1.41 卤0.09) times and (2.12 卤0.17) times higher than that in Ctrl group, and the expression of HIF-1 伪 in IO group was higher than that in IR group. The percentage of early erythrocytes in IO group was lower than that in Ctrl group, and there was no significant difference (P0.05). In IR group, the percentage of late erythrocytes in IO group was lower than that in Ctrl group, and that in IO group was lower than that in IR group. There was significant difference in the number of BFU-E and CFU-E between). IR group and Ctrl group. The number of BFU-E and CFU-E in IO group was significantly lower than that in IR group (P0.05). The expression of HIF-1 伪-related signal pathway molecule (PI3K/AKT/m TOR) in both). IR group and IO group was significantly higher than that in Ctrl group (P0.05). Application of m-TOR inhibitor RAPA significantly decreased the expression of HIF-1 伪 and the expression of related signal molecules, the difference was statistically significant (P0.05). BFU-E significantly increased, the difference was statistically significant (P0.05), the number of CFU-E increased, but there was no statistical difference (P0.05); early red blood cells and late erythrocytes increased slightly, there was no statistical difference (P0.05). Conclusion 1. Iron overload can damage erythrocytic hematopoiesis of bone marrow. By constructing iron overload animal model, it was found that iron overload could affect the ability of erythropoiesis colony formation (CFU-E,BFU-E) of primordial cells and the ratio of early and late primordial erythrocytes in iron overload mice. In particular, the effect on late red blood cells was significant, and the damage was aggravated by irradiation. 2. The expression of HIF-1 伪 in bone marrow mononuclear cells of iron overloaded mice was significantly increased, suggesting that HIF-1 伪 was related to the damage of erythroid hematopoiesis of bone marrow induced by iron overload. HIF-1 伪 and its related signal transduction pathways were studied. It was found that iron overload significantly increased the PI3K,AKT,m TOR expression of signal transduction molecules in HIF-1 伪 related pathways. 3. The damage of hemopoietic function of erythroid bone marrow induced by iron overload is reversible. By inhibiting the expression of HIF-1 伪 after the application of m-TOR inhibitor rapamycin, the damage effect of iron overload on erythroid hematopoiesis of bone marrow was partially restored. This suggests that the damage of erythrocytic hematopoiesis induced by iron overload may be alleviated by inhibition of HIF-1 伪.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R55
本文编号：2439122
[Abstract]:Aim to study the expression of hypoxia inducible factor (hypoxia inducible factor-1 伪 (HIF-1 伪) in bone marrow cells of irradiated mice with iron overload and its effect on erythroid hematopoiesis and its mechanism. Method 1. Twenty C57BL/6 mice were randomly divided into 4 groups. All the mice in the latter 3 groups were exposed to 4Gy 137Cs 纬-ray with the dose rate of 1.0 Gy / min. The mice in IO group and RAPA group were given intraperitoneal injection of iron dextran 25mg/ml (0.2ml/ only), with the exception of the latter three groups receiving 4Gy 137Cs 纬-ray irradiation at a dose of 1.0 Gy 路min ~ (- 1). The rats in Ctrl group and IR group were given intraperitoneal injection of saline (0.2ml/) for 4 weeks. The cumulative dose of iron in IR group and RAPA group was added to 50mg.RAPA group, and the body weight of rapamycin was given once a week for 4 weeks. Two months later, the cervical vertebrae were dislocated and the samples were taken. 2. Flow cytometry for detection of variable iron pool (labile iron pool,LIP in bone marrow mononuclear cells and Prussian blue staining for bone marrow cells flick to verify iron overload model. 3. Hemopoietic colony forming ability of hemopoietic stem progenitor cells was detected by semi-solid methylcellulose method: CFU-E (clony-forming units-erythroid, erythroid colony forming unit), BFU-E (burst clony-forming units-erythroid, burst erythroid hematopoietic colony formation); Flow cytometry was used to detect the number and proportion of early erythrocytes (Ter119 CD71) and late erythrocytes (Ter119 CD71-) in bone marrow. Real-time fluorescence quantitative PCR was used to detect the expression of HIF-1 伪 and HIF-1 伪 signal pathway molecules (PI3K/AKT/m TOR) m RNA) in bone marrow cells and ELISA to detect the expression level of serum EPO. Results the expression of HIF-1 伪 in IR group and IO group was (1.41 卤0.09) times and (2.12 卤0.17) times higher than that in Ctrl group, and the expression of HIF-1 伪 in IO group was higher than that in IR group. The percentage of early erythrocytes in IO group was lower than that in Ctrl group, and there was no significant difference (P0.05). In IR group, the percentage of late erythrocytes in IO group was lower than that in Ctrl group, and that in IO group was lower than that in IR group. There was significant difference in the number of BFU-E and CFU-E between). IR group and Ctrl group. The number of BFU-E and CFU-E in IO group was significantly lower than that in IR group (P0.05). The expression of HIF-1 伪-related signal pathway molecule (PI3K/AKT/m TOR) in both). IR group and IO group was significantly higher than that in Ctrl group (P0.05). Application of m-TOR inhibitor RAPA significantly decreased the expression of HIF-1 伪 and the expression of related signal molecules, the difference was statistically significant (P0.05). BFU-E significantly increased, the difference was statistically significant (P0.05), the number of CFU-E increased, but there was no statistical difference (P0.05); early red blood cells and late erythrocytes increased slightly, there was no statistical difference (P0.05). Conclusion 1. Iron overload can damage erythrocytic hematopoiesis of bone marrow. By constructing iron overload animal model, it was found that iron overload could affect the ability of erythropoiesis colony formation (CFU-E,BFU-E) of primordial cells and the ratio of early and late primordial erythrocytes in iron overload mice. In particular, the effect on late red blood cells was significant, and the damage was aggravated by irradiation. 2. The expression of HIF-1 伪 in bone marrow mononuclear cells of iron overloaded mice was significantly increased, suggesting that HIF-1 伪 was related to the damage of erythroid hematopoiesis of bone marrow induced by iron overload. HIF-1 伪 and its related signal transduction pathways were studied. It was found that iron overload significantly increased the PI3K,AKT,m TOR expression of signal transduction molecules in HIF-1 伪 related pathways. 3. The damage of hemopoietic function of erythroid bone marrow induced by iron overload is reversible. By inhibiting the expression of HIF-1 伪 after the application of m-TOR inhibitor rapamycin, the damage effect of iron overload on erythroid hematopoiesis of bone marrow was partially restored. This suggests that the damage of erythrocytic hematopoiesis induced by iron overload may be alleviated by inhibition of HIF-1 伪.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R55
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