铁过载诱导ROS生成对造血干祖细胞功能的影响

发布时间：2018-05-12 14:00

本文选题：铁过载 + 活性氧物质　；参考：《天津医科大学》2012年硕士论文

【摘要】：目的：体外分离并培养单个核细胞(MNC),建立铁过载造血细胞模型,探讨铁过载对造血细胞内活性氧物质(ROS)水平的影响以及ROS升高对造血干祖细胞造血功能的影响及作用机制。方法： 1.体外分离培养骨髓及脐血来源的MNC,通过在培养液中添加枸橼酸铁铵(FAC),诱导造血干祖细胞铁过载,从而建立铁过载造血细胞模型。并通过检测细胞内可变铁池(LIP)水平验证这一模型的建立。 2.分别采用流式细胞术检测铁过载造血细胞的ROS水平、凋亡水平、各系造血细胞计数,采用甲基纤维素半固体培养法检测造血集落形成,采用western blot方法检测ROS相关信号因子p38MAPK/p-p38MAPK和AKT表达的水平。 3.分别用去铁胺(DFO)祛铁或抗氧化剂N-乙酰半胱氨酸(NAC)、谷胱甘肽(GSH)清除过多的ROS后,检测上述指标的变化。结果： 1.在体外培养骨髓来源的MNC过程中加入不同浓度(50、200、400μmol/L)FAC培养不同时间(2、4、8、12、24、36、48h),发现骨髓造血细胞内LIP水平升高,且具有时间和浓度依赖性,在含400μmol/L FAC的培养液中培养24h时LIP水平达到最高。并且在400μmol/L FAC浓度下,培养骨髓造血细胞24h后骨髓造血细胞内总的ROS、髓系细胞和红系细胞内ROS水平显著升高,分别为对照组的1.77、1.75和2.12倍。分别用DFO和NAC处理后发现,细胞内ROS水平明显降低(p0.05)。 2.在体外培养脐血来源的MNC过程中加入不同浓度FAC (50、100、200、400μmol/L)培养不同时间(6、12、24h),发现ROS水平随着FAC的浓度的增加和培养时间的延长而变化,且在200μmol/L FAC的培养液中培养24h时ROS水平达到最高。用抗氧化剂NAC和GSH联合FAC处理细胞发现,抗氧化剂处理组细胞内总的ROS以及髓系细胞和红系细胞内的ROS明显低于FAC组(p值均小于0.05)。此外,在200μmol/L FAC的浓度下,培养脐血MNC24h后细胞内总的LIP、髓系细胞和红系细胞内LIP水平显著高于对照组(p0.05),但抗氧化剂NAC和GSH对铁过载细胞内LIP均无明显影响。 3.对ROS相关信号因子的检测发现与对照组相比,FAC组p38MAPK/p-p38MAPK、AKT的表达明显增强,用DFO和NAC处理后其表达均明显减弱。 4.对骨髓来源造血干祖细胞功能的检测发现FAC组细胞凋亡比例[(24.80±2.99)%]较对照组[(8.90±0.96)%]显著升高(p0.05)；造血集落形成单位(CFU-E、CFU-GM、BFU-E和CFU-mix)计数明显低于对照组(p值均小于0.05)；CD34+细胞比例[(0.39±0.07)%]较对照组[(0.91±0.12)%]也显著降低(p0.05)。对脐血造血干祖细胞造血功能的检测发现：FAC组细胞凋亡比例[(20.90±3.45)%]明显高于对照组[(9.20±1.29)%](p0.05)；造血集落形成单位(CFU-E、BFU-E、CFU-GM、CFU-mix)计数明显低于对照组(其中CFU-E、 BFU-E、CFU-GM差异具有统计学意义(p0.05)；CD34-、CD33+、GlyA+细胞比例及细胞计数均显著低于对照组(p值均小于0.05)；以上这些损伤均可通过DFO和NAC处理而部分恢复。结论：细胞外铁可以进入造血干祖细胞内,造成细胞内LIP水平升高,诱导细胞铁过载。铁过载通过诱导细胞内ROS生成增加影响造血干祖细胞造血功能(如造血干祖细胞集落形成、细胞凋亡和造血干祖细胞计数),这与ROS相关的信号分子p38MAPK/p-p38MAPK和AKT介导的信号传导通路改变有关,且这些损伤均可以通过祛铁和抗氧化处理减轻。因此,铁过载通过诱导造血干祖细胞内ROS水平升高进而影响造血细胞凋亡,增殖分化等,清除细胞内多余的铁和ROS均有利于维持造血干祖细胞的功能。
[Abstract]:Objective : To study the effect of iron overload on the level of active oxygen species ( ROS ) in hematopoietic cells and the effect of ROS on the hematopoietic function of hematopoietic progenitor cells .

Method :

1 . In vitro isolation and culture of MNC from bone marrow and cord blood source , the iron overload of hematopoietic stem progenitor cells was induced by adding ferric citrate ( FAC ) to the culture solution , thus establishing an iron overload hematopoietic cell model .

2 . Flow cytometry was used to detect the level of ROS , the level of apoptosis , the count of hematopoietic cells in each system , and the formation of hematopoietic colony was detected by the semi - solid culture method of methyl cellulose , and the level of ROS - related signal factor p38MAPK / p - p38MAPK was detected by western blot .

3 . After removing excessive ROS from iron or antioxidant N - acetyl cysteine ( NAC ) and glutathione ( GSH ) respectively , the changes of these indexes were detected .

Results :

1 . Different concentrations ( 50 , 200 , 400 渭mol / L ) FAC were added to culture bone marrow - derived MNC for different time ( 2 , 4 , 8 , 12 , 24 , 36 , 48 h ) .

2 . Different concentrations of FAC ( 50 , 100 , 200 , 400 渭mol / L ) were added to culture cord blood in vitro for different time ( 6 , 12 , 24 h ) . The ROS level was highest when the concentration of FAC increased and the culture time was prolonged .

3 . Compared with the control group , the expression of p38MAPK / p - p38MAPK was significantly increased in FAC group compared with the control group , and the expression of p38MAPK / p - p38MAPK was significantly decreased after treatment with DFO and NAC .

4 . Detection of hematopoietic stem progenitor cells from bone marrow showed that the percentage of apoptosis in FAC group was significantly higher than that in control group ( 24.80 卤 2.99 ) % , which was significantly higher than that in control group ( 8.90 卤 0.96 ) % ( p < 0.05 ) .
CFU - E , CFU - GM , BFU - E and CFU - mix were significantly lower in CFU - E , CFU - GM , BFU - E and CFU - mix than in the control group ( p < 0.05 ) .
The percentage of CD34 + cells ( 0.39 卤 0.07 ) % was significantly lower than that in control group ( 0.91 卤 0.12 ) % ( p < 0.05 ) .
CFU - E , BFU - E , CFU - GM , CFU - mix were significantly lower in CFU - E , BFU - E and CFU - GM ( p < 0.05 ) .
The percentage of CD34 - , CD33 + , GlyA + cells and cell count were significantly lower than those in control group ( p < 0.05 ) .
All of these lesions can be partially recovered by treatment with DFO and NAC .

Conclusion :

Iron overload can increase the level of ROS in hematopoietic stem cells ( such as hematopoietic stem progenitor cell colony formation , apoptosis and hematopoietic stem progenitor cell count ) .

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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