虾青素对储存早期去白细胞悬浮红细胞的影响研究

目的 探讨抗氧化剂虾青素(ASTA)对储存早期去白细胞悬浮红细胞超微结构,氧化应激水平,包括活性氧族(ROS)与丙二醛(MDA)含量,以及2,3-二磷酸甘油酸(DPG)含量的影响.方法 自2013年5～6月于河北省血液中心制备的去白细胞悬浮红细胞制剂中,采用简单随机抽样方法抽取6份去白细胞悬浮红细胞制剂标本为研究对象.将每份去白细胞悬浮红细胞制剂平均分装至4个空血袋内,按照随机数字表法随机选择其中1袋纳入对照组(n=6),另外3袋分别加入抗氧化剂ASTA,最后调节ASTA终浓度分别为5、10及20 μmol/L,并分别将其纳入5μmol/L ASTA组(n=6)、10 μmol/L ASTA组(n=6)及20 μmol/L ASTA组(n=6).对照组去白细胞悬浮红细胞制剂内未加入抗氧化剂ASTA,只加入等量二甲亚砜(DMSO).将4组去白细胞悬浮红细胞均于2～6℃冰箱内保存.于储存后第7及14天时,采用扫描电子显微镜观察4组去白细胞悬浮红细胞的超微结构,荧光酶标仪测定去白细胞悬浮红细胞内ROS含量,硫代巴比妥酸比色法测定去白细胞悬浮红细胞内MDA含量,紫外测试法测定去白细胞悬浮红细胞内2,3-DPG含量.统计学分析4组去白细胞悬浮红细胞储存后第7及14天时,ROS、MDA及2,3-DPG含量变化.结果 ①扫描电子显微镜下观察4组去白细胞悬浮红细胞储存后第7和14天时,红细胞的超微结构变化发现,5、10及20μmol/L ASTA组去白细胞悬浮红细胞的正常双凹圆盘状红细胞比例均高于对照组,而棘状红细胞比例均低于对照组,并且差异均有统计学意义(F=25.170、61.376,P=0.000);10 μmol/L ASTA组去白细胞悬浮红细胞的正常双凹圆盘状红细胞比例均高于5和20 μmol/L ASTA组,而棘状红细胞比例均低于上述两组,差异亦均有统计学意义(P＜0.05).其中,去白细胞悬浮红细胞储存后第7天时,5、10及20 μmol/L ASTA组及对照组的双凹圆盘状红细胞和棘状红细胞比例分别为77.4％与22.6％0、83.6％与16.4％、76.9％与23.1％、73.8％与26.2％;储存后第14天时,则分别为59.4％与40.6％、70.8％与29.2％、58.3％与41.7％、52.8％％与47.2％.②去白细胞悬浮红细胞储存后第7和14天时,4组去白细胞悬浮红细胞内ROS含量比较,差异均有统计学意义(F=13.530、27.515,P=0.000).去白细胞悬浮红细胞储存后第7和14天时,5、10及20μmol/L ASTA组去白细胞悬浮红细胞内ROS含量均明显低于对照组,10 μmol/L ASTA组去白细胞悬浮红细胞内ROS含量亦明显低于5和20 μmol/L ASTA组,并且差异均有统计学意义(P＜0.05);而5和20 μmol/L ASTA组去白细胞悬浮红细胞内ROS含量比较,差异无统计学意义(P＞0.05).其中,去白细胞悬浮红细胞储存后第7和14天时,对照组去白细胞悬浮红细胞内ROS含量均为最高,分别为(204.3±28.7)相对荧光强度/mgHb与(245.2±15.8)相对荧光强度/mgHb;而10 μmol/L ASTA组去白细胞悬浮红细胞内ROS含量均为最低,分别为(148.1±5.5)相对荧光强度/mgHb和(156.0±13.8)相对荧光强度/mgHb.③去白细胞悬浮红细胞储存后第7和14天时,4组去白细胞悬浮红细胞内MDA含量比较,差异均有统计学意义(F=20.698、21.398,P=0.000).去白细胞悬浮红细胞储存后第7和14天时,5、10及20μmol/L ASTA组去白细胞悬浮红细胞内MDA含量均明显低于对照组,10 μmol/L ASTA组去白细胞悬浮红细胞内MDA含量亦明显低于5和20 μmol/LASTA组,5μmol/L ASTA组去白细胞悬浮红细胞内MDA含量亦明显低于20 μmol/L ASTA组,并且差异均有统计学意义(P＜0.05).其中,去白细胞悬浮红细胞储存后第7和14天时,对照组去白细胞悬浮红细胞内MDA含量均为最高,分别为(23.6±5.1)μmol/mgHb与(33.0±4.4)μmol/mgHb;而10 μmol/LASTA组去白细胞悬浮红细胞内MDA含量均为最低,分别为(7.3±2.6) μmol/mgHb和(15.5±3.2)μmol/mgHb.④去白细胞悬浮红细胞储存后第7和14天时,4组去白细胞悬浮红细胞内2,3-DPG含量比较,差异均有统计学意义(F=15.376、17.443,P=0.000).去白细胞悬浮红细胞储存后第7和14天时,5、10及20 μmol/L ASTA组去白细胞悬浮红细胞内2,3-DPG含量均明显高于对照组,10μmol/LASTA组去白细胞悬浮红细胞内2,3-DPG含量均显著高于5和20 μmol/L ASTA组,并且差异均有统计学意义(P＜0.05);而5和20 μmol/L ASTA组去白细胞悬浮红细胞内2,3-DPG含量比较,差异无统计学意义(P＞0.05).其中,去白细胞悬浮红细胞储存后第7和14天时,对照组去白细胞悬浮红细胞内2,3-DPG含量均为最低,分别为(517.1±106.2)μg/L与(417.6±62.9)μg/L;而10 μmol/L ASTA组去白细胞悬浮红细胞内2,3-DPG含量均为最高,分别为(789.1±48.2)μg/L和(677.5±90.8)μg/L.结论 ASTA可通过降低储存早期去白细胞悬浮红细胞氧化应激水平,延缓去白细胞悬浮红细胞超微结构改变的速度,减缓去白细胞悬浮红细胞内2,3-DPG含量减少的进度.去白细胞悬浮红细胞保存液中,ASTA的最佳处理浓度是否为10 μmol/L,仍有待进一步研究证实.

Research of effect of astaxanthin on suspended red blood cells without leukocyte during early storage

Objective To study the effect of astaxanthin (ASTA) on morphological structure,oxidative stress level,induding content of reactive oxygen species (ROS) and malondialdehyde (MDA),and content of 2,3-diphosphoglycerate (DPG) in suspended red blood cells without leukocyte during early storage.Methods From May to June 2013,6 bags of suspended red blood cells without leukocyte which were prepared at Hebei Blood Center were collected as research objects in this study by random sampling method.Then the suspended red blood cells without leukocyte of each bag were randomly divided into four groups.The ASTA was added into preservation solution of 3 groups,and the final content of ASTA was 5,10 and 20 μmol/L,respectively,and these 3 groups were then enrolled into 5 μmol/L ASTA group,10 μmol/L ASTA group and 20 μmol/L ASTA group.As the last group,only equivalent volume of dimethyl sulfoxide (DMSO) was added into the reservation solution,and this group was enrolled into control group.The suspended red blood cells without leukocyte of four groups were stored at 2-6 ℃.On day 7 and 14 of storage,the morphological structure of suspended red blood cells without leukocyte was observed by scanning electron microscopy.The content of ROS in suspended red blood cells without leukocyte was detected by fluorescence microplate reader.The content of MDA in suspended red blood cells without leukocyte was detected by thiobarbituric acid method.The content of 2,3-DPG in suspended red blood cells without leukocyte was detected by ultraviolet test method.The content changes of ROS,MDA and 2,3-DPG in suspended red blood cells without leukocyte on day 7 and 14 of storage were analyzed by statistical methods.Results ① On day 7 and 14 of storage,the percentages of double concave disc suspended red blood cells without leukocyte in 5,10 and 20 μtmol/L ASTA group were all higher than that in control group,and the percentages of suspended red blood cells without leukocyte with spines were all lower than that in control group,and all the differences were statistically significant (F=25.170,61.376;P =0.000).The percentage of double concave disc suspended red blood cells without leukocyte in 10 μmol/L ASTA group was higher than those in 5 and 20 μmol/L ASTA group,and the percentage of suspended red blood cells without leukocyte with spines in 10 μmol/L ASTA group was lower than those in 5 and 20 μmol/L ASTA group,and all the differences were statistically significant (P＜0.05).On day 7 of storage,the percentages of double concave disc suspended red blood cells and suspended red blood cells without leukocyte with spines in 5,10,20 μmol/L ASTA group and control group were 77.4％ and 22.6％,83.6％ and 16.4％,76.9％ and 23.1％,73.8％ and 26.2％,respectively.On day 14 of storage,the percentages were 59.4％ and 40.6％,70.8％ and 29.2％,58.3％ and 41.7％,52.8％ and 47.2％,respectively.②On day 7 and 14 of storage,the contents of ROS in suspended red blood cells without leukocyte of four groups were compared,and both the differences were statistically significant (F=13.530,27.515;P=0.000).On day 7 and 14 of storage,the contents of ROS in suspended red blood cells without leukocyte of 5,10 and 20 μmol/L ASTA group were all significantly lower than that of control group,and the content of ROS in suspended red blood cells without leukocyte of 10 μmol/L ASTA group was also significantly lower than those of 5 and 20 μmol/L ASTA group,and all the differences were statistically significant (P＜0.05).There was no significant difference in the content of ROS in suspended red blood cells without leukocyte between 5 and 20 μmol/L ASTA group (P＞0.05).On day 7 and 14 of storage,thecontents of ROS in control group were (204.3±28.7) relative fluorescence intensity/mgHb and (245.2± 15.8) relative fluorescence intensity/mgHb,respectively,and both of them were the highest among four groups.And the contents of ROS in 10 μmol/L ASTA group were (148.1 ±5.5) relat ive fluorescence intensity/mgHb and (156.0± 13.8) relative fluorescence intensity/mgHb,respectively,and both of them were the lowest among four groups.③On day 7 and 14 of storage,the contents of MDA in suspended red blood cells without leukocyte of four groups were compared,and both the differences were statistically significant (F=20.698,21.398;P =0.000).On day 7 and 14 of storage,the contents of MDA in suspended red blood cells without leukocyte of 5,10 and 20 μmol/L ASTA group were all significantly lower than that of control group,and the content of MDA in suspended red blood cells without leukocyte of 10 μmol/L ASTA group was also significantly lower than those of 5 and 20 μmol/L ASTA group,and the content of MDA in suspended red blood cells without leukocyte of 5 μmol/L ASTA group was also significantly lower than that of 20μmol/L ASTA group,and all the differences were statistically significant (P＜ 0.05).On day 7 and 14 of storage,the contents of MDA in control group were (23.6±5.1) μmol/mgHb and (33.0±4.4) μmol/mgHb,respectively,and both of them were the highest among four groups.And the contents of MDA in 10 μmol/L ASTA group were (7.3 ± 2.6) μmol/mgHb and (15.5±3.2) μmol/mgHb,respectively,and both of them were the lowest among four groups.④On day 7 and 14 of storage,the contents of 2,3-DPG in suspended red blood cells without leukocyte of four groups were compared,and the difference was statistically significant (F=15.376,17.443;P=0.000).On day 7 and 14 of storage,the contents of 2,3-DPG in suspended red blood cells without leukocyte of 5,10 and 20 μmol/L ASTA group were all significantly higher than that of control group,and the content of 2,3-DPG in suspended red blood cells without leukocyte of 10μmol/L ASTA group was also significantly higher than those of 5 and 20 μmol/L ASTA group,and all the differences were statistically significant (P＜0.05).There was no significant difference in the content of 2,3-DPG in suspended red blood cells without leukocyte between 5 and 20 μmol/L ASTA group (P＞0.05).On day 7 and 14 of storage,the contents of 2,3-DPG in control group were (517.1±-106.2) μg/L and (417.6±62.9) μg/L,respectively,and both of them were the lowest among four groups.And the contents of 2,3-DPG in 10 μmol/L ASTA group were (789.1± 48.2) μg/L and (677.5±90.8) μtg/L,respectively,and both of them were the highest among four groups.Conclusions During early storage,ASTA could delay the change speed of morphological structure and slow down the decreasing progress of 2,3-DPG content in suspended red blood cells without leukocyte by decreasing the oxidative stress level.But whether 10 μtmol/L is the best treatment content of ASTA in suspended red blood cells without leukocyte reservation solution,it still needs further study to confirm.