贮存式自体全血与单纯红细胞输注对家兔骨髓造血干细胞功能的影响

Effect of preoperative autologous whole blood and simple red blood cells donation and transfusion on the function of rabbit bone marrow hematopoietic stem cells

目的探讨贮存式自体全血和红细胞成分对骨髓造血干细胞衰老相关指标的影响。方法健康雄性新西兰家兔30只,3~6月龄,体重2.5~3.0 kg。采用随机数表法将其分为五组:对照组(C组)、单纯手术组(S组)、手术采血组(ABS组)、贮存式自体全血输注组(PAB组)和贮存式自体红细胞输注组(PARB)组,每组6只。C组家兔麻醉后,不做任何处理,按时点采集骨髓标本,其余各组均开腹行肝叶切除,ABS组、PAB组和PARB组于术前1周采血贮存,S组和ABS组术中输液补充血容量,PAB组和PARB组术中分别输注贮存的自体全血和自体红细胞。于麻醉诱导后手术前(T_(1))、术后6 h(T_(2))和术后24 h(T_(3))采集骨髓样本测定家兔CD34^(+)细胞百分比、活性氧(ROS)浓度、衰老细胞比例及衰老相关蛋白(p53、p21)的表达情况。结果与T_(1)时比较,T_(2)、T_(3)时S组、ABS组、PAB组和PARB组的CD34^(+)细胞百分比明显升高,ROS浓度、衰老细胞比例明显降低,T_(2)时ABS组,T_(2)、T_(3)时S组、PAB组和PARB组p53、p21蛋白含量明显降低(P<0.05)。与T_(2)时比较,T_(3)时S组、ABS组、PAB组和PARB组的CD34^(+)细胞百分比明显升高,T_(3)时PARB组衰老细胞比例明显降低,T_(3)时ABS组、PAB组和PARB组p53蛋白含量明显升高,T_(3)时ABS组和PAB组p21蛋白含量明显升高(P<0.05)。与C组比较,T_(2)、T_(3)时S组,T_(1)—T_(3)时ABS组、PAB组和PARB组的CD34^(+)细胞百分比明显升高,ROS浓度、衰老细胞比例及p53、p21蛋白含量明显降低(P<0.05)。与S组比较,T_(1)时ABS组,T_(1)—T_(3)时PAB组和PARB组CD34^(+)细胞百分比明显升高,T_(1)—T_(3)时ABS组、PAB组和PARB组的ROS浓度明显降低,T_(1)、T_(2)时ABS组,T_(1)—T_(3)时PAB组和PARB组衰老细胞比例和p53、p21蛋白含量明显降低(P<0.05)。与ABS组比较,T_(2)、T_(3)时PAB组和PARB组的CD34^(+)细胞百分比明显升高,T_(1)—T_(3)时PAB组和PARB组的ROS浓度明显降低,T_(2)、T_(3)时PAB组和PARB组衰老细胞比例和p53、p21蛋白含量明显降低,T_(1)时PARB组p53、p21蛋白含量明显升高(P<0.05)。与PAB组比较,T_(3)时PARB组的CD34^(+)细胞百分比明显升高,T_(2)、T_(3)时PARB组的ROS浓度明显降低,T_(2)时PARB组的衰老细胞比例明显降低(P<0.05)。结论贮存式自体输血在一定程度逆转骨髓细胞衰老表型,促进CD34^(+)细胞生成,从造血干细胞的角度而言,采集并输注红细胞成分比全血更加有益。

Objective To study and evaluate the effect of autologous whole blood and red blood cells donation and transfusion on aging-related indexes of bone marrow hematopoietic stem cells.Methods Thirty healthy male New Zealand rabbits,aged 3-6 months,weighing 2.5-3.0 kg,were randomly divided into 5 groups(n=6):control group(group C),simple surgery group(group S),simple blood collection group(group ABS),stored autologous whole blood transfusion group(group PAB),and the storage autologous red blood cell transfusion group(group PARB).After anesthetized,the rabbits in group C were collected bone marrow specimens on time without other treatment.Other groups of rabbits underwent hepatectomy.And 10%of whole blood storage was collected from groups ABS,PAB and PARB one week before surgery.Then,rabbits in groups S and ABS were infused with lactated ringer s solution to supplement blood volume during operation,while the rabbits in groups PAB and PARB were infused with stored autologous whole blood and autologous red blood cells.The percentage of bone marrow CD34^(+)cells,Ros concentration,proportion of senescent cells and the expression of senescence-associated proteins(p53,p21)in hematopoietic stem cells were detected after induction of anesthesia before surgery(T_(1)),6 hours after surgery(T_(2))and 24 hours after surgery(T_(3)).Results Compared with T_(1),the percentage of CD34^(+)cells were increased while the ROS concentration and the proportion of senescent cells were decreased significantly in groups S,ABS,PAB and PARB T_(2) and T_(3),and the expression of p53 and p21 proteins was significantly reduced in group ABS at T_(2) and in groups S,PAB,and PARB at T_(2) and T_(3) respectively.Compared with T_(2),the percentage of CD34^(+)cells in groups S,ABS,PAB and PARB was significantly increased when the proportion of senescent cells in group PARB was significantly reduced at T_(3),moreover,the p53 protein expression in groups ABS,PAB and PARB and the expression of p21 protein in groups ABS and PAB were significantly increased at T_(3)(P<0.05).Compared with group C,the percentage of CD34^(+)cells were significantly increased in group S at T_(2),T_(3) and in groups ABS,PAB and PARB at T_(1)-T_(3),while the ROS concentration,the proportion of senescent cells,and the expression of p53 and p21 proteins were significantly reduced(P<0.05),Compared with group S,the percentage of CD34^(+)cells were increased significantly in group ABS at T_(1),as well as in groups PAB and PARB at T_(1)-T_(3),and the ROS concentration in groups ABS,PAB and PARB at T_(1)-T_(3) were decreased significantly,in addition,the proportion of senescent cells and the expression of p53 and p21 proteins were significantly reduced in group ABS at T_(1) and T_(2),and in groups PAB and PARB at T_(1)-T_(3) respectively(P<0.05).Compared with group ABS,the percentage of CD34^(+)cells in groups PAB and PARB were increased from T_(2) and T_(3),while the ROS concentration in groups PAB and PARB were decreased at T_(1)-T_(3),similarly,the proportion of senescent cells and the expression of p53 and p21 proteins in groups PARB and PARB were significantly reduced at T_(2) and T_(3),while the expression of p53 and p21 proteins in group PARB were significantly increased at T_(1)(P<0.05).Compared with group PAB,the percentage of CD34^(+)cells in group PARB was significantly increased at T_(3),while the ROS concentration in group PARB was significantly reduced at T_(2) and T_(3),and the proportion of senescent cells in group PARB was significantly reduced at T_(2)(P<0.05).Conclusion Storage of autologous blood can reverse the aging phenotype of bone marrow cells to a certain extent,significantly promote the production of CD34^(+)cells in response to blood loss stimulation.And from the perspective of hematopoietic stem cells,collecting and transfusion of red blood cell components is more beneficial than doing whole blood.