粒细胞集落刺激因子对CD34+骨髓干细胞在心肌内归巢的影响

            作者：冯金华，李玉光，石刚刚，侯玉清，曹世平

【关键词】  干细胞；心肌梗死；归巢；粒细胞集落刺激因子

   【Abstract】 AIM: To explore the possible effects of granulocyte colonystimulating factor (GCSF) on the homing of CD34+ bone marrow stem cell (BMSC) to myocardium. METHODS: Seventeen adult mongrel dogs were randomly assigned to 4 groups. Group 1 (GCSF and MI, n=5) were treated with 10 μg/(kg・d)×5 d of GCSF injection sc and then myocardial infract operation; group 2 (GCSF and sham operation, n=4), treated with 10 μg/(kg・d)×5 d of GCSF injection sc and sham operation; group 3 (nonGCSF and MI, n=4) were injected with 10 μg/(kg・d)×5 d sc normal saline and underwent myocardial infarct operation; group 4 (nonGCSF and sham operation, n=4) were injected with 10 μg/(kg・d)×5 d sc normal saline and get sham operation. Thoracotomy was performed on the left thorax of each animal and left anterior descending artery was exposed and separated about 1 cm. In group 1 and 3 the separated arteries were occluded for 3 h and then reopened, while the other 2 groups were not treated in this way. Every dogs received catheterizing in right femoral artery into the root of aorta arch and a topmodified pigtail catheter cannulating through left jugular superficial vein into coronary venous sinus. 5 mL blood from each animal aorta and coronary sinus were harvested for two times, in which erythrocytes were lysed. Mononuclear cells were separated with lymphocyte separating solution and CD34+ BMSC were clarified and counted by flow cytometry. Data were calculated and managed with SPSS 10.0 statistical package. RESULTS: The amount of the CD34+ stem cells homing to myocardium in each group were (183.0±9.2)×104/L, (71.1±9.2)×104/L, (82.3±9.5)×103/L and (78.5±9.8)×103/L, respectively. We found after comparing the data of each group that the difference among every group was very significant (P=0.000, respectively) except between group 3 and 4 (P=0.912). CONCLUSION: GCSF could not only vastly mobilize the BMSC into periphery blood, but also could increase the CD34+ stem cells homing to myocardium independent of myocardial infarct.

   【Keywords】 stem cell; myocardial infarction; homing; granulocyte colonystimulating factor

   【摘要】 目的：观察粒细胞集落刺激因子（GCSF）对CD34+骨髓干细胞（BMSC）在心肌内归巢的影响. 方法：成龄杂种犬17只，随机分4组. A组5只：接受10 μg/（kg・d），5 d皮下注射和实验性心梗；B组4只：接受10 μg/（kg・d），5 d皮下注射和假手术；C组4只：接受10 μg/（kg・d），5 d生理盐水皮下注射和实验性心梗；D组4只：接受10 μg/（kg・d），5 d生理盐水皮下注射和假手术，对每只动物股动脉插管至主动脉根部，左颈浅静脉插管至冠状静脉窦，分别于造型前后抽血5 mL（抽复管），裂解红细胞，分离单个核细胞，流式细胞仪测定CD34+细胞数，数据分析用SPSS 10.0统计软件包. 结果：4个实验组CD34+ BMSC心肌内归巢数分别为：（183.0±9.2）×104个/L,（71.1±9.2）×104个/L,（82.3±9.5）×103个/L和（78.5±9.8）×103/L，除C，D组之间差别无统计学意义（P=0.912）外，其余各组间均有统计学意义（均为P=0.000). 结论：GCSF不仅能动员BMSC大量释放入外周血，同时还能增加CD34+ BMSC在心肌内的归巢.

   【关键词】 干细胞；心肌梗死；归巢；粒细胞集落刺激因子

    0引言

   心肌梗死（myocardial infarction, MI）已成为第一位致死性疾病，以往认为坏死心肌无法治愈，现在研究发现干细胞能在体内外分化成心肌细胞［1-2］，自体骨髓干细胞（bone marrow stem cell, BMSC）能在心梗时得到动员［3-4］，并归巢（homing）到梗死部位分化心肌细胞，然而正常情况下外周血干细胞含量很低［5］，远达不到修复坏死心肌所需要的浓度，使用粒细胞集落刺激因子（granulocyte colonystimulating factor, GCSF）动员骨髓可以使大量BMSC进入外周血，促进其分化成心肌和血管内皮细胞，达到再生和修复作用，很显然只有归巢到靶部位的干细胞才有可能分化成心肌和血管内皮细胞，因此干细胞的归巢决定了心肌的再生. 我们对GCSF能否影响干细胞的归巢进行了探讨.

   1材料和方法

   1．1材料成龄杂种犬17只，雌雄不限，体质量17.5~24.7（平均21.2±2.2）kg，随机分为4组，A组（GCSF+MI）；B组（GCSF+sham）；C组（NS+MI）;D组（NS+sham）.

   1．2方法A组（5只）接受GCSF（商品名惠尔血，麒麟昆鹏公司生产）10 μg/(kg・d), 5 d皮下注射和MI手术；B组（4只）接受GCSF 10 μg/（kg・d），5 d皮下注射和假手术；C组（4只）接受10 μg/(kg・d), 5 d生理盐水皮下注射和MI手术；D组（4只）接受10 μg/(kg・d), 5 d生理盐水皮下注射和假手术.

   MI手术： 每只动物左侧开胸，暴露并分离左冠脉前降支近端约1 cm，阻断3 h后放开，制作MI模型，MI的判断用心电监护和心肌声学造影（图1~4），对每只动物股动脉插管至主动脉根部（冠状动脉窦）、左颈浅静脉插管至冠状窦，分别于造型前后抽冠状动脉和冠状静脉窦血5 mL（均抽复管），裂解红细胞，淋巴细胞分离液分离单个核细胞，流式细胞仪（美国BD公司）测CD34+骨髓单个核细胞数.

   统计学处理： 所有数据采用SPSS 10.0统计软件包统计处理，计量资料以x±s表示，多组间比较采用方差分析，以P<0.05为统计学差异判断标准.

    2结果

   不同实验组归巢前（冠状动脉窦血）CD34+ BMSC含量和数量比较见表1.

   4个组比较后发现在GCSF动员的基础上发生MI，CD34+ BMSC在心肌内归巢的数量大大增加，只用GCSF动员而不发生MI的动物（B组）CD34+ BMSC的归巢数有所降低（P=0.000），而未经GCSF动员组（C,D组）即使发生MI，CD34+ BMSC在心肌内的归巢数也微乎其微.表1归巢前冠状动脉血CD34+ BMAC含量及数量比较

   3讨论

   自体BMSC因不引起免疫排斥反应、取材方便、无伦纠纷等特有的优势在临床应用中倍受青睐，研究发现急性心肌梗死本身即可引发自体BMSC动员［3,5］，使外周血含量增加，促进坏死组织的自发修复，但这种应急性反应所致骨髓动员意义到底多大尚无定论. 本研究显示单纯MI组外周血CD34+ BMSC含量（222±12）×104/L与无心肌梗死组（224±10）×104/L比无明显差别（P=0.958），两个组发生归巢的CD34+ BMSC数也无显著性差异（82±10）×103/L比（78±10）×103/L（P=0.912），提示单纯MI本身对干细胞动员和归巢的影响不起决定作用.

   干细胞向心肌或血管内皮细胞方向的分化首先需要归巢到心肌内，至于BMSC是如何通过血管内皮迁移到心肌靶部位的目前尚不得而知，但是已经有研究发现局部组织以及MI后侵润的炎性细胞所分泌的活性细胞因子对梗死后心肌再生可能起一定的作用［6-7］. Kuethe等［8］给心肌梗死患者每日应用GCSF 10 μg/(kg・d)共7 d，3 mo后发现患者心室射血分数平均增加40%，Sugano等［9］在实验性心肌梗死的动物身上也得出了类似的结果. Fukuhara等［10］研究显示静脉应用GCSF后梗死边缘区BMSC的数量明显增多、反映心肌再生的肌钙蛋白重链等的基因表达也明显增多，提示GCSF的作用靶点可能是在BMSC向坏死心肌靶部位的迁移归巢或是定向分化这两个环节中. 本研究我们通过精确测定证实，GCSF确实大大提高了CD34+ BMSC在心肌内发生归巢的程度，所以我们认为GCSF是通过促进CD34+ BMSC在心肌内的归巢而发挥其促心肌细胞再生作用；经过进一步的对比我们还发现在GCSF动员的基础上发生心肌梗死的心肌内CD34+ BMSC归巢的数量远远超过没有发生心肌梗死的心肌，而在未经GCSF动员的心肌内即使发生心肌梗死CD34+ BMSC归巢的数量也无显著增加，说明GCSF对CD34+ BMSC归巢的影响是不受MI干预的.

      【】

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