干细胞动员促进心肺复苏后大鼠脑损伤的修复

The mobilization of stem cells promote the recovery of the ischeInia brain injury after cardiopulmonary resuscitation

目的观察粒细胞集落刺激因子（granulocyte colony-stimulating factor，G-CSF）与AMD3100动员自身骨髓干细胞治疗复苏后脑缺血损伤的效果，并探讨其作用机制。方法在中山大学心肺脑复苏研究所建立窒息法心肺复苏大鼠动物模型。56只SD大鼠随机（随机数字法）分4组：G—CSF单独动员组、G—CSF＋AMD3100联合动员组、单纯复苏组和假手术组。通过NDS评分、纸带移除实验、头颅MR扫描影像分析等方法评价干细胞自体动员对复苏后神经功能的影响；采用ELISA法检测脑组织中血管内皮细胞生长因子（VEGF）含量、TUNEL法检测脑组织中神经细胞凋亡、免疫荧光法检测脑组织毛细血管密度变化。结果复苏后3d时，G—CSF＋AMD3100组NDS评分（61．4±10．7）显著高于单纯复苏组（49．9±10．4）（P〈0．05），纸带移除时间为（85．5±28．9）s，显著短于单纯复苏组（148．1±23．8）s与G—CSF组（118．5±30．4）S（P〈0．05）；脑MRI显示的脑损伤严重程度两个干细胞动员组低于单纯复苏组；G-CSF＋AMD3100组的神经细胞凋亡率（0．23±0．06）显著低于G—CSF组（0．34±0．08）（P〈0．05），而两者均显著低于单纯复苏组（0．44±0．09）（P〈0．05）。在复苏后3d与6d，G-CSF＋AMD3100组脑组织中VEGF质量浓度（pg／mL）分别为（106．2±23．3）与（79．9±18．4），G—CSF组脑组织VEGF质量浓度（pg／mL）分别为（50．6±13．7）与（73．9±16．6），均显著高于单纯复苏组（23．1±10．2）与（36．2±12．8）（P〈0．05）。G—CSF＋AMD3100组在复苏后3d时的脑毛细血管密度（351．8±67．9）个／高倍视野，显著高于G—CSF组（301．4±77．3）个／高倍视野与单纯复苏组（250．4±48．0）个／高倍视野（P〈0．05）。在复苏后6d时，G—CSF组的脑毛细血管密度较3d时明显升高，为（348．4±76．7）个／高倍视野（P〈0．05），G—CSF＋AMD3100组为（344．1±65．7）个／高倍视野，与3d时比较无明显变化。结论干细胞动员显著改善了复苏后大鼠神经功能状况，联合动员后神经功能恢复更加显著且恢复时间早于单独动员。干细胞动员对脑损伤的修复作用机制可能与抑制神经细胞凋亡、促进VEGF分泌及损伤区新生血管生成有关。

Objective To explore the therapeutic potential and mechanism of stem ceils mobilized by granulocyte colony-stimulating factor （G-CSF） and AMD3100 to repair global cerebral ischemia injuries in a rat model of cardiac arrest （CA） and cardiopulmonary resuscitation （CPR）. Methods Cardiac arrest was induced by asphyxia. Fifty-six SD rats were randomly assigned into four groups： G-CSF group, G-CSF ＋ AMD3100 group, CPR control group and sham operated group. The animals were sacrificed at 3d and 6d after CPR respectively. The neurological status and morphological changes of damaged cerebrum, the apoptosis of nerve cells and vascular endothelial growth factor （VEGF） expressed in brain tissue and capillary density in hippocampus and temporal lobe cortex were measured and analyzed by means of neurological deficit score （NDS）, adhesive tape removal test （TRT）, ELISA, MRI and immunofluorescence. Results NDS in G-CSF ＋ AMD3100 group （61.4 ± 10. 7 ） was significantly higher than that in CPR control group （49. 9 ± 10. 4） at 3 d after CPR （P 〈0. 05）. And less time consumption for TRT found in G-CSF ＋ AMD3100 group （85.5 ±28.9） s rather than was in CPR control group （148. 1 ± 23.8） s and G-CSF group （ 118.5 ± 30. 4） s （P 〈 0. 05）. The severity of cerebral injury assessed by MRI was significantly milder at both 3 d and 6 d in the two stem cell mobilization groups. The apoptosis rate of nerve cells in G-CSF ＋ AMD3100 group （0. 23 ± 0. 06） was significantly lower than that in G-CSF group （0. 34 ± 0. 08） at 3 d after CPR, and that in both stem cell mobilization groups was lower than that in CPR control group （0.44 ± 0.09） （ P 〈 0. 05 ）. At 3 d and 6 d after CPR, the levels of VEGF in brain tissue were （106. 2 ±23.3） pg/mL and （79. 9 ± 18.4） pg/mL in G-CSF ＋ AMD3100 group, and were （50. 6 ± 13.7） pg/mL and （73.9 ± 16. 6） pg/mL in G-CSF group, which were both significantly higher than that in CPR control group （23.1 ± 10. 2） pg/mL and （ 36. 2 ± 12. 8 ） pg/mL （ P 〈 0.05 ）. At 3 d after CPR, the cerebral capillary density （351.8 ± 67.9） branches in every high power field （A/HPF） was significantly higher in G-CSF ± AMD3100 group than that （301.4 ±77. 3） A/HPF in G-CSF group and （250. 4 ±48. 0） A/HPF in CPR control group （P 〈 0. 05 ） . The cerebral capillary density in G-CSF group elevated to （348.4 ±76. 7） A/HPF at 6 d after CPR which was significantly higher than that at 3 d （P 〈0.05）, and there was no difference between that at 3 d and 6 d in G-CSF ＋ AMD3100 group. Conclusions The mobilization stem cells improve the impaired neurological function. The increased expression of VEGF in brain tissue, the neo-vascularization promoted by the mobilized stem cells and the inhibition of nerve cell apoptosis may be associated with the protective effects of the stem cell mobilization.