Guo Yinghua, Liu Changting, He Jianguo
Department of Respiratory Disease, General Hospital of Chinese PLA, Beijing 100853, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011 Jun;25(6):736-40.
To investigate the effect of combined therapy of granulocyte colony stimulating factor (G-CSF) and bone marrow mesenchymal stem cells (BMSCs) carrying hepatocyte growth factor (HGF) gene on the angiogenesis of myocardial infarction (MI) in rats and the mechanisms of the synergistic effect.
BMSCs were aspirated from the femur and tibia of 3-week-old Sprague Dawley (SD) male rats. The third generation of BMSCs were harvested and transfected with Ad-HGF. The MI models were established in 44 SD male rats (weighing 200-250 g) by ligating the left coronary artery. At 4 weeks after ligation, the shorting fraction (FS) of the left ventricle being below 30% was used as a criteria of model success. The BMSCs (5 x 10(7)/mL) transfected with Ad-HGF were transplanted into the infarct zone of 12 SD rats, and the expression of HGF protein was detected by Western blot method at 2, 7, and 14 days after transplantation. At 4 weeks, the other 32 SD rats were randomly divided into 4 groups (n = 8). The 0.1 mL normal saline was injected into the infarct zone in control group; 0.1 mL normal saline was injected combined with intraperitoneal injection G-CSF [100 microg/ (kg x d)] for 5 days in G-CSF group; 0.1 mL BMSCs (5 x 10(7)/mL) transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs (5 x 10(7)/mL) transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 microg/ (kg x d)] for 5 days in combined therapy group. At 2 weeks after transplantation, heart function was detected by cardiac ultrasound and hemodynamic analysis, and then myocardial tissue was harvested to analyse the angiogenesis of the infarct zone, and the expression of VEGF protein by immunofluorescence staining.
The expression of HGF protein in vivo was detected at 2 days and 7 days of BMSCs transfected with Ad-HGF transplantation. There was no significant difference in left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), dP/dtmax, and FS between G-CSF group and control group (P > 0.05). When compared with the control group, LVEDP decreased significantly; LVSP, FS, and dP/dtmax increased significantly (P < 0.05) in HGF group and combined therapy group. When compared with HGF group, FS and dP/dtmax increased significantly in combined therapy group (P < 0.05). Immunofluorescence staining showed that the vascular endothelial cells were observed in myocardial infarction border zone. The vascular density and the expression of VEGF protein were significantly higher in combined therapy group than in other 3 groups (P < 0.05).
The combined therapy of G-CSF and BMSCs carrying HGF gene has a synergistic effect and can enhance infarct zone angiogenesis through inducing the expression of VEGF protein.
探讨粒细胞集落刺激因子（G-CSF）与携带肝细胞生长因子（HGF）基因的骨髓间充质干细胞（BMSCs）联合治疗对大鼠心肌梗死（MI）血管生成的影响及其协同作用机制。
从3周龄雄性Sprague Dawley（SD）大鼠的股骨和胫骨中抽取BMSCs。收获第三代BMSCs并用Ad-HGF转染。通过结扎左冠状动脉在44只SD雄性大鼠（体重200-250 g）中建立MI模型。结扎后4周，以左心室缩短分数（FS）低于30%作为模型成功的标准。将用Ad-HGF转染的BMSCs（5×10⁷/mL）移植到12只SD大鼠的梗死区域，并在移植后2、7和14天通过蛋白质免疫印迹法检测HGF蛋白的表达。4周时，将另外32只SD大鼠随机分为4组（n = 8）。对照组在梗死区域注射0. mL生理盐水；G-CSF组在梗死区域注射0.1 mL生理盐水并腹腔注射G-CSF[100 μg/（kg·d）]，共5天；HGF组在梗死区域注射0.1 mL用Ad-HGF转染的BMSCs（5×10⁷/mL）；联合治疗组在梗死区域注射0.1 mL用Ad-HGF转染的BMSCs（5×10⁷/mL）并腹腔注射G-CSF[100 μg/（kg·d）]，共5天。移植后2周，通过心脏超声和血流动力学分析检测心脏功能，然后采集心肌组织分析梗死区域的血管生成情况，并通过免疫荧光染色检测VEGF蛋白的表达。
在用Ad-HGF转染的BMSCs移植后2天和7天检测到体内HGF蛋白的表达。G-CSF组与对照组之间的左心室收缩压（LVSP）、左心室舒张末期压力（LVEDP）、dP/dtmax和FS无显著差异（P>0.05）。与对照组相比，HGF组和联合治疗组的LVEDP显著降低；LVSP、FS和dP/dtmax显著升高（P<0.05）。与HGF组相比，联合治疗组的FS和dP/dtmax显著升高（P<0.05）。免疫荧光染色显示在心肌梗死边缘区观察到血管内皮细胞。联合治疗组的血管密度和VEGF蛋白表达明显高于其他3组（P<0.05）。
G-CSF与携带HGF基因的BMSCs联合治疗具有协同作用，可通过诱导VEGF蛋白表达增强梗死区域血管生成。
