粒细胞集落刺激因子联合携带肝细胞生长因子基因的BMSCs移植对心肌梗死大鼠血管重建的影响

EFFECT OF COMBINED THERAPY OF GRANULOCYTE COLONY STIMULATING FACTOR AND BONE MARROW MESENCHYMAL STEM CELLS CARRYING HEPATOCYTE GROWTH FACTOR GENE ON ANGIOGENESIS OF MYOCARDIAL INFARCTION IN RATS

目的研究粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)联合携带肝细胞生长因子(hepatocyte growth factor,HGF)基因的BMSCs移植对心肌梗死大鼠血管重建的影响,初步探讨作用机制。方法取3周龄雄性SD大鼠骨髓分离培养BMSCs,取第3代BMSCs以携带HGF基因的5型复制缺陷型腺病毒(Ad-HGF)感染。成年雄性SD大鼠44只,体重200～250 g,结扎左冠状动脉建立心肌梗死模型。造模4周后心脏超声检查,以左室短轴缩短率(shorting fraction,FS)<30%作为造模成功标准。取其中12只大鼠,于梗死心肌边缘注射0.1 mL Ad-HGF感染的BMSCs(5×107个/mL),2、7、14 d后用Western blot方法检测大鼠体内HGF蛋白的表达。将其余32只大鼠随机分为4组,每组8只:对照组注射0.1 mL生理盐水;G-CSF组注射0.1 mL生理盐水并于腹腔注射G-CSF 100μg(/kg.d)共5 d;HGF组注射0.1 mL Ad-HGF感染的BMSCs(5×107个/mL);联合治疗组注射0.1 mL Ad-HGF感染的BMSCs(5×107个/mL)并于腹腔注射G-CSF 100μg/(kg.d)共5 d。细胞移植后2周,行心功能和血流动力学检测,然后处死大鼠取心肌组织行免疫荧光双染后激光共聚焦显微镜下评价血管生成情况,Western blot检测VEGF蛋白表达。结果感染Ad-HGF的BMSCs移植2、7 d时在大鼠体内表达HGF蛋白。心功能及血流动力学检测显示,G-CSF组左室收缩压(left ventricular systolic pressure,LVSP)、左室舒张末期压力(left ventricularend-diastolic pressure,LVEDP)、LVSP上升/降低时间(dP/dtmax)、FS与对照组相比差异均无统计学意义(P>0.05);HGF组和联合治疗组与对照组相比,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升高(P<0.05)。免疫荧光双染显示心肌梗死交界区增生细胞是血管内皮细胞。联合治疗组血管密度明显高于其他3组(P<0.05),VEGF蛋白表达较其他3组明显增加(P<0.05)。结论 在大鼠心肌梗死4周时给予G-CSF联合携带HGF基因的BMSCs移植治疗,可明显改善心功能,促进心肌梗死边缘缺血区域的血管生成,其作用机制之一是增加了缺血心肌VEGF蛋白的表达。

Objective 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. Methods 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 μg/（kg.d）] for 5 days in G-CSF group; 0.1 mL BMSCs （5 × 10^7/mL） transfected with Ad-HGF was injected into the infarct zone in HGF group; 0.1 mL BMSCs （5 × 10^7/mL） transfected with Ad-HGF was injected combined with intraperitoneal injection G-CSF [100 big/（kg.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. Results 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）. Conclusion 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.