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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 effects of granulocyte colony stimulating factor (G-CSF) combined with BMSCs carrying hepatocyte growth factor (HGF) gene on the vascular remodeling after myocardial infarction in rats. Methods BMSCs were isolated from the bone marrow of 3-week-old male Sprague-Dawley rats. The 3rd passage BMSCs were infected with adenovirus type 5 (Ad-HGF) carrying HGF gene. Forty-four adult male Sprague-Dawley rats weighing 200-250 g were infused with left coronary artery to establish a myocardial infarction model. Echocardiography was performed 4 weeks after modeling, and the shortening fraction (FS) <30% was used as a success criterion. Twelve rats were injected with 0.1 mL Ad-HGF-infected BMSCs (5 × 107 cells / mL) at the margin of infarcted myocardium. The expression of HGF protein in rats was detected by Western blot after 2, 7 and 14 days. The remaining 32 rats were randomly divided into 4 groups of 8 rats in each group: the control group was injected with 0.1 mL of normal saline; G-CSF group was injected with 0.1 mL of normal saline and intraperitoneal injection of 100 μg of G-CSF (/kg.d) d, BMSCs (5 × 107 cells / mL) were injected into the HGF group with 0.1 mL of Ad-HGF, and 0.1 mL of Ad-HGF-infected BMSCs (5 × 107 cells / mL) /(kg.d) a total of 5 d. Two weeks after the transplantation, the cardiac function and hemodynamics were determined. Then the myocardial tissues of the rats were sacrificed and the expression of VEGF was evaluated by laser scanning confocal microscopy after immunofluorescence double staining. Results The HGF protein was expressed in rats at 2 and 7 days after transplantation of Ad-HGF-infected BMSCs. Left ventricular systolic pressure (LVSP), left ventricular left ventricular end diastolic pressure (LVEDP) and LVSP up / down time (dP / (P> 0.05). Compared with control group, LVEDP, LVSP, FS and dP / dtmax of HGF group and combination therapy group were significantly increased (P < 0.05). Compared with the HGF group, the FS and dP / dtmax of the combined treatment group increased (P <0.05). Double immunofluorescence staining showed that the border zone of myocardial infarction was endothelial cells. Compared with the other three groups, the vascular density in the combination therapy group was significantly higher than that in the other three groups (P <0.05). Conclusions G-CSF combined with HGF gene-transplanted BMSCs can improve cardiac function and promote angiogenesis in the ischemic region of myocardial infarction at 4 weeks after myocardial infarction in rats. One of the mechanisms is the increase of ischemic myocardium VEGF protein expression.