芪参益气滴丸联合骨髓间充质干细胞移植对心肌梗死小鼠血管新生及心功能的影响研究

Experimental study of Qishen Yiqi drop pill combined with bone marrow mesenchymal stem cell transplantation on angiogenesis and cardiac function in mice with myocardial infarction

目的:利用体外细胞分子生物学实验探讨芪参益气滴丸联合骨髓间充质干细胞移植对心肌梗死后小鼠血管生成及心功能的影响。方法:采用C57BL/6J的雄性小鼠,将60只小鼠采用数字随机法分为3组(n=20):心肌梗死组(MI+PBS组)、心肌梗死+间充质干细胞移植组(MI+MSCs组)和心肌梗死+间充质干细胞移植+芪参益气滴丸组(MI+MSCs+QSYQ组)。用蒸馏水将芪参益气滴丸制备为3.9 mg/mL浓度的药液,MI+MSCs+QSYQ组按0.1 mL·kg^(-1)·d^(-1)灌胃,MI+PBS组和MI+MSCs组均按等量生理盐水灌胃。各组小鼠经适应性连续喂养14 d后,采用开胸的方式结扎冠状动脉前降支建立MI模型,造模24 h后,从小鼠胫骨或腓骨中分离骨髓间充质干细胞,将骨髓间充质干细胞通过多点心肌内注射的方式注射到小鼠心肌梗死临近区域,心肌梗死组以同样方式注射等量PBS。在显微镜下观察骨髓间充质干细胞移植0 h及3、7、14 d的干细胞形态变化;21 d后应用彩色多普勒超声心动图进行心功能检测;造模完成21 d后,处死小鼠,留取心脏标本,用免疫组化染色和微血管密度的测定观察小鼠血管新生情况。结果:移植干细胞形态生长良好与细胞移植时间成正比,与MI+PBS组比较,MI+MSCs组和MI+MSCs+QSYQ组骨髓间充质干细胞表面的CD90.2和Ly6A呈高表达,CD31和CD117几乎不表达。干细胞移植21 d,MI+MSCs+QSYQ组LVDd、LVSd值明显低于MI+PBS组和MI+MSCs组,同时LVEF、LVFS均明显升高(P<0.05)。免疫组化定量分析结果提示MI+MSCs+QSYQ组的血管新生密度较其余两组明显增多,组间差异具有统计学意义(P<0.05)。结论:芪参益气滴丸联合骨髓间充质干细胞移植不仅能促进心肌梗死小鼠血管新生,而且对改善心功能具有积极作用。

Objective:To investigate the effects of Qishengyiqi drop pill combined with bone marrow mesenchymal stem cell transplantation on angiogenesis and cardiac function in mice after myocardial infarction through in vitro cell molecular biology experiments.Methods:The animals used in this experiment were male mice with C57BL/6J.Totally sixty mice were randomly divided into three groups(n=20):myocardial infarction group(MI+PBS),myocardial infarction+mesenchyme plasma stem cell transplantation group(MI+MSCs)and myocardial infarction+Qishenyiqi drip pill combined with mesenchymal stem cell transplantation group(MI+MSCs+QSYQ).Qishenyiqi dripping pills were prepared into a medicinal solution with a concentration of 3.9 mg/mL with distilled water.The MI+MSCs+QSYQ group was orally administered with 0.1 mL/kg/day,and the other two groups were orally administered with an equal amount of normal saline.Mice in each group were adaptively fed continuously for 2 weeks,and the myocardial infarction model was established by ligation of the anterior descending coronary artery by thoracic ligation.Twenty-four hours after the model was established,bone marrow mesenchymal stem cells were isolated from the tibia of the mice and injected intracardiacly.Bone marrow-derived mesenchymal stem cells were transplanted,and multiple injections were made around the myocardial infarction area of mice.The control group was injected with the same amount of PBS.0 h,3 days,7 days,and 14 days after cell transplantation,the stem cell morphology was observed under a microscope;on day 7 of cell transplantation,the expression of eGFP-positive cells was tracked with a fluorescence microscope;before modeling,14 and 21 days after cell transplantation,cardiac function was measured by echocardiography.After 21 days of modeling,the mice were sacrificed,and heart samples were taken.The angiogenesis of the mice was observed by immunohistochemical staining and microvascular density determination.Results:The morphological growth of transplanted stem cells was proportional to the time of cell transplantation.Compared with MI+PBS group,CD90.2 and Ly6A were highly expressed on the surface of bone marrow mesenchymal stem cells in MI+MSCs group and MI+MSCs+QSYQ group,while CD31 and CD117 were almost not expressed.On the 21st day after stem cell transplantation,the values of LVDd and LVSD in MI+MSCs+QSYQ group were significantly lower than those in MI+PBS group and MI+MSCs group.At the same time,LVEF and LVFS increased significantly.The results of quantitative immunohistochemical analysis showed that the angiogenesis density in the MI+MSCs+QSYQ group increased significantly,and the difference between the groups was statistically significant(P<0.05).Conclusion:Qishenyiqi dripping pills combined with bone marrow mesenchymal stem cell transplantation can not only promote angiogenesis in mice with myocardial infarction,but also plays a positive role in improving cardiac function.