超声微泡预处理对骨髓间充质干细胞向缺血心肌归巢的影响

Effects of ultrasound-exposed microbubbles pretreatment on bone marrow mesenchymal stem cells homing to ischemic myocardium

目的探讨超声微泡（UM）预处理对骨髓间充质干细胞（BMSCs）向缺血心肌归巢及梗死后心功能的影响。方法冠脉结扎法制备大鼠急性心肌梗死（AMI）模型，随机分为PBS组、干细胞治疗（SCT）组、超声＋干细胞治疗（USCT）组及uM＋干细胞治疗（UMSCT）组，分别经尾静脉注入PBS、干细胞、US预处理后的干细胞（USCT）和UM预处理后的干细胞（UMSCT）。48h后共聚焦显微镜检测BMSCs向缺血心肌的归巢情况；4周后超声心动图检测心功能，Masson染色观察局部梗死面积，免疫组化检测局部新生毛细血管密度（cD31）等。结果①归巢情况：BMSCs移植48h后，uMsCT组心肌缺血区荧光阳性细胞数显著多于USCT组和SCT组；②后期心功能：BMSCs移植4周后，uMSCT组的左室收缩功能强于USCT组和SCT组，而后二者间差异无统计学意义，但显著强于PBS组；③病理学检查：Masson染色显示USCT组的梗死面积百分比[（35．9±1．1）％]与SCT组[（36．5±1．3）％]差异无统计学意义（P〉0．05），但显著小于PBS组[（45．2±1．4）％，P〈0．05]。UMSCT组的梗死面积百分比[（25．8±1．O）％]显著小于USCT组和SCT组（P〈O．05）。免疫组化结果显示USCT组的新生毛细血密度（25．9±1．3）与SCT组（25．2±1．3）差异无统计学意义（P〉0．05），但显著多于PBS组（17．6±1．1，P〈0．05）。UMSCT组的新生毛细血密度（33．2±1．6）显著多于USCT组和SCT组（P〈0．05）。结论UM预处理可促进经静脉移植的BMSCs向心肌缺血区的归巢，进一步促进毛细血管的新生，减少梗死面积，改善AMI后心功能。

Objective To explore the effects of pretreatment of bone marrow mesenchymal stem cells （BMSCs） by ultrasound-exposed microbubbles （UM） on both homing to ischemic myocardium and cardiac function after acute myocardial infarction （AMI）. Methods Rats of AMI model established by ligation of left anterior descending coronary artery were divided into four groups randomized., phospho-buffered saline （PBS） group, stem cells treatment （SCT） group, ultrasound and stem cells treatment （USCT） group, and UM stem cells treatment （UMSCT） group, and each group was injected with PBS, stem cells, US-pretreated stem cells and UM-pretreated stem cells through the caudal veins after AMI respectively. Homing of BMSCs to the ischemic myocardium was examined by confocal microscopy at 48 h after implantation, and cardiac function was examined by ultrasonic cardiogram （UCG） after 4 weeks. Masson staining was used to examine the changes of local ischemic cardiac tissues, and immunohistochemistry was used to detect the density of local neo-capillaries （CD31）. Results 1） The numbers of CM-Dil-positive cells counted under confocal microscopy in the ischemic myocardial tissues of each groups 48 hours after implantation were not the same：there was no significant difference of the numbers of positive cells between USCT group （19.67 ±2.08） and SCT group （18.67 ± 2.08）. However,the number of positive cells in the UMSCT group （39.33 ± 3.06） was larger than that in USCT group and SCT group （ P〈0.05）. 2） UCG examinations showed that there was no significant difference of left ventricular systole function between the USCT group [-LVEF （44.92± 2.77）%,LVFS （22.83± 1.79）] and SCT group [,LVEF （42.28 ±2.82）M,LVFS （21.52 ± 1.88） , P 〈0.05],but both were better than that in PBS group [,LVEF （20.52 ± 1.88） ,LVFS （9.55 ±0.85） , P 〈0.05]. The left ventricular systolic function in UMSCT group [LVEF （61.85 ± 3.15）%, LVFS （32.74 ± 2.45）%] was significantly higher than that in USCT group and SCT group （P 〈0.05）, while which was still significantly lower than that in pseudo-surgery group [,LVEF （75.88 ± 4.52）%, LVFS （42.76±2.88）%, P 〈0.05]. 3） Pathological examinations showed the percentages of AMI areas in the USCT group （35.9± 1.1%） were not different compared with that in SCT group [（36.5 ± 1.3）%, P 0.05],while both were significantly smaller than that in PBS group [（45.2 ± 1.4）%, P 〈0.05]. The percentages of AMI areas in the UMSCT group [（25.8 ± 1.0）%] were significantly smaller than that in USCT group and SCT group （ P〈0.05）. The density of neo-capillaries （25.9 ± 1.3） in USCT groups had no difference compared with that in SCT group （25.2 ± 1.3）, while both were significantly higher than that in PBS group （17.6± 1.1, P 〈0.05） ; the density of neo-capillaries （33.2 ± 1.6） was significantly higher in UMSCT group than that in both USCT group and SCT group （ P 〈0.05）, which were examined by immunohistochemistry. Conclusions Homing to ischemic myocardium of BMSCs transplanted intravenously could be promoted by UM pretreatment, which stimulates development of capillaries, reduces AMI areas,and improves the cardiac function after AMI.