脂肪来源干细胞移植治疗心肌梗死大鼠的实验研究
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摘要
[目的]研究脂肪来源干细胞移植治疗心肌梗死的可行性。
[方法]1、脂肪来源干细胞的培养和诱导分化：由SD大鼠的肠系膜脂肪获得脂肪来源间充质干细胞,培养后用10μmol·-1浓度的5氮杂胞苷(5-azacytidine)诱导,经透射电镜、扫描电镜、免疫组化等检测诱导后细胞的变化。2、脂肪来源干细胞的标记：超小超顺磁性氧化铁(ultrasmall superparamagnetic iron oxide, USPIO)40μg Fe/ml、多聚赖氨酸(poly-1-lysine, PLL)1.5μg/ml与ADSCs共孵育培养,普鲁士蓝染色和透射电镜验证标记有效性,细胞培养液检测VEGF浓度。3、心梗模型的建立及细胞移植术：采取结扎前降支的办法建立SD大鼠心肌梗死模型,经Micro-PET及MRI确认心梗。采取经胸心外膜注射法将ADMSCs移植于心肌梗死区。4、脂肪来源干细胞在心肌梗死区的存活及对实验动物心功能的影响。通过Micro-PET了解心梗后梗塞灶大小的变化；1.5T MR扫描仪对实验组(n=10)和空白对照组(n=10)以及心梗组大鼠(n=10)行磁共振成像,观察心肌信号、计算并比较两组大鼠的左室舒张末容积(left-ventricular end-diastolic volume, LVEDV).左室收缩末容积(left-ventricular end-systolic volume, LVESV)及左室射血分数(left-ventricular ejection fraction, LVEF),来了解移植前后心功能变化。病理组织学检查观察心肌结构和标记ADSCs的分布。
[结果]1、脂肪来源干细胞经10μmol·-1浓度的5氮杂胞苷(5-azacytidine)诱导后,透射电镜可见到明显肌丝,扫描电镜可看到细胞呈现心肌细胞样外观变化,免疫组化可见到诱导后细胞为肌红蛋白抗体所着染。2、普鲁士蓝染色显示USPIO标记脂肪来源干细胞的阳性率>99%,透射电镜下可见黑色氧化铁颗粒位于细胞溶酶体内,细胞培养液可以检测到VEGF。3、开胸结扎冠脉前降支成功构建心梗模型,实验组大鼠心脏于Micro-PET下行18F-FDG及68Ga-RGD显像显示心尖部无血流灌注,无心肌存活；移植细胞后梗塞区面积减小,有血管再生。MR检查显示FIESTA和FSPGR序列图像上可见左室前壁内发现信号降低和室壁运动异常,并于2D MDE序列图像上发现实验组心肌内出现延迟强化。心梗组LVEDV为：0.62±0.37ml, LVESV为0.28±0.36ml, LVEF为40.57±3.03%,移植USPIO标记的ADSCs细胞组LVEDV为：0.51±0.31ml, LVESV为0.27±0.38ml,LVEF为50.94±59%,以上数据除LVESV外心梗组均低于移植细胞组,有显著差异(P<0.05),而两组之间LVESV无明显差异(P>0.05)。空白对照组的LVEDV为：0.44±0.30ml, LVESV为0.20±0.23ml, LVEF为62.84±3.65%,与移植USPIO标记的ADSCs细胞组相比,移植USPIO标记的ADSCs细胞组的以上数据均低于空白对照组,有显著差异(P<0.05)。空白对照组的数据与心梗组的数据相比较,心梗组的数据均低于空白对照组,有显著差异(P<0.05)。4、病理组织学检查于梗死心肌周边发现局灶性氧化铁颗粒沉积。
[结论]脂肪来源干细胞可经5-氮杂胞苷诱导转化为心肌样细胞,移植后可存活于心肌梗死区并能改善实验动物的心功能。
[Objective] To investigate the feasibility inducing adipose-derived stem cells into myogenic cells in vitro and a potential treatment of ischemic heart disease with transplantation of adipose-derived stem cells.
[Methods]1Adipose-derived stem cells are taken from Sprague Dawley (SD)rat's mesenteric adipose tissue and cultured, third-passaged cells were exposed to10μmol·1-1dilutions of5-azacytidine for24hours. Transmission Electron microscopy, Scanning Electron microscopy, immunohistochemistry were used to demonstrate the characteristics of the cells.2ADSCs were incubated with culture medium containing40μg/ml USPIO and1.5μg/ml poly-1-lysine (PLL) for24h. The distribution of iron particles in cells was determined by Prussian blue staining and transmission electron microscopy (TEM).3A model myocardial infarction was created by ligation of the distal left anterior descending artery in SD rat. The labeled ADSCs were injected transthoracic into the myocardium. In vivo.4The infraction area was assessed by Micro-PET and MR. MR imaging was performed with FIESTA Cine, FSPGR Cine and2D MDE sequences for all groups. The left-ventricular end-diastolic volume (LVEDV), left-ventricular end-systolic volume (LVESV) and left-ventricular ejection fraction (LVEF) were calculated on Report Card Workstastion to assess the cardiac function. Postmortal study was carried out to determine the distribution of USPIO particles in the myocadium with Prussian blue stain.
[Results] The myofiber in adipose-derived stem cells was demonstrated by transmission electron microscopy after ADSCs exposed to5-aza, which was stained by immunohistochemistry of anti-myoglobin antibody. The cardiac function of the Sprague Dawley rat was improved and the infraction area was reduced after the transplantation of adipose-derived stem cells.
[Conclusion] Adipose-derived stem cells can differentiate into myogenic cells in vitro after5-azacytidine exposure. Transplantation adipose-derived stem cells may improve cardiac function of ischemic heart disease and shrink the infraction area of the heart in Sprague Dawley rat.
[方法]1、脂肪来源干细胞的培养和诱导分化：由SD大鼠的肠系膜脂肪获得脂肪来源间充质干细胞,培养后用10μmol·-1浓度的5氮杂胞苷(5-azacytidine)诱导,经透射电镜、扫描电镜、免疫组化等检测诱导后细胞的变化。2、脂肪来源干细胞的标记：超小超顺磁性氧化铁(ultrasmall superparamagnetic iron oxide, USPIO)40μg Fe/ml、多聚赖氨酸(poly-1-lysine, PLL)1.5μg/ml与ADSCs共孵育培养,普鲁士蓝染色和透射电镜验证标记有效性,细胞培养液检测VEGF浓度。3、心梗模型的建立及细胞移植术：采取结扎前降支的办法建立SD大鼠心肌梗死模型,经Micro-PET及MRI确认心梗。采取经胸心外膜注射法将ADMSCs移植于心肌梗死区。4、脂肪来源干细胞在心肌梗死区的存活及对实验动物心功能的影响。通过Micro-PET了解心梗后梗塞灶大小的变化；1.5T MR扫描仪对实验组(n=10)和空白对照组(n=10)以及心梗组大鼠(n=10)行磁共振成像,观察心肌信号、计算并比较两组大鼠的左室舒张末容积(left-ventricular end-diastolic volume, LVEDV).左室收缩末容积(left-ventricular end-systolic volume, LVESV)及左室射血分数(left-ventricular ejection fraction, LVEF),来了解移植前后心功能变化。病理组织学检查观察心肌结构和标记ADSCs的分布。
[结果]1、脂肪来源干细胞经10μmol·-1浓度的5氮杂胞苷(5-azacytidine)诱导后,透射电镜可见到明显肌丝,扫描电镜可看到细胞呈现心肌细胞样外观变化,免疫组化可见到诱导后细胞为肌红蛋白抗体所着染。2、普鲁士蓝染色显示USPIO标记脂肪来源干细胞的阳性率>99%,透射电镜下可见黑色氧化铁颗粒位于细胞溶酶体内,细胞培养液可以检测到VEGF。3、开胸结扎冠脉前降支成功构建心梗模型,实验组大鼠心脏于Micro-PET下行18F-FDG及68Ga-RGD显像显示心尖部无血流灌注,无心肌存活；移植细胞后梗塞区面积减小,有血管再生。MR检查显示FIESTA和FSPGR序列图像上可见左室前壁内发现信号降低和室壁运动异常,并于2D MDE序列图像上发现实验组心肌内出现延迟强化。心梗组LVEDV为：0.62±0.37ml, LVESV为0.28±0.36ml, LVEF为40.57±3.03%,移植USPIO标记的ADSCs细胞组LVEDV为：0.51±0.31ml, LVESV为0.27±0.38ml,LVEF为50.94±59%,以上数据除LVESV外心梗组均低于移植细胞组,有显著差异(P<0.05),而两组之间LVESV无明显差异(P>0.05)。空白对照组的LVEDV为：0.44±0.30ml, LVESV为0.20±0.23ml, LVEF为62.84±3.65%,与移植USPIO标记的ADSCs细胞组相比,移植USPIO标记的ADSCs细胞组的以上数据均低于空白对照组,有显著差异(P<0.05)。空白对照组的数据与心梗组的数据相比较,心梗组的数据均低于空白对照组,有显著差异(P<0.05)。4、病理组织学检查于梗死心肌周边发现局灶性氧化铁颗粒沉积。
[结论]脂肪来源干细胞可经5-氮杂胞苷诱导转化为心肌样细胞,移植后可存活于心肌梗死区并能改善实验动物的心功能。
[Objective] To investigate the feasibility inducing adipose-derived stem cells into myogenic cells in vitro and a potential treatment of ischemic heart disease with transplantation of adipose-derived stem cells.
[Methods]1Adipose-derived stem cells are taken from Sprague Dawley (SD)rat's mesenteric adipose tissue and cultured, third-passaged cells were exposed to10μmol·1-1dilutions of5-azacytidine for24hours. Transmission Electron microscopy, Scanning Electron microscopy, immunohistochemistry were used to demonstrate the characteristics of the cells.2ADSCs were incubated with culture medium containing40μg/ml USPIO and1.5μg/ml poly-1-lysine (PLL) for24h. The distribution of iron particles in cells was determined by Prussian blue staining and transmission electron microscopy (TEM).3A model myocardial infarction was created by ligation of the distal left anterior descending artery in SD rat. The labeled ADSCs were injected transthoracic into the myocardium. In vivo.4The infraction area was assessed by Micro-PET and MR. MR imaging was performed with FIESTA Cine, FSPGR Cine and2D MDE sequences for all groups. The left-ventricular end-diastolic volume (LVEDV), left-ventricular end-systolic volume (LVESV) and left-ventricular ejection fraction (LVEF) were calculated on Report Card Workstastion to assess the cardiac function. Postmortal study was carried out to determine the distribution of USPIO particles in the myocadium with Prussian blue stain.
[Results] The myofiber in adipose-derived stem cells was demonstrated by transmission electron microscopy after ADSCs exposed to5-aza, which was stained by immunohistochemistry of anti-myoglobin antibody. The cardiac function of the Sprague Dawley rat was improved and the infraction area was reduced after the transplantation of adipose-derived stem cells.
[Conclusion] Adipose-derived stem cells can differentiate into myogenic cells in vitro after5-azacytidine exposure. Transplantation adipose-derived stem cells may improve cardiac function of ischemic heart disease and shrink the infraction area of the heart in Sprague Dawley rat.
引文
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