【摘要】 目的:为保护濒死心肌提供机会窗口,对比观察经冠脉移植自体骨髓单个核细胞或间充质干细胞后,实验性急性心肌梗死动物心功能变化及心肌组织核转录因子кB、心肌细胞凋亡情况。方法:实验于2005-03/2006-11在河北省人民医院实验中心完成。选用24只雄性冀中白猪,随机数字表法分为4组:正常对照组、模型组、单个核细胞组、间充质干细胞组,6只/组。①24只猪均以盐酸氯胺酮200mg臀部肌肉注射麻醉后,分别于各自右侧股骨抽取骨髓20mL,采用Fercoll法分离获得骨髓单个核细胞,加入胶体金溶液,培养12～16h待用。分离过程中取出含有骨髓单个核细胞成分的细胞层,常规培养传代,每3d换液1次,贴壁生长细胞即为骨髓间充质干细胞,加入胶体金溶液,培养24h待用。②除正常对照组外,其余各组均经导管球囊封闭第一对角支以远的前降支,复制猪急性心肌梗死模型。单个核细胞组、间充质干细胞组均于造模后立即开通前降支,分别经球囊注入预先分离的骨髓单个核细胞6×108个、间充质干细胞6×108个。模型组造模后于梗死1h开通前降支,经球囊注入磷酸盐缓冲液10mL。③各组分别于术前及术后4周经心脏超声检测心功能,取材行病理学检查、心肌组织核转录因子кB的免疫组织化学检测及心肌细胞凋亡检测。结果:24只雄性白猪均进入结果分析。①心功能变化:术前各组左心室收缩末内径、左心室舒张末内径、左心室射血分数、短轴缩短率基本相似。移植术后4周,正常对照组、单个核细胞组、间充质干细胞组左心室舒张末内径均明显低于模型组(F=4.68,P=0.01),左心室射血分数及短轴缩短率均明显高于模型组(F=5.14,P=0.01;F=3.32,P=0.04),各组左心室收缩末内径差异无显著性意义(F=1.64,P=0.21)。②心肌组织病理学改变:电镜下单个核细胞组、间充质干细胞组在梗死边缘区可见有胶体金颗粒的不成熟的心肌细胞,胞质中散在肌丝结构,肌丝排列紊乱不规则。③心肌组织核转录因子кB阳性率表达:与模型组比较,单个核细胞组、间充质干细胞组的梗死边缘区核转录因子кB阳性率明显降低(F=25.59,P=0.0001);正常心肌区核转录因子кB阳性率亦明显降低(F=18.20,P=0.0001)。④心肌细胞凋亡检测结果:与模型组比较,单个核细胞组、间充质干细胞组在心肌梗死区细胞凋亡率均明显降低(F=6.63,P=0.0027),梗死边缘区细胞凋亡率亦明显降低(F=36.07,P=0.0001)。正常心肌区单个核细胞组细胞凋亡率与模型组基本相似(F=9.69,P=0.004),但间充质干细胞组有所降低。⑤心功能与心肌细胞凋亡及心肌组织NF-кB的相关性:急性心肌梗死4周时,左心室射血分数与心肌细胞凋亡、心肌组织核转录因子кB均呈负相关(r=0.613,P=0.001;r=-0.437,P=0.033)。心肌细胞凋亡与心肌组织核转录因子кB呈正相关(r=0.672,P=0.002)。结论:经冠脉移植骨髓单个核细胞和间充质干细胞均可改善实验性急性心肌梗死动物的心功能,与梗死边缘区核转录因子кB表达降低及心肌细胞凋亡减少有关。骨髓单个核细胞移植的促血管增生作用优于间充质干细胞移植。更多还原

【Abstract】 AIM: To protect the ischemic myocardium to be infarct, we study the effects of intracoronary autologous bone marrow mononuclear cells and mesenchymal stem cells on the cardiac function and the changes of myocardium nuclear factor (NF)-кB and myocardium apoptosis of acute myocardial infarction. METHODS: The experiment was undertaken in Experimental Center, Hebei Provincial People’s Hospital from March 2005 to November 2006. Twenty-four male white swines were selected and randomly divided into 4 groups, namely normal control group, model group, bone marrow mononuclear cells group and mesenchymal stem cells group, 6 in each group. ①Twenty-four swines was anaesthetized by intramuscularly injection 200 mg Ketamine, then 20 mL marrow was extracted from right thighbone. According Fercoll method autologous bone marrow mononuclear cells were gotten and mixed with colloid gold solution, cultured for 12-16 hours. Intercellular layer containing bone marrow mononuclear cells was obtained in separation process. Routine culture and passage was performed, change the liquor once every 3 days. Adhered cells were mesenchymal stem cells and mixed with colloid gold solution, cultured for 24 hours. ②Except normal control group, transcatheter blocking anterior descending branch made acute myocardial infarction models. Anterior descending branch was opened after establishing models, and 6×108 bone marrow mononuclear cells and 6×108 mesenchymal stem cells were infused into the criminal artery through Over-The-Wire balloon in the bone marrow mononuclear cells group and mesenchymal stem cells group. One hour after acute myocardial infarction anterior descending branch was opened in the model group. 10 mL phosphate buffer saline was inserted into sacculus sphaericus. ③Cardiac function ultrasound, pathology, immunohistochemistry of myocardial NF-кB and myocardial apoptosis were performed before operation and 4 weeks after operation in each group. RESULTS: Twenty-four male white swines entered the result analysis. ①cardiac function: Left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and shortening fraction were similar mostly in each group before operation. Four weeks after transplantation, LVEDD was obviously lower in the normal control group, bone marrow mononuclear cells group and mesenchymal stem cells group than the model group (F = 4.68,P = 0.01), while LVEF and shortening fraction were markedly higher than those in the model group (F = 5.14,P = 0.01;F = 3.32,P = 0.04). There was no significant difference of LVESD in each group (F =1.64,P = 0.21). ②pathological change of myocardium: Under electron microscope, immature myocardial cells containing colloid gold solution around infracted edge and disordered irregular myoneme structure in kytoplasm appeared in the bone marrow mononuclear cells group and mesenchymal stem cells group. ③positive expression of NF-кB in myocardium: In the margin area beside infarct myocardium the positive expression of NF-кB was significantly lower either in bone marrow mononuclear cells group and mesenchymal stem cells group as compared with the model group (F = 25.59,P = 0.000 1). The positive expression also obviously decreased in normal myocardium (F = 18.20,P = 0.000 1). ④apoptosis of myocardial cells: Compared with the model group, the myocardial apoptotic index in infarct zone was remarkably decreased in the bone marrow mononuclear cells group and mesenchymal stem cells group (F = 6.63,P = 0.002 7). The index significantly reduced in infarct and marginal zone (F = 36.07,P = 0.000 1). The apoptotic rate was similar in normal myocardium between bone marrow mononuclear cells group and model group (F = 9.69,P = 0.004). But it decreased in the mesenchymal stem cells group. ⑤correlation of cardiac function and myocardial apoptosis and NF-кB: At week four, LVEF had a negative correlation to myocardial apoptotic index and myocardial NF-кB(r = 0.613,P = 0.001;r =-0.437,P = 0.033). Myocardial apoptotic index had a positive correlation to myocardial NF-кB (r = 0.672,P = 0.002). CONCLUSION: Intracoronary transplant autologous bone marrow mononuclear cells and mesenchymal stem cells can improve cardiac function in acute myocardial infarction swine, which is associated with decreasing of expression of NF-кB and myocardial apoptosis. The effects of bone marrow mononuclear cells transplantation on angioginesis are surpassed to those of mesenchymal stem cell transplantation.更多还原