骨髓干细胞动员与梗死心肌修复及移植静脉桥预后的实验研究
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摘要
背景：冠状动脉搭桥(冠脉搭桥)可改善心肌梗死区的血液供应，促进骨髓干细胞动员，但由于外周血中骨髓干细胞数量有限，其在心肌梗死(心梗)区促进组织血运重建及心肌再生的潜能目前还没有受到重视。本实验从三方面研究冠脉搭桥与骨髓干细胞动员相结合治疗缺血性心脏病的可行性。
第一部分为临床实验。目的：研究冠脉搭桥术后骨髓干细胞动员规律及影响因素。材料与方法：符合实验要求的36例冠心病患者，采用流式细胞仪检测非体外循环冠脉搭桥术后外周血CD34~+细胞计数变化，间接反应骨髓干细胞动员情况。结果：冠心病患者外周血中存在少量CD34~+细胞，术后24小时达到峰值，48-72小时恢复到术前水平；CD34~+细胞动员与血清心肌酶及血管内皮生长因子呈高度正性相关。
第二部分为心梗治疗实验。目的：研究骨髓干细胞动员对梗死心肌修复及心梗后心功能变化的影响。材料与方法：新西兰大白兔24只，随机均分为两组，结扎实验动物前降支制作急性心肌梗死模型，实验组术后24小时开始给予重组人粒细胞集落刺激因子(100ug／Kg)皮下注射，1次／日，连续10天，对照组实验动物给予等量生理盐水皮下注射，骨髓干细胞动员情况以实验动物外周血有核细胞计数及单个核细胞比例表示，观察实验动物心功能变化及梗死区病理改变。结果：结扎实验动物前降支可成功制作急性心肌
骨髓十细胞动员’J梗死心肌修复及移植静脉桥预后的实验研究
梗死模型，重组人粒细胞集落刺激因子可动员兔骨髓干细胞入外周
血，实验组与对照组相比，心肌梗死面积降低、心梗区小血管密度
增加、左室射血分数改善。
第三部分为静脉桥移植实脸。目的:研究骨髓干细胞动员对移
植静脉桥近期通畅率及吻合口内膜增生的影响。材料与方法:新西
兰大白兔24只，随机均分为实验组和对照组，全身麻醉下颈部正
中切口，8一0无创带针棉纶线行动一静脉端侧吻合，同时建立双侧颈
动一静脉桥移植模型，其中一侧静脉桥移植前用0 .5%胰蛋白酶消化
掉内皮细胞，实验组动物骨髓干细胞动员及检测同第二部分，观察
移植静脉桥近期通畅率及内膜增生情况。结果:内皮完整移植静脉
桥在实验组和对照组均有较高通畅率;去内皮移植静脉桥通畅率明
显降低，实验组通畅率67%，对照组通畅率30%，实验组明显高于
对照组;病理检查示所有移植静脉桥吻合口内膜均有不同程度增
生，去内皮移植静脉桥内膜增生程度较重，但实验组去内皮移植静
脉桥再内皮化完全，吻合口内膜增生程度明显轻于对照组;统计分
析结果表明骨髓干细胞动员、内皮结构完整及两者的交互作用有利
于降低植静脉桥吻合口内膜增生程度。
结论:骨髓干细胞动员有利于梗死心肌修复，改善心功能;同
时对移植静脉桥有保护作用，可促进移植静脉桥再内皮化，提高近
期通畅率，预防吻合口内膜增生引起的再狭窄。由此可见，冠脉搭
桥与骨髓干细胞动员相结合可提高缺血性心脏病的临床疗效。
Background: Coronary artery bypass graft can improve blood supply to infarcted myocardial areas and facilitate bone marrow stem cell mobilization. But the potency of bone marrow stem cells on infarcted myocardium repair is often omitted because there are only a few stem cells in the peripheral blood circulation. The feasibility of combined coronary artery bypass graft and bone marrow stem cell mobilization for ischemia heart disease treatment was studied in three parts.
Part I. Objective: To investigate the pattern and the affecting factors of bone marrow stem cell mobilization after off-pump coronary artery bypass graft. Material and methods: According to experimental requirements, 36 cases of coronary artery disease were chosen for this study. The CD34+ cell counts were made by flow cytometer after off-pump coronary artery bypass. The increase of CD34+ cell counts can represent bone marrow stem cells mobilization indirectly. Results: There were a few CD34+ cells in the peripheral blood circulation in patients with coronary artery diseases before operation. The CD34+ cell counts significantly increased, peaking at 24 hours after operation, and turn back to pre-operation level at 48-72 hours after operation. There are highly positive correlations between CD34+ cell mobilization and serum myocardium enzyme and vascular endothelial growth factor.
Part II. Objective: To further investigate the effects of bone marrow stem cell mobilization on infarcted myocardium and cardiac function. Material and methods: New Zealand rabbits, 24 cases, were divided equally into two groups at random. Acute myocardial infarction model was induced by ligating left anterior descending branch of coronary artery with 4-0 noninvasive medical sutures. In the test group recombinant human granulocyte colony-stimulating factor was given by subcutaneous injection 24 hours after operation, once per day in successive 10 days. In the control group saline was given in the same way. Bone marrow stem cell mobilization was reflected in karyote counts and mononuclear cell proportion in peripheral blood. Results: Acute myocardial infarction model was made
successfully by left anterior descending branch ligation. The ejection fraction of left ventricular in the test group was improved compared to control group, with decreased infarcted myocardial areas and increased small vessel density in the infarcted myocardial areas.
Part III. Objective: To investigate the effects of bone marrow stem cell mobilization on grafted vein bridge prognosis and intima proliferation of anastomosis. Material and methods: New Zealand rabbits, 24 cases were divided equally into test group and control group at random. Cervical middle incision was made under general anesthesia. Bilateral vein bridges by end to side anastomosis were made to the common carotid arteries with 8-0 Dacron sutures. Vascular endothelium of one grafted vein bridge was digested by 0.5% trypsin. The way of bone marrow stem cell mobilization was the same as that in Part II. Results: The grafted vein bridges with intact endothelium had higher rate of patency, and the lower patent rate was seen in grafted vein bridges with complete endothelial denudation. The patent rate of the grafted vein bridges with complete endothelial denudation was 67% versus 30% between test group and control group. Pathological examination showed that there were various degrees of intima proliferation in
all anastomosises of grafted vein bridges. The intima proliferation of anastomosis in grafted vein bridges with complete endothelial denudation was more severe than grafted vein bridges anastomosis with intact endothelium. In the test group reendothelization hi grafted vein bridges with complete endothelial denudation occurred completely and the anastomosis had relatively lower intima proliferation than that in the control group. Statistic analysis demonstrated that bone marrow stem cell mobilization and intact endothelium and their interaction might prove advantageous to reduce the degree of anastomosis intima proliferation.
Concl
第一部分为临床实验。目的：研究冠脉搭桥术后骨髓干细胞动员规律及影响因素。材料与方法：符合实验要求的36例冠心病患者，采用流式细胞仪检测非体外循环冠脉搭桥术后外周血CD34~+细胞计数变化，间接反应骨髓干细胞动员情况。结果：冠心病患者外周血中存在少量CD34~+细胞，术后24小时达到峰值，48-72小时恢复到术前水平；CD34~+细胞动员与血清心肌酶及血管内皮生长因子呈高度正性相关。
第二部分为心梗治疗实验。目的：研究骨髓干细胞动员对梗死心肌修复及心梗后心功能变化的影响。材料与方法：新西兰大白兔24只，随机均分为两组，结扎实验动物前降支制作急性心肌梗死模型，实验组术后24小时开始给予重组人粒细胞集落刺激因子(100ug／Kg)皮下注射，1次／日，连续10天，对照组实验动物给予等量生理盐水皮下注射，骨髓干细胞动员情况以实验动物外周血有核细胞计数及单个核细胞比例表示，观察实验动物心功能变化及梗死区病理改变。结果：结扎实验动物前降支可成功制作急性心肌
骨髓十细胞动员’J梗死心肌修复及移植静脉桥预后的实验研究
梗死模型，重组人粒细胞集落刺激因子可动员兔骨髓干细胞入外周
血，实验组与对照组相比，心肌梗死面积降低、心梗区小血管密度
增加、左室射血分数改善。
第三部分为静脉桥移植实脸。目的:研究骨髓干细胞动员对移
植静脉桥近期通畅率及吻合口内膜增生的影响。材料与方法:新西
兰大白兔24只，随机均分为实验组和对照组，全身麻醉下颈部正
中切口，8一0无创带针棉纶线行动一静脉端侧吻合，同时建立双侧颈
动一静脉桥移植模型，其中一侧静脉桥移植前用0 .5%胰蛋白酶消化
掉内皮细胞，实验组动物骨髓干细胞动员及检测同第二部分，观察
移植静脉桥近期通畅率及内膜增生情况。结果:内皮完整移植静脉
桥在实验组和对照组均有较高通畅率;去内皮移植静脉桥通畅率明
显降低，实验组通畅率67%，对照组通畅率30%，实验组明显高于
对照组;病理检查示所有移植静脉桥吻合口内膜均有不同程度增
生，去内皮移植静脉桥内膜增生程度较重，但实验组去内皮移植静
脉桥再内皮化完全，吻合口内膜增生程度明显轻于对照组;统计分
析结果表明骨髓干细胞动员、内皮结构完整及两者的交互作用有利
于降低植静脉桥吻合口内膜增生程度。
结论:骨髓干细胞动员有利于梗死心肌修复，改善心功能;同
时对移植静脉桥有保护作用，可促进移植静脉桥再内皮化，提高近
期通畅率，预防吻合口内膜增生引起的再狭窄。由此可见，冠脉搭
桥与骨髓干细胞动员相结合可提高缺血性心脏病的临床疗效。
Background: Coronary artery bypass graft can improve blood supply to infarcted myocardial areas and facilitate bone marrow stem cell mobilization. But the potency of bone marrow stem cells on infarcted myocardium repair is often omitted because there are only a few stem cells in the peripheral blood circulation. The feasibility of combined coronary artery bypass graft and bone marrow stem cell mobilization for ischemia heart disease treatment was studied in three parts.
Part I. Objective: To investigate the pattern and the affecting factors of bone marrow stem cell mobilization after off-pump coronary artery bypass graft. Material and methods: According to experimental requirements, 36 cases of coronary artery disease were chosen for this study. The CD34+ cell counts were made by flow cytometer after off-pump coronary artery bypass. The increase of CD34+ cell counts can represent bone marrow stem cells mobilization indirectly. Results: There were a few CD34+ cells in the peripheral blood circulation in patients with coronary artery diseases before operation. The CD34+ cell counts significantly increased, peaking at 24 hours after operation, and turn back to pre-operation level at 48-72 hours after operation. There are highly positive correlations between CD34+ cell mobilization and serum myocardium enzyme and vascular endothelial growth factor.
Part II. Objective: To further investigate the effects of bone marrow stem cell mobilization on infarcted myocardium and cardiac function. Material and methods: New Zealand rabbits, 24 cases, were divided equally into two groups at random. Acute myocardial infarction model was induced by ligating left anterior descending branch of coronary artery with 4-0 noninvasive medical sutures. In the test group recombinant human granulocyte colony-stimulating factor was given by subcutaneous injection 24 hours after operation, once per day in successive 10 days. In the control group saline was given in the same way. Bone marrow stem cell mobilization was reflected in karyote counts and mononuclear cell proportion in peripheral blood. Results: Acute myocardial infarction model was made
successfully by left anterior descending branch ligation. The ejection fraction of left ventricular in the test group was improved compared to control group, with decreased infarcted myocardial areas and increased small vessel density in the infarcted myocardial areas.
Part III. Objective: To investigate the effects of bone marrow stem cell mobilization on grafted vein bridge prognosis and intima proliferation of anastomosis. Material and methods: New Zealand rabbits, 24 cases were divided equally into test group and control group at random. Cervical middle incision was made under general anesthesia. Bilateral vein bridges by end to side anastomosis were made to the common carotid arteries with 8-0 Dacron sutures. Vascular endothelium of one grafted vein bridge was digested by 0.5% trypsin. The way of bone marrow stem cell mobilization was the same as that in Part II. Results: The grafted vein bridges with intact endothelium had higher rate of patency, and the lower patent rate was seen in grafted vein bridges with complete endothelial denudation. The patent rate of the grafted vein bridges with complete endothelial denudation was 67% versus 30% between test group and control group. Pathological examination showed that there were various degrees of intima proliferation in
all anastomosises of grafted vein bridges. The intima proliferation of anastomosis in grafted vein bridges with complete endothelial denudation was more severe than grafted vein bridges anastomosis with intact endothelium. In the test group reendothelization hi grafted vein bridges with complete endothelial denudation occurred completely and the anastomosis had relatively lower intima proliferation than that in the control group. Statistic analysis demonstrated that bone marrow stem cell mobilization and intact endothelium and their interaction might prove advantageous to reduce the degree of anastomosis intima proliferation.
Concl
引文
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2. Luttun A, Carmeliet G, Carmeliet P. Vascular progenitors: from biology to treatment. Trends Cardiovasc Med. 2002;12: 88-96.
3. Rafii S, Heissig B, Hattori K. Efficient mobilization and recruitment of marrow-derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors. Gene Ther. 2002;9: 631-641.
4. Chambers DJ. Vascular endothelial growth factor and coronary artery surgery: implications for improved endothelial protection! European Heart Journal. 2000;21: 1649-1651.
5. Takahashi T, Kalka C, Masuda H, et al. Ischemia and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovasculariation. Nat Med. 1999;5: 434-438.
6. Moore MA, Hattori K, Heissig B, et al, Mobilization of endothelial and hematopoietic stem progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin- 1. Ann NY Acad Sci. 2001 ;938: 36-45.
7. Badorff C, Brandes RP, Popp R, et al. Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation. 2003;107: 1024-1032.
8. Condorelli G, Borello U, De Angelis L, et al. Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration. Proc Natl Acad Sci USA. 2001;14: 2540-2548.
9. Kawamoto A, Tkebuchava T, Yamaguchi JI, et al. Intramyocardial transplantation of autologous endothelial progenitor cell for therapeutic neovascularization of myocardial ischemia. Circulation. 2003; 107: 461-468.
10. Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001; 7: 430-436.
11. Kamihata H, Matsubara H, Nishiue T, et al. Improvement of collateral perfusion and regional function by implantation of peripheral blood mononuclear cells into ischemic hibernating myocardium. Arterioscler Thromb Vasc Biol. 2002;22: 1804-1810.
12. Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded
endothelial progenitor cells for myocardial ischemia. Circulation. 2001;103: 634-637.
13. Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA. 2000;97: 3422-3427.
14. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18): 10344-9.
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