【作者】 李文斌；

【导师】 余国龙；

【作者基本信息】 中南大学 ， 心血管内科， 2010， 硕士

【摘要】 目的：探讨人脐血单个核细胞(HUMNCs)静脉多次移植对急性心肌梗死(AMI)后心肌胶原网络重构的影响。方法：自健康、足月分娩孕妇取脐血,分离并培养脐血单个核细胞(HUMNCs),60只健康中国家兔随机分为4组：(1)假手术组(n=15),冠脉前降支(LAD)过线不结扎,术后7d,9d,11d,13d,15d经耳缘静脉注入生理盐水500uL；(2)心肌梗死对照组(n=15),结扎冠脉前降支,术后7d,9d,11d,13d,15d同途径注入等量生理盐水；(3)HUMNCs单次静脉移植组(n=15),结扎冠脉前降支,术后7d,同途径注入含有3x106HUMNCs生理盐水500uL,9d,11d,13d,15d同途径注入等量生理盐水；(4)HUMNCs多次静脉移植组(n=15),结扎冠脉前降支,术后7d,9d,11d,13d,15d经耳缘静脉注入含有3×106 HUMNCs生理盐水500uL。术后1周,3周和5周行超声心动图检测左室舒张末内径(LVEDd)和左室射血分数(LVEF)；免疫组化检测心肌BrdU阳性细胞,HE染色和改良Masson’s三色染色观测心肌胶原病理变化。结果：(1)与假手术组比较,术后1周,3周,5周两细胞移植组和对照组超声LVEDd显著增加,LVEF显著降低,均有显著统计学意义(P<0.01)；与对照组比较,术后3周和5周两细胞移植组LVEDd减低,LVEF增加,有统计学意义(P<0.05)；与单次细胞移植组比较,多次细胞移植组术后3周LVEDd,LVEF改善,但无统计学意义(P>0.05),但术后5周LVEF显著改善,并有统计学意义(P<0.05)；(2)免疫组化示经静脉单次与多次移植组的梗死周边区均存在BrdU阳性细胞,且多次移植组BrdU阳性细胞计数多于单次移植组；改良Masson’s三色染色结果显示,术后3周及5周假手术组心肌细胞间有少量蓝色的、呈条索状与心肌纤维平行排列的胶原纤维；与假手术组比较,术后3周对照组及两细胞移植组梗死区及非梗死区胶原密度显著增加,梗死区域胶原纤维部分融合,排列较紊乱,心肌基本组织结构破坏；术后5周两细胞移植组梗死周边区域心肌细胞间胶原含量较对照组明显减少,梗死区域胶原纤维融合较少,排列较有序,且与单次细胞移植组比较,多次细胞移植组进一步改善。结论：(1)多次静脉移植人脐血单个核细胞迁移并存活至急性心肌梗死梗死周边区域,数量多于单次移植,伴心功能明显改善。(2)经静脉单次与多次移植人脐血单个核细胞均降低心肌梗死区域内胶原纤维的沉积和融合,促进心肌胶原网络有序排列,且多次移植优于单次移植。(3)多次静脉移植脐血单个核细胞改善急性心肌梗死后心脏功能、阻抑心肌胶原纤维重构,其疗效优于单次静脉移植。更多还原

【Abstract】 Objective:To investigate the influence of multiple transplantation intravenously on the remodeling of cardiac collagen network after myocardial infarction by human umbilical cord blood mononuclear cells (HUMNCs)Methods:HUMNCs were harvested, cultured and expanded from umbilical cord blood of healthy full-term pregnant females. 60 Chinese rabbits were divided into four groups randomly. The sham operation group (n=15) was intravenously injected with normal saline 7d,9d, 11d,13d,15d after the non-ligation of the LAD. The control group (n=15), which had undergone ligation of the left anterior coronary artery (LAD), was intravenously injected with normal saline 7d,9d, 11d,13d,15d after operation. The single transplantation group (n=15) were intravenously injected with human umbilical cord blood mononuclear cells (HUMNCs) labeled with bromodexyuridine (BrdU) 7d after operation, and normal saline 9d, 11d,13d,15d after the ligation of the LAD. The multiple transplantation group (n=15) were intravenously injected with human umbilical cord blood mononuclear cells (HUMNCs) labeled with BrdU 7d,9d,11d, 13d,15d after the ligation of the LAD. Echocardiography examination was performed to measure left ventricular end-diastolic dimension(LVEDd) and left ventricular ejection fraction (LVEF) at 1 week,3 weeks and 5 weeks after operation. The rabbits were sacrificed for immunohistochemistry staining to identify the survival of transplanted cells (anti-BrdU monoclonal antibody), and for HE staining and Masson’s trichrome staining to observe the structure of the cardiac collagen network.Results:(1) Transthoracic echocardiography at 1 week,3 weeks and 5 weeks after the operation showed a significant enlargement of left ventricular end-diastolic dimension (LVEDd) and left ventricular ejection fraction (LVEF) in the cell groups and the control group, compared to the sham operation group(P<0.01). At 3 weeks and 5 weeks after the operation, the both cell transplatation groups showed a less enlargement of left ventricular end-diastolic dimension (LVEDd) and less reduction of and left ventricular ejection fraction (LVEF), compared to the control group (P<0.05), and while the heart function of the multiple transplantation group was improved compared with the single transplantation group at 5 weeks after the operation (P<0.05).(2) The BrdU positive cells were found in the peri-myocardial infarction area in the both transplantation groups, and while the number of the BrdU positive cells in the multiple transplantation group are more than that in the single transplantation group. Masson’s trichrome staining showed that collagen fibers were arranged regularly in the myocardial tissue of the sham operation group’s rabbits at 3 and 5 weeks after operation. Compared to the sham operation group, the counts of the collagen fiber in the control group and the cell transplatation groups were significantly increased at 3 weeks after the operation, and the collagen fibers were fused partially, and arranged irregularly, the basic structure of myocardium was destroyed. The collagen fibers in the multiple cell transplantation group were arranged more regularly, compared to in the single group.Conclusion:(1) The human umbilical cord blood mononuclear cells (HUMNCs) delivered by intravenous injection could migrate to the infarct region and survive in the peri-myocardial infarction area, and the number of the transplanted cells in the myocardium in the multiple transplantation group was increased, associated with more improvement of the heart function, compared to the single transplantation group.(2) The single and multiple intravenous transplantation of human cord blood mononuclear cells could reduce the collagen deposition and integration of the collagen fuse, and promote orderly arrangement of myocardial collagen network in the myocardial infarction region. Compared with the single-cell transplantation group, the multiple cell transplantation group further improved.(3) The multiple intravenously transplanted cord blood mononuclear cell transplantation for myocardial infarction was superior to improve cardiac function and inhibit myocardial collagen remodeling, compared to single intravenously transplanted human cord blood mononuclear cells.更多还原