【作者】 唐洁；

【导师】 齐向前； 陈元禄； 黄云洲； 薛玉良； 张健；

【作者基本信息】 天津医科大学 ， 内科学， 2008， 博士

【摘要】 背景：心肌梗死(MI)是心肌的急性坏死,病情凶险,病死率高。传统观念认为,心肌细胞坏死后不能再生,成纤维细胞增生修复,形成瘢痕组织,并逐步发生心室重构,发展为顽固性心力衰竭。目前的药物治疗、介入治疗和冠脉旁路移植术能够恢复一定的血供,但均不能充分补充有效工作的心肌,无法改善MI的远期预后。近年来,细胞移植治疗心肌梗死为重建受损心肌带来新的希望。虽然相关基础实验及临床研究比较广泛,但研究结果均不一致,存在争议。细胞移植后的分化问题、细胞移植能否长期持久地改善心脏功能、改善缺血心功能的机制以及细胞移植是否引起心律失常这些问题均不明确。目的：1.结扎犬冠状动脉左前降支建立心肌梗死模型。2.观察骨髓单个核细胞悬液(BM-MNCs)的制备及经冠脉注射的可行性。3.观察经冠脉移植BM-MNCs对梗死后心功能的影响。4.观察荧光染料CM-Dil标记BM-MNCs的效率及荧光在体的时间。5.观察标记的BM-MNCs能否在梗死心肌内存活并且分化成为心肌样细胞。6.观察BM-MNCs移植对血管形成的影响及对血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、基质金属蛋白酶-9(MMP-9) mRNA表达的影响。7.观察BM-MNCs移植后心肌的有效不应期有无改变。8.观察BM-MNCs移植有无导致恶性心律失常发生的潜在风险。9.观察BM-MNCs移植后能否纠正缝隙连接排列紊乱而减少心律失常的发生。方法：1.杂种犬16只,随机分为移植组(n=10)和对照组(n=6)两组,结扎冠状动脉前降支建立犬心肌梗死模型。2.移植组的实验犬全麻后无菌条件下于髂前/后上嵴抽取骨髓25-30ml, ficoll淋巴细胞分离液密度梯度离心法分离得BM-MNCs, CM-Dil标记待移植的BM-MNCs,用倒置相差荧光显微镜观察细胞数目和标记效率,调整细胞浓度为3×107-1×108/ml。3.结扎冠脉后2h恢复血流,于冠脉结扎点远端插入套管针,向套管内注射制备好的BM-MNCs,对照组移植等量生理盐水,最后完全结扎LAD。术后予抗生素3天,饲养观察6周。4.所有实验犬于梗死后2h(细胞移植前)、移植后6周行血流动力学检查,测量左室收缩压、左室舒张末压；以热稀释法测量心排血量。5.所有实验犬于梗死后2h(细胞移植前)、移植后6周进行超声心动图检查,测量左室舒张末内径、左室收缩末内径、左室舒张末容积、左室收缩末容积、射血分数、短轴缩短率和每搏量。6.移植后6周,氯化钾注射法处死犬,取梗死区、梗死周边区心肌,观察CM-Dil示踪的细胞的分布,免疫荧光染色检测CM-DiI示踪的细胞是否表达心肌细胞特异性蛋白-心肌β-肌球蛋白重链(cardiac myosin heavy chain,β-MHC)、连接蛋白43(Connexin 43, Cx43)。vWF免疫组化染色,计算毛细血管密度。RT-PCR检测VEGF、bFGF和MMP-9的mRNA表达。7.梗死后6周测定心脏不同部位的有效不应期,免疫组化染色检测Connexin 43的表达。结果：1.BM-MNCs经CM-DiI标记后,细胞膜着色,紫外光激发呈现红色。标记效率为95%-100%。2.细胞移植治疗6周后,在BM-MNCs移植治疗的犬心肌组织均可见到CM-DiI标记的红色细胞；细胞移植组的心肌MHC、Connexin43免疫荧光染色均为阳性,呈现绿色荧光；与CM-Dil标记的红色荧光供体细胞进行叠加,可见βMHC、Connexin 43染色双阳性的细胞,呈现黄色荧光。3.对照组心肌梗死后6周和心肌梗死后2h比较,各项血流动力学指标均无显著改善(P>0.05)；而在移植组,细胞移植后6周LVEDP较梗死后2h(细胞移植前)明显降低,差异有显著性(5.1±3.07 vs 9.2±4.34,P<0.05)；与梗死后6周的对照组比较,LVEDP亦明显降低,差异有显著性(5.1±3.07 vs 11.67±3.42,P<0.01)。移植组细胞移植后6周CO较心肌梗死后2h(细胞移植前)明显升高,差异有显著性(3.1±0.89 vs 2.14±0.49,P<0.01)；与梗死后6周的对照组比较,CO亦明显升高,差异有显著性(3.1±0.89 vs 2.39±0.43,P<0.05)。余指标无显著变化(P>0.05)。4.对照组心肌梗死后6周和心肌梗死后2h比较,各项超声心动指标均无显著改善(P>0.05)；移植组细胞移植后6周,ESD、ESV、EDV、LVEF、FS、SV均较移植前有明显改善,与梗死后6周的对照组相比,亦有明显改善(P<0.05),其中移植组EF较对照组提高7%左右,余指标无显著变化(P>0.05)。5.BM-MNCs移植后6周,移植组有明显的血管新生,梗死边缘区新生血管数量,,移植组明显高于对照组(19.32±2.47 vs 9.47±1.28,P<0.01),而两组的梗死中心区的新生血管数量无显著性差异(3.44±0.51 vs 3.07±0.3,P>0.05)。6.移植组促血管生长因子VEGF188的mRNA表达水平显著高于对照组(1.16±0.38 vs 0.45±0.15,P<0.01)；移植组VEGF164的mRNA表达水平显著高于对照组(1.17±0.3 vs 0.42±0.17,P<0.01)；移植组促血管生长因子bFGF的mRNA表达水平显著高于对照组(0.90±0.21 vs 0.63±0.28,P<0.05)；移植组基质金属蛋白酶MMP-9的mRNA表达水平显著低于对照组(0.59±0.18 vs 0.93±0.3,P<0.05)。7.移植组与对照组相比,梗死6周测得梗死区、梗死周边区和正常区有效不应期无明显差别(85±9.26 vs90±7.07ms,P>0.05)(87.78±9.37 vs 90±7.07,P>0.05)(85±11.95 vs 88.33±9.4,P>0.05)；移植组和对照组正常心肌区之间Connexin43表达无显著性差异(3543.7±446.00 vs 3431.67±421.54,P>0.05)；移植组梗死边缘区Cx43较对照组明显增多(2312.50±412 vs 1356.17±332.73,P<0.05),但仍明显少于正常区相应部位(2312.50±412 vs 3543.7±446.00,P<0.05)；在梗死中心区移植组和对照组比较差异无显著性(327.00±98.67 vs 311.33±78.74,P>0.05)。结论：1.荧光染料CM-DiI标记BM-MNCs细胞效率高,荧光在活体细胞内存在的时间大于6周,可成功示踪移植的BM-MNCs。2. CM-DiI示踪的细胞能在梗死心肌内存活并且分化成为具有心肌细胞特异性标记-β-MHC的心肌样细胞,并同时表达Connexin 43。3.经冠脉内注射移植自体BM-MNCs可以改善急性心肌梗死后心脏收缩和舒张功能。4.经冠脉BM-MNCs移植可以促进血管生成,提高促血管生长因子VEGF188、VEGF164和bFGF的mRNA表达水平,减少基质金属蛋白酶MMP-9的mRNA表达水平。5.心肌梗死急性期行BM-MNCs移植后,移植细胞对于心梗恢复期心律失常的发生具有一定程度的改善作用,同时未观察到明显的致心律失常源性。更多还原

【Abstract】 Background:Myocardial infarction (MI) causes acute necrosis of cardiomyocytes. It is very dangerous and has a high rate of death. It is considered traditionally that cardiomyocytes can’t regenerate after they are dead. Then Fibroblasts proliferate and repair the myocardium and the myocardium changes into scar tissue. It leads to left ventricular (LV) remodeling, which ultimately results in heart failure. Current therapeutic modalities including pharmacological treatment, percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG) mainly aim at improving the blood supply, but can’t provide viable cardiomyocytes and can’t well improve long-term prognosis of MI. Cell transplantation offers a new promise of rebuilding the damaged myocardium. Both experimental studies and clinical trials widely are carried out however the results of them are not consistent. It is not clear if the transplanted cells can differentiate into cardiomyocytes and if this therapy can improve one’s heart function in the long term. The mechanism underlying this therapeutic effect is not fully understood and whether the cell transplantation causes arrhythmia is unclear.Objective:1.Left anterior descending coronary (LAD) artery ligation was used to produce AMI in hybrid canine.2.To explore the method of bone marrow mononuclear cell isolation and the feasibility of BM-MNCs being intracoronarily infused into infarction related artery.3. To investigate the effects of intracoronary autologous bone marrow mononuclear cells transplantation on cardiac function of AMI.4. To evaluate the labeling efficiency of CM-DiI and the lasting time of the fluorescence.5. To observe the survival and the differentiation of grafted bone marrow cells(BM-MNCs) in host myocardium.6. To abserve the effects of BM-MNCs transplantation on angiogenesis and the mRNA expression of cytokines such as VEGF、bFGF and MMP-9.7. To observe whether BM-MNCs transplantation can change the ERP of myocardium.8. To observe whether BM-MNCs transplantation can potentially cause arrhythmia.9. To observe whether the BM-MNCs transplantation can alter the spatial distribution of Connexins, which is important for arrhythmia genesis after MI.Methods:1.Sixteen hybrid canines were randomly divided into transplantation group(n=10) and control group(n=6). Left anterior descending coronary (LAD) artery ligation was used to produce AMI model.2. About 25-30ml of bone marrow was aspirated from the dogs’ iliac bone of the transplantation group. Bone marrow mononuclear cell (BM-MNCs) suspension was prepared by density centrifugation using ficoll. The BM-MNCs labeled with CM-DiI which are observed in fluorescence microscope are prepared with the concentration of 3 X 107- 1 X 108/ml.3. BM-MNCs suspension was intracoronarily infused into infarction related artery 2 hour after AMI with needle and saline of the same volume was injected for the control dogs.4.We detected LVSP、LVEDP and used Swan-Ganz catheter to detect CO 2 hour after the ligation and 6 weeks after the BM-MNCs transplantation.5. To evaluate the heart function, we used echocardiography to detect LVESD、LVEDD、LVESV、LVEDV、FS、EF、SV 2 hour after the ligation and 6weeks after the BM-MNCs transplantation. 6. Canines are killed and histological examination was performed. The expression of Myosin heavy chain and Connexin 43 was assessed by immunofluorescence staining. Capillary density were assessed by vWF immunohistochemical staining; The mRNA levels of VEGF, bFGF and MMP-9 within infarct area were determined by RT-PCR 7. days after MI.7 The ERP of different areas in myocardium was assessed 6 weeks after cell transplantation, The expression of Connexin 43 was assessed by immunohistochemical staining.Results:1. The implanted cells showed clear membrane red fluorescence.2.6 weeks after the BM-MNCs transplantation, CM-Dil labeled BM-MNCs were mainly located within periinfarct and infarct area. Some BM-MNCs were positive for MHC and Cx43. Combined "CM-Dil and FITC" in images were observed.3. There isn’t a significant difference of Hemodynamic indexs between 2 hours after the ligation and 6 weeks after the BM-MNCs transplantation in the control group (P>0.05). Compared to 2 hours after the ligation, LVEDP decreased significantly (5.1±3.07 vs 9.2±4.34, P<0.05) 6 weeks after MI in transplantation group. Compared to control group, LVEDP decreased significantly (5.1±3.07 vs 11.67±3.42, P<0.01) 6 weeks after MI. Compared to 2 hour after the ligation, CO increased significantly (3.1±0.89 vs 2.14±0.49, P<0.01) 6 weeks after MI in transplantation group. Compared to control group, CO increased significantly (3.1±0.89 vs 2.39±0.43, P<0.05)6 weeks after MI in transplantation group.4. There is not a significant difference of all the echocardiography index in control group between 2 hours after the ligation and 6 weeks after MI(P>0.05). Compared to 2 hours after the ligation, ESD、ESV、EDV、LVEF、FS、SV all improved in transplantation group 6 weeks after MI(P<0.05) and the same results exsists compared to the control group 6 weeks after MI(P<0.05). Compared control group, LVEF increased 7% in transplantation group. 5.Transplantation group increased the density of capillary significantly within periinfarct area 6 weeks after MI (19.32±2.47 vs 9.47±1.28, P<0.01), meanwhile within infarct area the density had no significant difference(3.44±0.51 vs 3.07±0.3, P>0.05).6. Transplantation group increase the mRNA level of VEGF 188、VEGF164 and bFGF significantly(1.16±0.38 vs 0.45±0.15, P<0.01) (1.17±0.3 vs 0.42±0.17, P<0.01) (0.90±0.21 vs 0.63±0.28, P<0.05) and decrease mRNA level of MMP-9 significantly (0.59±0.18 vs 0.93±0.3, P<0.05) 7. Compared control group, the ERP of infarct area、periinfarct area and normal area in transplantation group has no significant difference (85±9.26 vs 90±7.07ms, P>0.05) (87.78±9.37 vs 90±7.07, P>0.05) (85±11.95 vs 88.33±9.4, P>0.05). The expression of Cx43 in normal area is similar between transplantation group and control group(3543.7±446.00 vs 3431. 67±421.54, P>0.05). The expression of Cx43 in transplantation group in periinfarct area was significantly higer than that in control group((2312.50±412 vs 1356.17±332.73, P<0.05), but was still much less than in normal area(2312.50±412 vs 3543.7±446.00, P<0.05). The expression of Cx43 in infarct area is similar between transplantation group and control group (327.00±98.67 vs 311.33±78.74, P>0.05).Conclusions:1. The labeling efficiency of CM-DiI was more than 95% and the lasting time of the fluorescence in the host was more than 6 weeks. CM-DiI could be a good tracer for the BM-MNCs.2. The implanted BM-MNCs could survive in the infarcted lesion and differentiate into cells expressing MHC.and Cx43.3. The left ventricular systolic and diastolic function could be improved by BM-MNCs implantation.4. The BM-MNCs transplantation can increase capillary density, especially in the border zone of the myocardial infarction. BM-MNCs transplantation can increase the mRNA level of VEGF188、VEGF164 and bFGF, at the same time, it decreases the mRNA level of MMP-9.5. The spatial distribution of Cx43 gap junction was ameliorated by BM-MNCs transplantation, and it may ameliorate the arrhythmic susceptibility.更多还原