脂肪间充质干细胞治疗急性心肌缺血损伤的实验研究

发布时间：2018-05-29 01:58

本文选题：心肌梗死 + 脂肪间充质干细胞　；参考：《北京协和医学院》2013年博士论文

【摘要】：本课题研究分为两个部分。第一部分：研究探索人和小型猪脂肪间充质干细胞获取、培养、增殖及应用CM-Dil标记干细胞的方法和可行性。研究人脂肪间充质干细胞体外分化的能力。研究探讨经CM-Dil标记的人和小型猪脂肪间充质干细胞对小型猪急性心梗后心肌缺血损伤的治疗效果。在第一部分的研究结果的基础上,为了进一步研究人和小型猪的脂肪间充质干细胞治疗小型猪急性心梗后心肌缺血损伤的机制,进行了第二部分的研究。研究探索干细胞移植后在梗死区存活、分化及旁分泌作用的情况。第一部分：脂肪间充质干细胞治疗急性心梗后的心室重构与心功能评价目的：心肌梗死的主要特点是心脏的血供减少,导致有功能的心脏细胞坏死。已经成为目前社会主要致病和致死的原因。心脏仅有很有限的自我更新的能力,心肌梗死的不良预后会导致心功能衰竭。脂肪间充质干细胞是一种新兴的干细胞,具有分化为多胚层组织细胞的能力。本研究探讨培养、增殖和CM-Dil标记脂肪间充质干细胞的可行性,观察其体外分化的情况。研究探索人和小型猪脂肪间充质干细胞移植后,对小型猪急性心梗后心室重构的治疗效果和心脏功能的评价。方法：收集人和小型猪的脂肪组织,体外培养、增殖脂肪间充质干细胞并且用CM-Dil标记。流式细胞分析仪分析培养出的人脂肪干细胞表面细胞标志物。应用特殊培养基体外诱导人脂肪间充质干细胞分化。手术结扎左冠状动脉前降支制作小型猪急性心梗模型,随机分为五组。1.空白组(n=6),直接关胸,不予任何处理。2.对照组(n=6),心梗区及边缘注射PBS。3.实验组一(n=6),心梗区及边缘注射人脂肪间充质干细胞。4.实验组二(n=6),心梗区及边缘注射同种异体脂肪间充质干细胞。5.实验组三(n=6),心梗区及边缘注射自体脂肪间充质干细胞。 1.手术前、手术后2小时和手术后6周心脏超声学检查。 2.手术后2小时和手术后6周心肌灌注核素扫描检查。 3.手术后6周心梗区心肌组织Masson三色染色观察存活心肌量。统计学分析比较上述检测结果数据。结果人和小型猪的脂肪间充质干细胞均生长良好,CM-Dil染色明显。流式细胞分析仪检测发现培养的人脂肪间充质干细胞符合国际细胞治疗协会(ISCT)对间充质干细胞的细胞表型做出的规定。并且可以体外分化为成熟脂肪细胞和软骨细胞。干细胞移植6周后,实验组一,实验组二和实验组三舒张期梗死区室壁厚度,收缩期梗死区室壁厚度及心室壁增厚率明显高于空白组和对照组(p0.001)。干细胞移植6周后,实验组一、实验组二和实验组三心肌灌注面积较前均有改善,且三组之间灌注面积改善无明显差异(p=0.98)。空白组和对照组心肌灌注面积较前具有恶化,两组之间无明显差异(p=0.69)。实验组一、实验组二和实验组三存活心肌量较空白组和对照组明显增加,差异具有显著统计学意义(p0.001)。实验组三存活心肌量较实验组一、二稍好,差异没有统计学意义(p=0.555)。结论人和小型猪脂肪间充质干细胞获取,培养及扩增简单,易于操作。移植脂肪间充质干细胞后不仅可以阻止左心室扩张、梗死区室壁变薄和心室重构,还可以改善梗死区缺血心肌的血液灌注。第二部分：脂肪间充质干细胞修复急性心肌缺血损伤作用的机制目的在第一部分的基础上,研究探讨脂肪间充质干细胞移植后修复急性心肌缺血损伤作用的机制。包括分化为成熟心肌细胞和血管内皮细胞的能力；旁分泌作用分泌细胞因子的能力；抑制细胞凋亡的能力。方法结扎左冠状动脉前降支制作小型猪急性心梗模型,随机分为五组。1.空白组(n=6),直接关胸,不予任何处理。2.对照组(n=6),心梗区及边缘注射PBS。3.实验组一(n=6),心梗区及边缘注射人脂肪间充质干细胞。4.实验组二(n=6),心梗区及边缘注射同种异体脂肪间充质干细胞。5.实验组三(n=6),心梗区及边缘注射自体脂肪间充质干细胞。6周后进行以下检测： 1) RT-PCR检测VEGF、 vWF、 TGF-β3、 HGF、 CXCR4及SDF-1mRNA表达水平,量化并分析比较结果。 2)vWF免疫组化染色观察新生血管密度,TUNEL染色观察细胞凋亡情况,量化并分析比较结果。 3)免疫荧光染色观察干细胞移植后存活细胞量,量化并分析比较结果；观察分化成心肌细胞和血管内皮细胞的情况；观察新生心肌与原有存活心肌之间形成闰盘连接的情况以及促进梗死区细胞增殖的情况。结果 1)实验组一、实验组二和实验组三VEGF、 vWF、 TGF-β3、 HGF、 CXCR4及SDF-1mRNA表达水平较空白组和对照组明显增加(p0.001),三个实验组之间差异没有统计学意义(p=0.97)。 2)实验组一、实验组二和实验组三新生血管密度较空白组和对照组明显增加(p0.001)。三个实验组之间差异没有统计学意义(p=0.86)。实验组一、实验组二和实验组细胞凋亡数明显低于较空白组和对照组(p0.001),三个实验组之间差异没有统计学意义(p=0.58)。 3)三个实验组都有较大量脂肪干细胞存活,实验组二和实验组三存活细胞量明显高于实验组一p0.001)。实验组二和实验组三中移植的脂肪间充质干细胞可分化成心肌细胞和血管内皮细胞,而且新生心肌之间可以形成闰盘连接。未观察到实验组一干细胞分化的情况。实验组一、实验组二和实验组三都观察到梗死区有明显的细胞增殖,而且发现CM-Dil阴性细胞增殖的情况。结论脂肪间充质干细胞移植后可以增加梗死区新生血管密度,抑制细胞凋亡；通过分泌细胞因子促进梗死区新生血管生成,动员自身的干细胞,诱导其分化修复心肌损伤。同种异体和自体脂肪间充质干细胞移植后可以分化成心肌细胞和血管内皮细胞,并且形成闰盘连接。
[Abstract]:This study was divided into two parts . The first part : The study explored the method and feasibility of obtaining , culturing , proliferating and applying CM - Dil - labeled stem cells to human and small pig fat mesenchymal stem cells .

The first part : The purpose of the evaluation of ventricular remodeling and cardiac function after treatment of acute myocardial infarction with adipose - derived mesenchymal stem cells :

The main feature of myocardial infarction is that the blood supply of the heart is reduced , which leads to the necrosis of cardiac cells . The heart has limited self - renewal ability , and the poor prognosis of myocardial infarction can lead to heart failure .

Method :

The adipose tissue of human and minipigs was collected , cultured in vitro , expanded to mesenchymal stem cells and labeled with CM - Dil . Flow cytometry was used to analyze the surface cell markers of human adipose - derived stem cells . The differentiation of human adipose - derived mesenchymal stem cells was induced by special culture medium .

The acute myocardial infarction model of small pigs was made by operation ligation of left anterior descending branch of left coronary artery . The model was divided into five groups : 1 . blank group ( n = 6 ) , direct closure and no treatment . 2 . The control group ( n = 6 ) , myocardial infarction area and edge injected PBS . 4 . The experimental group ( n = 6 ) , myocardial infarction area and the edge injected with allogeneic fat mesenchymal stem cells . 5 . The experimental group 3 ( n = 6 ) , myocardial infarction area and edge injected autologous fat mesenchymal stem cells .

1 . Pre - operative , 2 - hour post - surgery , and 6 - week post - operative cardiac ultrasound examination .

2 . After 2 hours after surgery and 6 weeks after surgery , the myocardial perfusion radionuclide scan was examined .

3 . The myocardial tissue of myocardial tissue in the myocardial tissue of 6 weeks after operation was used to observe the survival myocardium . Statistical analysis compared the data of the above - mentioned detection results .

Results

The mesenchymal stem cells of human and minipigs were well - grown , and CM - Dil staining was clear . The flow cytometry showed that cultured human adipose - derived mesenchymal stem cells were in line with the cell phenotype of mesenchymal stem cells by the International Association of Cellular Therapy ( ISCT ) .

After 6 weeks of stem cell transplantation , the thickness of ventricular wall , wall thickness and wall thickness of ventricular wall in experimental group 1 , experimental group 2 and experimental group were significantly higher than those in blank group and control group ( p 0.001 ) .

After 6 weeks of transplantation of the stem cells , the myocardial perfusion area of the experimental group 1 , the experimental group 2 and the experimental group was improved , and there was no significant difference between the three groups ( p = 0.98 ) . The myocardial perfusion area in the blank group and the control group was worse than that in the control group , there was no significant difference between the two groups ( p = 0.69 ) .

There was no significant difference between the experimental group and the experimental group ( p = 0.555 ) .

Conclusion

Human and small pig fat mesenchymal stem cells are obtained , cultured and amplified simply , and are easy to operate . After transplantation of the mesenchymal stem cells , the left ventricle can be prevented from expanding , the wall thinning of the infarct zone and the remodeling of the ventricle can be prevented , and the blood perfusion of the ischemic myocardium in the infarct zone can be improved .

The second part : the mechanism of the repair of acute myocardial ischemia injury by adipose - derived mesenchymal stem cells

Purpose

On the basis of the first part , the mechanism of repairing acute myocardial ischemia injury after transplantation of adipose - derived mesenchymal stem cells was investigated .
The ability to secrete cytokines by parasecretory action ;
Ability to inhibit apoptosis .

method

The acute myocardial infarction model of small pigs was made by ligation of left anterior descending branch of left coronary artery . The model was divided into five groups . 1 . Blank group ( n = 6 ) , myocardial infarction , no treatment . 2 . The control group ( n = 6 ) , myocardial infarction area and the edge injected with PBS . 4 . The experimental group 2 ( n = 6 ) , myocardial infarction area and edge injected autologous fat mesenchymal stem cells . 5 . The experimental group 3 ( n = 6 ) , myocardial infarction area and edge injected autologous fat mesenchymal stem cells . The following tests were performed after 6 weeks :

1 ) RT - PCR was used to detect the expression level of VEGF , VWF , TGF - 尾3 , HGF , CXCR4 and SDF - 1 mRNA , quantify and analyze the results .

2 ) Immunocytochemistry staining was used to observe the density of neovascularization , TUNEL staining was used to observe the apoptosis of the cells , quantify and analyze the results .

3 ) observing the survival cell quantity after the stem cell transplantation by immunofluorescence staining , quantifying and analyzing the comparison result ;
Observe the differentiation into myocardial cells and vascular endothelial cells ;
To observe the relationship between the neonatal myocardium and the original viable myocardium , and to promote the proliferation of the cells in the infarct zone .

Results

1 ) There was no significant difference between the three experimental groups ( p = 0.97 ) . There was no significant difference between the three experimental groups ( p = 0.97 ) in the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the

2 ) There was no significant difference between the three experimental groups ( p = 0.86 ) . There was no significant difference between the three experimental groups ( p = 0.86 ) . There was no significant difference between the experimental groups ( p = 0 . 58 ) .

3 ) There were a lot of adipose - derived stem cells in the three experimental groups , and the three survival cells in the experimental group and the experimental group were significantly higher than that in the experimental group ( P < 0.001 ) . The adipose - derived mesenchymal stem cells transplanted in the experimental group 2 and the experimental group were differentiated into cardiomyocytes and vascular endothelial cells , and the intercalated discs could be formed between the neonatal myocardium . The experimental group 1 , the experimental group 2 and the experimental group 3 observed the obvious cell proliferation in the infarct zone , and found that the CM - Dil - negative cell proliferation was observed .

Conclusion

After transplantation of adipose - derived mesenchymal stem cells , the density of neovascular endothelial cells in the infarct zone can be increased , and the apoptosis of the cells can be inhibited ;
It is possible to differentiate into cardiomyocytes and vascular endothelial cells after transplantation of allogeneic and autologous adipose - derived mesenchymal stem cells , and to form intercalated disk connections .
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22

【参考文献】