冠脉结合多次静脉移植间充质干细胞治疗慢性缺血性心脏病的实验研究

发布时间：2018-05-05 10:14

本文选题：间充质干细胞 + 细胞移植　；参考：《中国人民解放军军事医学科学院》2015年硕士论文

【摘要】：缺血性心脏病(ischemic heart disease,IHD)是威胁人类健康的主要杀手之一,具有极高的致死率和致残率[1]。尽管多数患者能从经皮冠状动脉腔内血管成形术(percutaneous transluminal coronary angioplasty,PTCA)、冠状动脉搭桥手术(coronary artery bypass graft,CABG)及药物治疗中获益,但仍然有相当数量的慢性缺血性心脏病(chronic ischemic heart disease,CHD)患者,他们因为冠脉病变严重、弥漫等各种原因无法进行手术治疗,且药物治疗效果往往不理想[2]。这部分CHD患者目前缺乏有效的干预措施,临床上也被称作“无选择权”患者。干细胞研究的进展为这类患者带来了希望。近年来,大量基础[3,4]和临床研究[5,6]显示,干细胞移植可以减少心肌细胞凋亡,促进新生血管形成,改善心室重构,提高心功能,显示出良好的应用前景。其中间充质干细胞(mesenchymal stem cells,MSCs)因其来源广泛,操作简便,具有免疫豁免效应等优势而受到研究人员的广泛青睐。现有的研究表明,MSCs治疗急性心肌缺血安全有效。但MSCs在治疗慢性心肌缺血方面的研究尚少。目前,国内外尚未见到在大动物模型上研究联合移植MSCs治疗CHD的报道。MSCs有多种来源,现有的研究多采用骨髓来源间充质干细胞(bone marrow-derived mesenchymal stem cells,BM-MSCs)。但BM-MSCs具有取材有创伤,年龄较大供体的MSCs增殖能力有限等缺点[7,8]。因此,研究人员开始探讨多种其他来源MSCs治疗心脏病的可行性。其中脐带来源间充质干细胞(umbilical cord-derived mesenchymal stem cells,UC-MSCs)因其取材无创伤,无伦理限制,细胞增殖和分化能力强,能分泌多种促血管生成因子等优势,成为替代BM-MSCs的较为理想的细胞来源[9]。目前已经有大量采用UC-MSCs治疗多种疾病的临床前或临床研究报道,包括肝硬化[10]、糖尿病[11]、系统性红斑狼疮[12]、移植物抗宿主病(graft-versus-host disease,GVHD)[13]等,但在心脏病的研究方面尚处于起步阶段。目前尚未见到采用UC-MSCs治疗CHD的研究报道。因此,本研究拟建立猪慢性缺血性心脏病动物模型,通过冠脉结合两次静脉输注的方式移植异体猪骨髓来源间充质干细胞(pig bone marrow-derived mesenchymal stem cells,p BM-MSCs)和人脐带来源间充质干细胞(human umbilical cord-derived mesenchymal stem cells,h UC-MSCs)进行干预,采用冠脉造影、心脏超声、单光子发射计算机断层成像(single-photon emission computed tomography,SPECT)和病理组织学等方法综合评价骨髓和脐带来源MSCs移植对CHD的治疗作用,从而为今后的临床应用提供依据。本研究首先采用Ficoll密度梯度法分离p BM-MSCs,相差显微镜观察显示,p BM-MSCs呈梭形,旋涡状或鱼尾状生长。流式检测结果表明,p BM-MSCs表达CD44、CD29、CD90,而CD14、CD34、CD45、CD166、HLA-DR表达呈阴性。诱导分化实验发现,p BM-MSCs能够分化为脂肪细胞、成骨细胞、软骨细胞。然后我们采用酶消化法分离h UC-MSCs,相差显微镜下观察,细胞呈梭形,旋涡状或平行排列,流式检测结果显示,细胞表达CD29、CD44、CD73、CD90、CD105和HLA-ABC,而CD34、CD45、HLA-DR表达呈阴性。诱导分化结果显示,h UC-MSCs可向脂肪细胞、成骨细胞和软骨细胞的分化。最后我们着重探讨了p BM-MSCs和h UC-MSCs对慢性缺血性心脏病大动物模型的治疗作用。选取30只小型猪,将Ameroid动脉缩窄环置于猪左冠回旋支(left circumflex coronary artery,LCX)建立慢性缺血性心脏病动物模型,4周后通过冠脉造影和心电图对模型进行评价;存活的20只动物随机分为p BM-MSCs组(n=8)、h UC-MSCs组(n=6)和对照组(n=6),治疗组采用冠脉结合两次静脉输注的方式移植经过CM-Di I标记的p BM-MSCs和h UC-MSCs进行干预,对照组则输注生理盐水。细胞移植前和移植后4周,冠脉造影检测冠脉侧枝循环,心脏超声检测心功能和心脏结构指标,SPECT检测心肌血流灌注。动物处死后取心脏行2,3,5-氯化三苯基四氮唑(2,3,5-triphenyl-2H-tetrazolium chloride,TTC)染色计算梗死面积,并选取梗死边缘区的组织制备病理切片,检测心肌细胞纤维化、凋亡、新生血管形成等情况,并观察移植细胞的植入和转归。建模后4周,存活的20只猪冠脉造影显示LCX均完全闭塞,心电图可见Ⅰ、AVL和(或)Ⅱ、Ⅲ、AVF和(或)V4-V6导联ST段压低和(或)T波改变。细胞移植后4周,冠脉造影结果显示:对照组和细胞治疗组均有侧枝血管形成,但与治疗前相比,对照组Rentrop评分增加无统计学意义,而p BM-MSCs组和h UC-MSCs组结果则有显著差异(p0.01),表明细胞移植促进了侧枝循环的形成;与对照组相比,p BM-MSCs组和h UC-MSCs组侧枝血管计数结果有显著差异(P0.05),但两个细胞治疗组之间无明显差别。心电图结果表明,p BM-MSCs和h UC-MSCs移植组心率(heart rate,HR)较治疗前无明显变化,而对照组HR明显增加(P0.01)。心脏超声:细胞治疗组的左室射血分数(Left ventricular ejection fraction,LVEF)较治疗前均有升高,p BM-MSCs组从53.91%±6.72%升至58.19±7.10%(p0.05),h UC-MSCs组从56.12±2.86%升至61.32±3.23%(p0.05),而对照组的LVEF较治疗前下降;与对照组相比,细胞治疗组LVEF的升高具有显著差异(P0.05),但p BM-MSCs组与h UC-MSCs组间相比无差异;与对照组相比,两个细胞治疗组的梗死区收缩期室壁增厚率(systolic thickening fraction in the infarcted left ventricular wall,WTh F)升高均有统计学差异(P0.05);此外,对照组的左室收缩末期容积(left ventricular end-systolic volume,LVESV)和左室舒张末期容积(left ventricular end-diastolic volume,LVEDV)较治疗前均明显增大(P0.05),而p BM-MSCs组和h UC-MSCs组治疗前后未见明显改变。SPECT结果显示:对照组和细胞治疗组心肌血流灌注均较治疗前有所改善;但p BM-MSCs组(p0.01)和h UC-MSCs组(p0.05)改善较对照组明显,且二者之间无显著差异。病理组织学结果显示,TTC染色:p BM-MSCs组和h UC-MSCs组梗死面积分别为7.89±2.62%和8.35±1.88%,比对照组(14.52±4.85%)显著减小(p0.05);HE染色:细胞移植组炎性评分显著低于对照组(p0.05);Masson三色染色显示细胞移植组的胶原容积分数(collagen volume fraction,CVF)明显小于对照组(p0.05);血管计数:p BM-MSCs和h UC-MSCs移植组微血管密度显著高于对照组(p0.05);TUNEL(terminal deoxynucleotidyl transferase d UTP nick end labeling)法检测凋亡,p BM-MSCs组(11±3cells/100 cells)和h UC-MSCs组(13±4 cells/100cells)凋亡的心肌细胞数量明显少于对照组(24±3 cells/100 cells)(p0.01)。荧光显微镜下梗死边缘区可见有CM-Di I标记的细胞,免疫荧光染色显示p BM-MSCs组和h UC-MSCs组均可见移植后的MSCs部分分化为血管内皮细胞,未观察到其向心肌细胞分化。RT-q PCR显示,与对照组相比,p BM-MSCs组的促血管生成素(angiogenin,Ang)表达增高(p0.05),h UC-MSCs组的Ang(p0.01)和血管内皮生长因子(vascular endothelial growth factor,VEGF)(p0.05)表达增高,而白介素-6(interleukin-6,IL-6)、成纤维细胞生长因子(fibroblast growth factor,FGF)、胸腺肽β4(thymosin beta 4,Tβ4)的表达无显著改变。本研究成功分离培养了p BM-MSCs和h UC-MSCs;并将Ameroid动脉缩窄环置入猪LCX成功建立猪慢性缺血性心脏病模型;经冠脉结合两次静脉输注p BM-MSCs及h UC-MSCs治疗研究结果表明,p BM-MSCs及h UC-MSCs均能有效减少梗死面积,减轻炎症反应,减少心肌细胞凋亡和心肌纤维化,促进侧枝循环形成及血管新生,改善心肌血流灌注,改善心室重构,提高心功能,且两种细胞的治疗作用无显著差异。
[Abstract]:Ischemic heart disease (IHD) is one of the major killer of human health, with high mortality and disability rate [1]., although most patients can from percutaneous transluminal coronary angioplasty (percutaneous transluminal coronary angioplasty, PTCA), coronary artery bypass surgery (coronary artery) CABG) and the benefit of drug treatment, but there are still a considerable number of patients with chronic ischemic heart disease (chronic ischemic heart disease, CHD) who are unable to perform surgical treatment because of serious coronary lesions, diffuse and other reasons, and the effect of the drug treatment often fails to think of [2]., a part of the CHD patient currently lacking effective interventions. The bed is also known as "no choice". The progress of stem cell research has brought hope to these patients. In recent years, a large number of basic [3,4] and clinical studies [5,6] show that stem cell transplantation can reduce myocardial apoptosis, promote angiogenesis, improve ventricular remodeling, raise cardiac function, and show good prospects for application. Mesenchymal stem cells (MSCs) is widely favored by researchers because of its extensive origin, simple operation and immunity immunity effect. The present study shows that MSCs is safe and effective in the treatment of acute myocardial ischemia. However, there are few studies on the treatment of chronic myocardial ischemia by MSCs. At present, it has not been seen at home and abroad at home and abroad. There are a variety of sources for the study of the combined transplantation of MSCs for the treatment of CHD in the animal model. The existing studies are mostly using bone marrow derived mesenchymal stem cells (bone marrow-derived mesenchymal stem cells, BM-MSCs). But BM-MSCs has the disadvantages of being traumatic, and the MSCs colonization of older donors is limited. The feasibility of various other sources of MSCs for the treatment of heart disease is discussed. Among them, the umbilical cord derived mesenchymal stem cells (umbilical cord-derived mesenchymal stem cells, UC-MSCs) are ideal for replacing BM-MSCs because of their noninvasive, non ethical limitations, strong cell proliferation and differentiation, and the ability to secrete a variety of angiogenic factors. Cell source [9]. now has a large number of pre clinical or clinical reports on the use of UC-MSCs for various diseases, including liver cirrhosis [10], diabetic [11], systemic lupus erythematosus [12], graft-versus-host disease, GVHD [13] and so on, but it is still in the initial stage of heart disease research. UC-MSCs for the treatment of CHD is reported. Therefore, this study is to establish a porcine chronic ischemic heart disease animal model, transplanting allogenic pig bone marrow derived mesenchymal stem cells (pig bone marrow-derived mesenchymal stem cells, P BM-MSCs) and human umbilical cord derived mesenchymal stem cells (human umbilical) by two intravenous infusion of coronary artery. Ord-derived mesenchymal stem cells, H UC-MSCs) intervention, using coronary angiography, cardiac ultrasound, single photon emission computed tomography (single-photon emission computed tomography, SPECT) and histopathology to evaluate the therapeutic effect of MSCs transplantation on bone marrow and umbilical cord sources for future clinical applications. Firstly, the Ficoll density gradient method was used to separate P BM-MSCs, and the phase microscope observation showed that P BM-MSCs was spindle shaped, vortexed or fish tail like growth. The flow test results showed that P BM-MSCs expressed CD44, CD29, CD90, while CD34, CD34, and CD90 were negative. Adipose cells, osteoblasts, chondrocytes. Then we use enzyme digestion method to separate h UC-MSCs. Under phase contrast microscope, the cells are spindle shaped, vortexed or parallel arrangement. Flow cytometry results show that cells express CD29, CD44, CD73, CD90, CD105 and HLA-ABC, and CD34, CD45, HLA-DR expression is negative. The differentiation of adipose cells, osteoblasts and chondrocytes. Finally, we focused on the therapeutic effect of P BM-MSCs and H UC-MSCs on the large animal model of chronic ischemic heart disease. 30 small pigs were selected and the Ameroid artery coarctation ring was placed in the left circumflex branch of the pig (left circumflex coronary artery, LCX) to establish a chronic ischemic heart disease animal model. The model was evaluated by coronary angiography and electrocardiogram after 4 weeks. The 20 surviving animals were randomly divided into P BM-MSCs group (n=8), H UC-MSCs group (n=6) and control group (n=6). The treatment group was transplanted with CM-Di I marked P BM-MSCs and h, and the control group was transfused with saline. Coronary collateral circulation was detected by coronary angiography before and 4 weeks after transplantation. Cardiac function and cardiac structure were detected by echocardiography. Myocardial perfusion was detected by SPECT. The infarct area was calculated by 2,3,5- chlorination of three phenyl tetrazoles (TTC) after the death of the animals, and the marginal area of the infarct was selected. Pathological sections were prepared to detect myocardial fibrosis, apoptosis, neovascularization, and the implantation and outcome of the transplanted cells. 4 weeks after modeling, 20 surviving porcine coronary angiography showed that LCX was completely obliterate, and the electrocardiogram showed I, AVL and (or) II, AVF and (or) V4-V6 lead ST segment depression and (or) T wave changes. Cell transplantation After 4 weeks, the results of coronary angiography showed that there were collateral vessels in both the control group and the cell therapy group, but the Rentrop score in the control group was not statistically significant compared with the control group, while the results in the P BM-MSCs group and the H UC-MSCs group were significantly different (P0.01), indicating that the cell transplantation promoted the formation of the collateral circulation; compared with the control group, the P BM-MSCs group and the control group were compared with the control group. There was a significant difference in the collateral vessel count results in the H UC-MSCs group (P0.05), but there was no significant difference between the two cell treatment groups. The ECG results showed that the heart rate (heart rate, HR) in the P BM-MSCs and H UC-MSCs transplantation group was not significantly higher than that before the treatment, while the control group was significantly increased (P0.01). Ricular ejection fraction, LVEF) was higher than that before treatment, P BM-MSCs group rose from 53.91% + 6.72% to 58.19 + 7.10% (P0.05), H UC-MSCs group rose from 56.12 + 2.86% to 61.32 + 3.23% (P0.05), while LVEF in control group was lower than before treatment, and the increase of LVEF in cell therapy group was significantly different from that of control group. There was no difference between the Cs groups. Compared with the control group, the thickening rate of the systolic ventricular wall of the two cell therapy group (systolic thickening fraction in the infarcted left ventricular wall, WTh F) was statistically different. And the left ventricular end diastolic volume (left ventricular end-diastolic volume, LVEDV) was significantly higher than before the treatment (P0.05), while the P BM-MSCs group and the H UC-MSCs group had no significant changes in.SPECT results before and after treatment: the myocardial perfusion in the control group and the cell treatment group was better than that before the treatment. .05) improved compared with the control group, and there was no significant difference between the two. The histopathological results showed that the infarct area of the P BM-MSCs group and the H UC-MSCs group was 7.89 + 2.62% and 8.35 + 1.88%, respectively, compared with the control group (14.52 + 4.85%), respectively (P0.05); HE staining: the inflammatory score of the cell transplantation group was significantly lower than that of the control group (P0.05); Masson tricolor staining was found. The collagen volume fraction (collagen volume fraction, CVF) in the color display cell transplantation group was significantly smaller than that of the control group (P0.05), and the blood vessel count: the microvascular density in the P BM-MSCs and H UC-MSCs transplantation group was significantly higher than that of the control group (P0.05). The number of apoptotic cardiomyocytes in group 100 cells) and H UC-MSCs group (13 + 4 cells/100cells) was significantly less than that of the control group (24 + 3 cells/100 cells) (P0.01). There was CM-Di I labeled cells in the marginal area of the infarct under the fluorescence microscope. The immunofluorescence staining showed that the P BM-MSCs group and the H group were all differentiated into vascular endothelial cells. .RT-q PCR showed that the expression of angiopoietin (angiogenin, Ang) in the P BM-MSCs group was higher than that of the control group (P0.05), and the expression of Ang (P0.01) and vascular endothelial growth factor in the H UC-MSCs group was higher than that of the control group. The expression of fibroblast growth factor (FGF) and thymosin beta 4 (thymosin beta 4, T beta 4) had no significant changes. This study successfully isolated and cultured P BM-MSCs and H UC-MSCs, and succeeded in establishing a porcine chronic ischemic heart disease model by placing the contraction ring of the Ameroid artery. The results of MSCs therapy show that both P BM-MSCs and H UC-MSCs can effectively reduce infarct size, reduce inflammatory response, reduce myocardial apoptosis and myocardial fibrosis, promote the formation of collateral circulation and angiogenesis, improve myocardial perfusion, improve ventricular remodeling and improve cardiac function, and there is no significant difference in the therapeutic effect of two kinds of cells.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541

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