3D类心肌组织与ALGINATE水凝胶用于大鼠心肌梗死修复的研究
发布时间：2019-07-09 19:23
【摘要】：心肌梗死是由于心血管局部堵塞导致心肌局部供血不足而引发的心肌坏死，由于心肌组织的再生潜能极其有限，而且用于心脏移植的供体器官严重缺乏，心肌梗死和相关的心血管疾病是全世界最首要的死亡原因。目前对心肌梗死的治疗手段包括再灌注心肌、缓解疼痛、抗心律失常、抗休克、并发症治疗等，尤其是介入治疗技术的突飞猛进使很多急性心肌梗死患者避免了死亡，但临床治疗对心肌梗死后的心力衰竭仍力不从心，原因是心梗诱发的心室重构过程中心肌细胞的缺失和细胞外基质降解，心室壁变薄，室壁的力学性能下降，导致心功能降低，甚至心力衰竭的发生，因此研究用于心肌梗死治疗的新策略具有重要意义。大量研究表明，利用组织工程的方法将由生物材料与心肌细胞研制的组织工程心肌或活细胞移植或注射到受影响的心肌组织中，对于改善受损心肌的功能具有重要意义，为心肌梗死的治疗开辟了新的途径。然而这种治疗方案目前仍然处于动物实验的尝试阶段。本研究针对心梗诱发的心室重构过程中心肌细胞的缺失和细胞外基质的降解，以海藻酸钠水凝胶为基本材料，，分别设计体外心肌组织工程构建3D类心肌组织和注射水凝胶的原位心肌组织工程的方案对心肌梗死的损伤进行修复治疗，取得了较好的结果，为心肌梗死的治疗开辟了新的途径。 海藻酸钠是一种多糖类的高分子材料，因其有良好的生物相容性和抗凝血性，故在组织工程和心肌组织工程中具有广泛的应用，尤其作为支架材料在骨组织、心肌组织中应用较多。鉴于心肌梗死的病理过程中心肌细胞的缺失和常规三维支架材料难以在体外构建高密度的类心肌组织的难点，本研究采用微囊化技术在体外构建结构致密的三维类心肌组织。首先利用不同比例的胶原研制海藻酸钠/胶原复合微囊，该复合微囊的细胞相容性得到显著改善，适于原代大鼠心肌细胞的贴附和增殖。二维培养的心肌细胞通常增殖能力较弱，而且容易失去心肌细胞特征，而包覆于该微囊内的心肌细胞能够持续处于增殖状态，且无需传代，并维持典型心肌细胞的特征。因此，该复合微囊技术具有维持乳鼠心肌细胞持续增殖的独特优势。利用该复合微囊对大鼠乳鼠原代心肌细胞进行三维培养，可以持续培养长达3个月时间，在微囊内形成了高细胞密度、有持续搏动功能的三维类心肌组织。为了进一步验证该类心肌组织在体内的活性，将该类心肌组织植入大鼠心肌梗死的模型，6w后检测大鼠的心功能证实，该三维类心肌组织能够显著改善心脏的射血分数和梗死部位的血管再生。 心肌梗死引发的心肌细胞外基质的缺失是导致心功能降低的一个关键问题，可注射凝胶的局部注射能改善心肌梗死区的力学性能，减少心衰的发生。由于目前多用钙离子交联的海藻酸钠水凝胶，虽然有功能改善，但是钙离子交联的水凝胶存在着力学性能差，体内不易降解，细胞亲和力差等不足。本文采用将海藻酸钠进行部分氧化，得到具有活性的醛基，进而与明胶分子中的游离氨基发生反应形成共价交联的水凝胶。将化学交联的可注射的海藻酸钠水凝胶与离子交联的水凝胶进行了系统的对比研究，共价交联的凝胶系统的力学性能、降解性能和细胞亲和力明显优于钙离子交联的水凝胶，从而总体上提高了心肌功能的恢复，该凝胶力学性能达到106Pa，凝固时间可调控于30-40min，孔隙率达到70%。化学交联的凝胶在体外、体内降解率加快，植入大鼠心肌梗死模型后，心功能恢复增强。组织学研究表明，化学交联的可注射凝胶在体内增加左心室室壁厚度，促进血管再生，并能抑制基质金属蛋白酶的活性。 总之，本论文针对心肌梗死的病理过程中心肌细胞损伤和细胞外基质损伤两个基本问题进行了研究。针对心肌细胞的损伤设计了海藻酸钠水凝胶三维微囊化技术成功构建了具有持续搏动功能的类心肌组织，而针对细胞外基质流失设计了化学交联的可注射凝胶，并应用于大鼠心肌梗死模型，实验证实：三维类心肌组织和可注射凝胶均能改善梗死区的心肌功能，为心肌梗死的治疗提供了新的途径。
文内图片：![心肌梗死的发病机制[20]](http://image.cnki.net/getimage.ashx?id=1014085261.nh0001)
图片说明：心肌梗死的发病机制[20]
[Abstract]:The myocardial infarction is the myocardial necrosis caused by the partial blood supply of the myocardium due to the partial blockage of the cardiovascular system, because the regeneration potential of the myocardial tissue is extremely limited, and the donor organ for the heart transplantation is severely deficient, Myocardial infarction and related cardiovascular diseases are the leading cause of death in the world. At present, the treatment of myocardial infarction includes the reperfusion of the myocardium, the alleviation of pain, the anti-arrhythmia, the anti-shock, the complication treatment, and the like, in particular the rapid development of the interventional treatment technology to avoid the death of many patients with acute myocardial infarction, but the heart failure of the heart failure after the myocardial infarction is still in the clinical treatment, the reason is that the loss of the myocardial cells and the degradation of the extracellular matrix during the ventricular remodeling induced by the myocardial infarction, the thinning of the ventricular wall, the mechanical property of the chamber wall are reduced, the heart function is reduced, and even the occurrence of the heart failure is caused, Therefore, it is of great significance to study the new strategy for the treatment of myocardial infarction. A large number of studies have shown that the method of tissue engineering is to transplant or inject tissue engineering myocardium or living cells developed by biological materials and myocardial cells into the affected myocardial tissue, which is of great significance for improving the function of the damaged myocardium, and opens up a new way for the treatment of myocardial infarction. However, such a treatment regimen is still at the experimental stage of the animal experiment. In this study, the loss of cardiac myocyte and the degradation of extracellular matrix in the process of ventricular remodeling induced by myocardial infarction were studied, and the sodium alginate hydrogel was used as the base material. The in-situ myocardial tissue engineering of the in vitro myocardial tissue engineering and the in situ myocardial tissue engineering of the injection hydrogel were designed to repair the myocardial infarction, and a good result was obtained, which opened up a new way for the treatment of myocardial infarction. Sodium alginate is a kind of high molecular material of polysaccharide, because it has good biocompatibility and anticoagulant effect, it has wide application in tissue engineering and myocardial tissue engineering, especially as scaffold material in bone tissue and myocardial tissue. In view of the lack of myocardial cells in the pathological process of myocardial infarction and the difficulty of the conventional three-dimensional scaffold materials in the construction of high-density myocardial tissue in vitro, this study uses the microencapsulation technology to construct a compact three-dimensional myocardial group in vitro The preparation method comprises the following steps of: firstly, developing a sodium alginate/ collagen composite microcapsule by using a different proportion of collagen, the cell compatibility of the composite microcapsule is remarkably improved, and the preparation method is suitable for the attachment and the increase of the cardiac muscle of the primary rat The two-dimensional cultured cardiomyocytes usually have weak proliferation ability and can easily lose the characteristics of the cardiac muscle cells, and the myocardial cells coated in the micro-capsules can be continuously in the proliferation state without passage, and the special cardiac muscle cells are maintained. Therefore, the composite micro-capsule technology has the unique advantages of maintaining the continuous proliferation of the neonatal rat cardiac muscle cells. a three-dimensional cardiac muscle group with high cell density and continuous beating function is formed in the microcapsule by the three-dimensional culture of the primary myocardial cells of the rat mammary rat by using the composite microcapsule, in order to further verify the activity of the myocardial tissue in the body, the myocardial tissue is implanted into the model of the rat myocardial infarction, the heart function of the rat is detected after 6w, and the three-dimensional myocardial tissue can obviously improve the ejection fraction of the heart and the blood vessel of the infarction site, The deletion of the extracellular matrix of the myocardial cells induced by myocardial infarction is a key problem in the reduction of cardiac function. The local injection of the injectable gel can improve the mechanical properties of the myocardial infarction area and reduce the heart failure. The calcium ion cross-linked hydrogel has poor mechanical property, is not easy to degrade in the body, and the cell affinity is poor due to the fact that the present multi-purpose calcium ion cross-linked sodium alginate hydrogel In this paper, the sodium alginate is partially oxidized to obtain the active aldehyde group, and then the covalent cross-linking is formed with the free amino group in the gelatin molecule. The mechanical properties, degradation performance and cell affinity of the covalently cross-linked gel system were better than that of calcium ion cross-linking. The hydrogel can improve the recovery of the myocardial function as a whole, the mechanical property of the gel reaches 106 Pa, the setting time can be controlled at 30-40 min, and the porosity is achieved. 70%. The chemical cross-linked gel is in vitro, the in vivo degradation rate is accelerated, and after the myocardial infarction model of the rat is implanted, the heart function is improved. The histological study shows that the chemical cross-linked injectable gel increases the wall thickness of the left ventricle in the body, promotes the regeneration of the blood vessel, and can inhibit the matrix metalloproteinases. In conclusion, this paper deals with two basic problems of myocardial cell injury and extracellular matrix injury in the pathological process of myocardial infarction In this paper, the three-dimensional microencapsulation of sodium alginate hydrogel was designed to successfully construct a type of myocardial tissue with the function of continuous pulsatility, and a chemical cross-linked injectable gel was designed for the loss of extracellular matrix and applied to the model of myocardial infarction in rats. The experimental results show that both the three-dimensional myocardial tissue and the injectable gel can improve the myocardial function of the infarct zone and provide the treatment for myocardial infarction.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22;R318.08
本文编号：2512383
文内图片：
图片说明：心肌梗死的发病机制[20]
[Abstract]:The myocardial infarction is the myocardial necrosis caused by the partial blood supply of the myocardium due to the partial blockage of the cardiovascular system, because the regeneration potential of the myocardial tissue is extremely limited, and the donor organ for the heart transplantation is severely deficient, Myocardial infarction and related cardiovascular diseases are the leading cause of death in the world. At present, the treatment of myocardial infarction includes the reperfusion of the myocardium, the alleviation of pain, the anti-arrhythmia, the anti-shock, the complication treatment, and the like, in particular the rapid development of the interventional treatment technology to avoid the death of many patients with acute myocardial infarction, but the heart failure of the heart failure after the myocardial infarction is still in the clinical treatment, the reason is that the loss of the myocardial cells and the degradation of the extracellular matrix during the ventricular remodeling induced by the myocardial infarction, the thinning of the ventricular wall, the mechanical property of the chamber wall are reduced, the heart function is reduced, and even the occurrence of the heart failure is caused, Therefore, it is of great significance to study the new strategy for the treatment of myocardial infarction. A large number of studies have shown that the method of tissue engineering is to transplant or inject tissue engineering myocardium or living cells developed by biological materials and myocardial cells into the affected myocardial tissue, which is of great significance for improving the function of the damaged myocardium, and opens up a new way for the treatment of myocardial infarction. However, such a treatment regimen is still at the experimental stage of the animal experiment. In this study, the loss of cardiac myocyte and the degradation of extracellular matrix in the process of ventricular remodeling induced by myocardial infarction were studied, and the sodium alginate hydrogel was used as the base material. The in-situ myocardial tissue engineering of the in vitro myocardial tissue engineering and the in situ myocardial tissue engineering of the injection hydrogel were designed to repair the myocardial infarction, and a good result was obtained, which opened up a new way for the treatment of myocardial infarction. Sodium alginate is a kind of high molecular material of polysaccharide, because it has good biocompatibility and anticoagulant effect, it has wide application in tissue engineering and myocardial tissue engineering, especially as scaffold material in bone tissue and myocardial tissue. In view of the lack of myocardial cells in the pathological process of myocardial infarction and the difficulty of the conventional three-dimensional scaffold materials in the construction of high-density myocardial tissue in vitro, this study uses the microencapsulation technology to construct a compact three-dimensional myocardial group in vitro The preparation method comprises the following steps of: firstly, developing a sodium alginate/ collagen composite microcapsule by using a different proportion of collagen, the cell compatibility of the composite microcapsule is remarkably improved, and the preparation method is suitable for the attachment and the increase of the cardiac muscle of the primary rat The two-dimensional cultured cardiomyocytes usually have weak proliferation ability and can easily lose the characteristics of the cardiac muscle cells, and the myocardial cells coated in the micro-capsules can be continuously in the proliferation state without passage, and the special cardiac muscle cells are maintained. Therefore, the composite micro-capsule technology has the unique advantages of maintaining the continuous proliferation of the neonatal rat cardiac muscle cells. a three-dimensional cardiac muscle group with high cell density and continuous beating function is formed in the microcapsule by the three-dimensional culture of the primary myocardial cells of the rat mammary rat by using the composite microcapsule, in order to further verify the activity of the myocardial tissue in the body, the myocardial tissue is implanted into the model of the rat myocardial infarction, the heart function of the rat is detected after 6w, and the three-dimensional myocardial tissue can obviously improve the ejection fraction of the heart and the blood vessel of the infarction site, The deletion of the extracellular matrix of the myocardial cells induced by myocardial infarction is a key problem in the reduction of cardiac function. The local injection of the injectable gel can improve the mechanical properties of the myocardial infarction area and reduce the heart failure. The calcium ion cross-linked hydrogel has poor mechanical property, is not easy to degrade in the body, and the cell affinity is poor due to the fact that the present multi-purpose calcium ion cross-linked sodium alginate hydrogel In this paper, the sodium alginate is partially oxidized to obtain the active aldehyde group, and then the covalent cross-linking is formed with the free amino group in the gelatin molecule. The mechanical properties, degradation performance and cell affinity of the covalently cross-linked gel system were better than that of calcium ion cross-linking. The hydrogel can improve the recovery of the myocardial function as a whole, the mechanical property of the gel reaches 106 Pa, the setting time can be controlled at 30-40 min, and the porosity is achieved. 70%. The chemical cross-linked gel is in vitro, the in vivo degradation rate is accelerated, and after the myocardial infarction model of the rat is implanted, the heart function is improved. The histological study shows that the chemical cross-linked injectable gel increases the wall thickness of the left ventricle in the body, promotes the regeneration of the blood vessel, and can inhibit the matrix metalloproteinases. In conclusion, this paper deals with two basic problems of myocardial cell injury and extracellular matrix injury in the pathological process of myocardial infarction In this paper, the three-dimensional microencapsulation of sodium alginate hydrogel was designed to successfully construct a type of myocardial tissue with the function of continuous pulsatility, and a chemical cross-linked injectable gel was designed for the loss of extracellular matrix and applied to the model of myocardial infarction in rats. The experimental results show that both the three-dimensional myocardial tissue and the injectable gel can improve the myocardial function of the infarct zone and provide the treatment for myocardial infarction.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22;R318.08
【参考文献】
相关期刊论文 前1条
1 陈红霞,高英茂,邴鲁军;急性大鼠心肌梗死实验模型的制备[J];中国组织化学与细胞化学杂志;2005年01期
本文编号：2512383
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