葛根素水凝胶在MSCs移植治疗心肌梗死中的作用及机制研究

发布时间：2018-06-01 02:48

本文选题：心肌梗死 + 干细胞　；参考：《南方医科大学》2016年博士论文

【摘要】：随着人们生活水平的提升,近年来心血管疾病,特别是心肌梗死的发病率呈逐年上升趋势,心肌梗死仍然是我国致死率和致残率最高的疾病之一。尽管近年来人们对心肌梗死的认识和诊疗水平都取得了长足进展,血管重建术(包括经皮冠脉成形术和冠脉旁路移植术)和相关药物的使用很大程度上降低心肌梗死患者的死亡率,但这些措施无法解决心肌梗死后心肌细胞丧失这一关键问题。干细胞移植治疗心肌梗死是最有希望使心肌再生的方法,是近十余年来该研究领域的热点,未来发展的主要方向之一。骨髓间充质干细胞(Mesenchymal stem cells, MSCs)由于其获取和扩增方便,有多向分化能力,可以自体移植及异体移植有免疫豁免功能等优点而倍受青睐,是近年来用于临床研究最常见的干细胞之一。但是,临床试验结果表明,干细胞移植治疗后远期获益不明显,全因死亡率等主要临床终点改善不佳。究其原因主要是干细胞移植后面临心肌局部血流的冲击和细胞坏死后产生的过氧化物损伤,导致干细胞的滞留率和存活率都很低。如能为移植的干细胞提供一个暂时的“安身的场所”,并对干细胞的功能进行调控,增强其抵抗心梗局部过氧化微环境损伤,则有希望提高干细胞的存活和滞留,从而发挥其应有的作用。可注射性心肌组织工程(Injectable cardiac tissue engineering)是将可注射性支架材料通过心内膜或心外膜局部注射到心肌病变位置进行治疗。一般是指具有三维网络样结构的水凝胶,具有一定的韧性和粘稠度,富含水分达95%以上,可以携带药物或者细胞进行治疗。研究表明,水凝胶可以作为细胞移植的载体,能够粘附包裹移植细胞,在一定程度上减少因为渗漏或者血流冲击造成的细胞流失,增加细胞在目标位点的滞留时间和数量而发挥其应有的作用；另外,用可注射性水凝胶进行心肌组织工程治疗可以增加室壁厚度,为心室壁提供一个机械支撑力,从而减少心室张力,改善心室重构,改善心功能,当水凝胶的机械力学性质类似于健康心肌,这个效果更显著。有生物学功能的生物材料能够根据心梗后的病理生理变化及干细胞功能需求进一步优化,近年来受到研究者的青睐,如促进血管新生的功能性多肽水凝胶、具有靶向作用的生物纳米材料、具有抗氧化作用的水凝胶等。冠脉梗塞后由于局部心肌缺血缺氧,或者由于血管重建再通导致的再灌注损伤,心肌细胞及成纤维细胞等在代谢过程中可以产生一系列活性氧簇(Reactive oxygen species, ROS),主要包括：O2-、H2O2、HO2·和·OH等,对心肌细胞和外源性移植的干细胞都可以造成严重损伤。如能开发出具有抗氧化作用的水凝胶,不仅能发挥水凝胶的机械支撑作用,而且可以有效降低梗死区局部ROS水平,保护细胞免受损伤,提高细胞移植后的存活率。葛根素(化学名为8-p-D-葡萄糖吡喃糖-7,4'-二羟基异黄酮,puerarin)是从野葛的干燥根中提取分离得到的主要活性成分,是一种异黄酮类化合物。研究显示：葛根素能够清除超氧化物阴离子,抑制由过氧化氢引起的红细胞溶血损伤和脂质过氧化物的生成；能保护过氧化诱导的内皮细胞和PC12细胞损伤,有很好的保护细胞,抗过氧化的作用；基础实验研究表明葛根素这种保护细胞与抗凋亡的作用的可能机制为：(1)葛根素分子可以提供质子耦合机体过量的ROS,直接破坏H2O2分子,降低细胞内的过氧化水平,从而减少其对细胞的损伤；(2)提高MSCs的内源性抗氧化酶,比如SOD酶、GSH-PX等酶的活性,提高细胞自身抗氧化防御系统对过氧化阴离子的清除能力；(3)葛根素能直接参与损伤细胞DNA的修复。因此,面对细胞移植后心梗局部的过氧化微环境损伤,葛根素有望能够调控干细胞移植后的存活。如能开发出葛根素类水凝胶进行心肌组织工程治疗,不仅能够为梗塞后心室提供机械支撑力,包载移植细胞减少细胞的流失,而且可以发挥葛根素抗氧化保护细胞的作用,进一步增加移植细胞的滞留和存活,使其发挥最大功效。载药水凝胶的构建一般包括化学键键合和物理包裹的形式,在前者中药物既成为药物释放的对象又是载体的一部分,能够明显提高药物包载率,但是较后者在材料的选择和制作工艺上有更高的要求。我们课题组在尝试采用葛根素键合多肽构建水凝胶的过程中发现,葛根素本身不进行任何的化学修饰就能够通过简易的工艺和促发方式自组装形成水凝胶。结合葛根素的化学结构及其他成胶体系的成胶原理,我们推测葛根素水凝胶(Puerarin hydrogel, PG)的成胶原理可能为：葛根素分子结构中既含有亲水的葡萄吡喃糖结构也有相对疏水的异黄酮结构,在常温下处于微溶物质,在加热下可以使其完全溶解,冷却时可以通过分子间苯环堆叠力、疏水作用力和氢键作用自组装形成具有三维纤维网络的胶体结构。葛根素水凝胶是迄今为止发现的第一种能自己形成小分子水凝胶的天然药物。葛根素水凝胶的发现具有明显的优势：(1)由于成份只有葛根素本身,因此其药物包载率是100%,是药物包载率最高的药物载体,其药物释放是通过胶体的缓慢降解实现；(2)制作工艺和成胶方式简便；(3)通过加热冷却后约数分钟成胶,为其可注射性创造条件。接着,课题组对葛根素水凝胶进行研究,主要包括水凝胶的物理化学性能表征和生物学相容性检测,体外观察葛根素水凝胶在过氧化环境下保护MSCs的能力,葛根素水凝胶携带MSCs体内移植大鼠心肌梗死模型的研究。研究分以下三部分进行：第一部分：葛根素水凝胶的物理化学性能表征和生物学相容性检测我们对葛根素水凝胶表征时观察到,其最低成胶浓度为1%,并且为均匀的透明头胶体,随着胶体浓度增高,其透明性有所减弱,6%时形成乳白色的不透明的水凝胶；通过透射电镜表征水凝胶的自组装形貌：葛根素水凝胶为三维网络状结构,各个浓度的水凝胶均形成较长的纳米纤维,纤维直径大约为10-50nm,1%、2%和4%形成的纤维长度及直径均相似；力学性能表征发现,葛根素水凝胶是非频率依赖性,而是应力依赖型胶体,2%和4%的葛根素水凝胶处于稳定状态时的力学强度约为10K.Pa,这与心肌组织的力学强度相似,适合进行心肌组织工程研究；体外药物释放实验发现：在生理环境下,2%和4%浓度的水凝胶在前12h以较快的速率释放葛根素药物,药物可以持续释放12h以上；细胞相容性实验表明：CCK-8检测结果显示葛根素水凝胶与心肌细胞和心肌成纤维细胞均有良好的细胞相容性,无明显细胞毒害作用；第二部分：葛根素水凝胶在过氧化环境下保护MSCs的能力研究及机制探讨本实验采用100μmol/L H2O2干预1h作为外源性ROS模拟体内心肌梗死后的过氧化环境。实验分5组进行：正常MSCs组、H2O2干预组、H2O2+2%PG组、H2O2+4%PG组和H2O2+6%PG组。通过检测细胞内的SOD活性、MDA含量和ROS荧光探针DHE的水平可以反映细胞内ROS的含量,与正常MSCs组比较,H202干预组细胞内MDA含量和SOD活性增加,DHE红色荧光数量明显增加,而2%、4%和6%浓度的葛根素水凝胶可以显著减少SOD活性和MDA含量,DHE红色荧光明显减少；用CCK-8检测细胞存活和增殖,培养基中LDH的释放反映细胞毒性的大小,AO/EB染色从细胞形态学上观察细胞的存活和凋亡,Annexin V-FITC/PI双染凋亡检测法检测细胞的凋亡率,这些检测结果表明H202诱导后细胞存活减少、死亡增加,2%、4%和6%的葛根素水凝胶均可明显降低氧化应激后MSCs的凋亡率,增加细胞存活；Western blot分析显示2%、4%和6%的葛根素水凝胶可使抑制细胞凋亡蛋白Bcl-2表达增加和促凋亡蛋白Bax和Caspase-3表达减少,从蛋白水平印证了细胞形态学的变化。这些结果均表明H202诱导细胞ROS的产生及细胞存活减少、凋亡增多,而通过2%、4%和6%的葛根素水凝胶培养细胞均可以抑制ROS的产生,保护细胞减少细胞凋亡,其机制可能是通过减少促凋亡蛋白Bax和Caspase-3的表达和增加抑制细胞凋亡蛋白Bcl-2的表达产生作用。第三部分：葛根素水凝胶在MSCs移植治疗大鼠心肌梗死中的作用及机制研究在本部分研究中,我们将可注射性葛根素水凝胶携带MSCs移植治疗大鼠心肌梗死模型,以观察其对移植细胞的滞留存活、大鼠心功能和心脏结构的改善作用。实验采用心脏超声对移植后心功能的变化进行评价；HE染色、Masson染色检测移植后心脏组织的形态学变化,包括室壁厚度,心梗面积和心肌纤维化程度；用免疫荧光评价心梗部位心肌和血管新生情况,Western blot检测抗凋亡蛋白和促凋亡蛋白的变化。结果发现：细胞移植后4周,4%PG+MSCs组较单纯MSCs组的细胞滞留及存活明显增加；移植治疗术后4周,心彩超心功能检测提示4%PG+MSCs组、MSCs组和4%PG水凝胶联合MSCs组均能改善左室射血分数(LVEF),增加心室短轴缩短率(FS),但4%PG+MSCs组改善更加明显；组织形态学检测显示4%PG、MSCs组和4%PG+MSCs组均能改善心梗后室壁厚度,减少心梗面积,减少心肌纤维化程度,减少心肌损伤,增加血管新生,增加心肌组织数量,且葛根素水凝胶联合MSCs治疗组的效果更加明显。综述所述,本研究成功制备葛根素水凝胶,其成份只有葛根素,是现今发现的第一种能自组装形成水凝胶的天然药物,其具有与心肌组织相近的力学强度,能够平稳释放葛根素药物达12小时以上,具有良好的心肌细胞和心肌成纤维细胞相容性；葛根素是一种强抗氧化剂,可以减少H2O2诱导的细胞ROS水平,增加细胞存活,减少细胞凋亡率及细胞凋亡相关蛋白的产生,其机制可能与其抑制凋亡有关；葛根素水凝胶能够提高移植细胞在心梗局部的滞留和存活,促进MSCs改善心功能和心室重构的治疗效果,其机制可能是通过抗氧化,减少细胞凋亡,促进血管新生,改善心梗微环境的作用修复心梗组织。本研究从干细胞移植治疗的不足这一关键临床问题出发,紧密结合生物材料学与心肌组织工程学,研制了葛根素水凝胶并对其抗过氧化、保护细胞的生物学效用进行研究,并包裹干细胞移植治疗动物心梗模型,阐明其改善心梗微环境与促进心梗修复的作用及可能机制,对于心肌组织工程研究中支架材料的选择和设计以及未来心肌梗死临床治疗具有重要指导意义。
[Abstract]:With the improvement of people's living standards, the incidence of cardiovascular disease, especially myocardial infarction, is increasing year by year. Myocardial infarction is still one of the most fatal diseases in our country. Although people have made great progress in the level of understanding and diagnosis of myocardial infarction in recent years, vascular reconstruction (including percutaneous coronary intervention) The use of coronary angioplasty and coronary bypass graft) and the use of related drugs greatly reduce the mortality of patients with myocardial infarction, but these measures do not solve the key problem of myocardial cell loss after myocardial infarction. Stem cell transplantation for myocardial infarction is the most promising method for cardiac muscle regeneration. It has been the study for more than ten years. Mesenchymal stem cells (MSCs), which is one of the most common stem cells used in recent years, is one of the most common stem cells for clinical research. The results of clinical trials showed that the long-term benefit of stem cell transplantation was not obvious, and the main clinical endpoints such as the death rate were not well improved. The main reason is that the stem cell transplantation faces the impact of local blood flow and the peroxide damage after cell necrosis, which leads to the low retention and survival rate of stem cells. The stem cells provide a temporary "safe place" and regulate the function of the stem cells to enhance their resistance to the injury of the partial peroxidation microenvironment of the myocardial infarction, so as to improve the survival and retention of the stem cells, and thus play its due role. Injectable cardiac tissue engineering is an injectable myocardium. Injectable scaffolds are treated by local injection of the endocardium or epicardium to the location of the myocardial lesion. Generally, it refers to a hydrogel with a three-dimensional network structure, with a certain toughness and consistency, more than 95% moisture content, and can be used to carry drugs or cells for treatment. Research shows that hydrogel can be used as a cell transplant. The carrier is able to adhere to the transplanted cells, to some extent, to reduce the loss of cells caused by leakage or blood flow impact, and to increase the cell's retention time and quantity at the target site. In addition, the use of injectable hydrogel for myocardial tissue engineering can increase the thickness of the ventricular wall and provide the ventricular wall. It provides a mechanical support to reduce ventricular tension, improve ventricular remodeling, and improve cardiac function. When the mechanical properties of hydrogel are similar to that of healthy myocardium, this effect is more significant. Biological materials with biological functions can be further optimized according to the pathophysiological changes after myocardial infarction and the demand for stem cell function. People have the favor, such as functional polypeptide hydrogels that promote angiogenesis, biological nanomaterials with targeted effects, hydrogels with antioxidative action, etc. after coronary infarction, cardiac myocytes and fibroblasts can be metabolized in the metabolic process due to ischemia and anoxia of local myocardium, or reperfusion injury caused by revascularization and revascularization. A series of Reactive oxygen species (ROS), including O2-, H2O2, HO2 and OH, can cause serious damage to both cardiac and exogenous stem cells. For example, the ability to develop hydrogel with antioxidant activity can not only play the mechanical support of hydrogel, but also effectively reduce the infarct area. Local ROS level protects cells from damage and improves survival after cell transplantation. Ge Gensu (chemical named 8-p-D- glucosamine -7,4'- two hydroxyl isoflavone, puerarin) is a major active component extracted from the dried root of Pueraria lobata, a isoflavone compound. Anions, which inhibit the hemolytic injury of erythrocytes and the formation of lipid peroxide caused by hydrogen peroxide, can protect endothelial cells and PC12 cells induced by peroxide, protect cells and resist peroxidation; basic experimental study shows that the possible mechanism of Puerarin protective cells and anti apoptosis is: ( 1) the Ge Gensu molecule can provide a proton coupled body overdose of ROS, which directly destroys H2O2 molecules, reduces the level of peroxidation in cells and reduces its damage to cells. (2) increase the activity of endogenous antioxidant enzymes, such as SOD enzymes, GSH-PX and other enzymes in MSCs, and improve the scavenging energy of the cell's own antioxidant defense system to the peroxidation anions. (3) (3) puerarin can be directly involved in the repair of damaged cells. Therefore, in the face of partial peroxidation microenvironment injury after transplantation, puerarin may be able to regulate the survival of stem cells after transplantation. For example, the development of Puerarin hydrogel for myocardial tissue engineering can not only provide mechanical support for the infarcted ventricles. The inclusion of transplanted cells reduces the loss of cells and can play the role of the antioxidant protective cells of puerarin to further increase the retention and survival of the transplanted cells, making it the most effective. The construction of the hydrogel usually includes the form of chemical bond bonding and physical encapsulation. In the former, the drug is the object of drug release. As a part of the carrier, it can obviously improve the drug loading rate, but the latter has a higher requirement on the selection and fabrication of the material. In the process of trying to construct the hydrogel with Puerarin bond polypeptide, we found that the puerarin itself can pass the simple process and the promoter without any chemical modification. In combination with the chemical structure of puerarin and the glue forming principle of other gelatine systems, we speculate that the gelation principle of Puerarin hydrogel (PG) may be: the structure of the puerarin molecular structure contains both the hydrophilic grape Piran structure and the hydrophobic isoflavone structure, which is in the micro solution at normal temperature. Substances, which can be completely dissolved in heating, can form a colloid structure with a three-dimensional fiber network through the intermolecular piled force, hydrophobic interaction and hydrogen bonding. The puerarin hydrogel is the first natural drug to form a small sub hydrogel. Now it has obvious advantages: (1) because the ingredient is only puerarin itself, the drug loading rate is 100%, the drug loading rate is the highest, its drug release is achieved through the slow degradation of colloid; (2) the preparation process and the gelling method is simple and simple; (3) through the heating and cooling for a few minutes after the gelation, for its injectable creation bar Then, the team studied the puerarin hydrogel, including the physical and chemical characterization and biological compatibility detection of the hydrogel, in vitro observation of the ability of the puerarin hydrogel to protect the MSCs under the peroxidation environment. The Ge Gensu hydrogel carried the rat model of myocardial infarction with MSCs in vivo. The study was divided into three parts. The first part: physical and chemical characterization and biological compatibility test of Puerarin hydrogel, we observed that the minimum gel concentration was 1% when the Ge Gensu hydrogel was characterized, and it was a uniform transparent head colloid, with the increase of colloid concentration, its transparency decreased and the opaque hydrogel formed in milk white at 6%. The self-assembled morphology of the hydrogel was characterized by transmission electron microscopy: the gelatin hydrogel was a three-dimensional network structure, and the hydrogels of each concentration formed longer nanofibers. The fiber diameter was about 10-50nm, the length and diameter of the fibers formed in 1%, 2% and 4% were all similar. The mechanical properties showed that the puerarin hydrogel was a non frequency dependence. It is a stress dependent colloid. The mechanical strength of the 2% and 4% puerarin hydrogels at a stable state is about 10K.Pa, which is similar to the mechanical strength of the myocardium. It is suitable for the study of myocardial tissue engineering. In vitro drug release experiments found that in the physiological environment, the 2% and 4% concentration hydrogels are released at a faster rate in the front 12h. Puerarin drugs could continuously release more than 12h, and the cytocompatibility test showed that the results of CCK-8 detection showed that the puerarin hydrogel had good cytocompatibility with myocardial cells and myocardial fibroblasts, and there was no obvious cytotoxic effect. The second part: the ability to protect MSCs under the peroxidation environment of Puerarin hydrogel. The study and mechanism discussion used 100 mol/L H2O2 intervention 1H as a exogenous ROS to simulate the peroxidation environment after myocardial infarction in vivo. The experiment was divided into 5 groups: normal MSCs group, H2O2 intervention group, H2O2+2%PG group, H2O2+4%PG group and H2O2+6%PG group. The activity of SOD, MDA content and ROS fluorescence probe could be reflected by the detection of the cell SOD activity. The intracellular ROS content was compared with the normal MSCs group. The intracellular MDA content and SOD activity increased in the H202 intervention group, and the number of DHE red fluorescence increased significantly. The 2%, 4% and 6% puerarin hydrogels could significantly reduce the SOD activity and MDA content, and the DHE red fluorescence decreased obviously; CCK-8 was used to detect the cell survival and proliferation, and the LDH release in the medium was reversed. The size of enantiomer toxicity, AO/EB staining was used to observe cell survival and apoptosis from cell morphology, and Annexin V-FITC/PI double staining apoptosis detection method to detect cell apoptosis rate. These results showed that H202 induced cell survival decreased and death increased. The 2%, 4% and 6% puerarin hydrogels could significantly reduce the apoptosis of MSCs after oxidative stress. Western blot analysis showed that 2%, 4% and 6% puerarin hydrogels could inhibit the increased expression of apoptotic protein Bcl-2 and decrease the expression of apoptotic protein Bax and Caspase-3. The changes of cell morphology were confirmed from protein level. These results showed that H202 induced the production of ROS in cell and the decrease of cell survival and apoptosis. Increase, and the 2%, 4% and 6% puerarin hydrogel cultured cells can inhibit the production of ROS and protect cells to reduce apoptosis. The mechanism may be by reducing the expression of apoptotic protein Bax and Caspase-3 and increasing the expression of apoptosis protein Bcl-2. The third part: the puerarin hydrogel is treated with MSCs transplantation In this part of this study, we used injectable puerarin hydrogel to carry MSCs transplantation in the treatment of rat myocardial infarction model in order to observe the survival of the transplanted cells, the function of heart and the structure of the heart in rats. The morphological changes of cardiac tissue after transplantation were detected by HE staining and Masson staining, including the thickness of the wall, the area of myocardial infarction and the degree of myocardial fibrosis. The changes of myocardial and vascular neovascularization were evaluated by immunofluorescence, and the changes of anti apoptotic protein and apoptotic egg white were detected by Western blot. The results were found to be 4 weeks after the transplantation, 4%PG+ The cell retention and survival in group MSCs was significantly higher than that in group MSCs, and 4 weeks after transplantation, cardiac function test suggested that group 4%PG+MSCs, MSCs group and 4%PG hydrogel combined MSCs group could improve left ventricular ejection fraction (LVEF) and shorten the short axis shortening rate (FS), but the improvement of the 4% PG+MSCs group was more obvious; histomorphological detection showed 4%P. G, group MSCs and group 4%PG+MSCs can improve the thickness of the ventricular wall, reduce the area of myocardial infarction, reduce the degree of myocardial fibrosis, reduce the myocardial damage, increase the angiogenesis and increase the number of myocardial tissue, and the effect of the puerarin hydrogel combined with the MSCs treatment group is more obvious. Puerarin, the first natural drug that can be self assembled to form a hydrogel, has a mechanical strength similar to that of the myocardium. It can smoothly release Puerarin for more than 12 hours and has a good compatibility between myocardial cells and myocardial fibroblasts. Puerarin is a strong antioxidant and can reduce H2O2 induction. Cell ROS level can increase cell survival, reduce cell apoptosis rate and apoptosis related protein production, and its mechanism may be related to its inhibition.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.22

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