通过增加骨髓间充质干细胞的定向归巢和存活提高其治疗急性心梗疗效的实验研究

发布时间：2018-04-19 14:27

  本文选题：急性心肌梗死 + 阿托伐他汀　； 参考：《北京协和医学院》2016年博士论文

【摘要】：口服强化阿托伐他汀-联合阿托伐他汀预处理提高骨髓间充质干细胞治疗急性心肌梗死疗效的实验研究目的：骨髓来源的间充质干细胞(mesenchymal stem cells, MSCs)是进行急性心肌梗死(acute myocardial infarction, AMI)后心肌修复的一种有效的干细胞来源,但疗效有限,原因之一是归巢并存活于梗死心肌的MSCs数量较少。基质细胞衍生因子1(stromal cell-derived factor-1, SDF-1)与其特异性受体趋化因子受体4 (CXCchemokine receptor 4, CXCR4)构成的SDF-1/CXCR4轴在MSCs归巢、定植到损伤部位参与修复的过程中发挥重要作用。我们的前期研究表明,口服强化阿托伐他汀(atorvastatin, ATV)能改善梗死微环境促进MSCs存活,而且ATV预处理能增加MSCs表面CXCR4的表达,提高MSCs的归巢能力。本研究旨在探讨口服强化ATV联合移植ATV预处理的MSCs (ATV-MSCs)是否可以进一步改善心梗后心功能,并明确其作用是否是通过SDF-1/CXCR4轴发挥的。方法：第一部分将6-8周龄雌性Sprague-Dawley大鼠随机分为假手术组(Sham)、心梗对照组(AMI)、 AMI后口服强化ATV组,分别于AMI后1天、1周和2周取材,通过免疫组化、RT-PCR和ELISA测定梗死周边心肌组织SDF-1的动态变化,选择SDF-1表达高峰作为第二部分移植MSCs或ATV-MSCs的时间点。第二部分将大鼠随机分为Sham组,AMI对照组,移植MSCs组,口服强化ATV组,移植ATV预处理的MSCs (ATV-MSCs)组,口服强化ATV联合移植MSCs (ATV+MSCs)组,同时口服强化ATV联合移植ATV预处理的MSCs (ATV+ATV-MSCs)组,ATV+ATV-MSCs+AMD3100 (SDF-1/CXCR4阻滞剂)组。采用结扎冠状动脉前降支的方法制作急性心肌梗死模型,梗死后1周经颈静脉注射CM-Dil标记的MSCs或ATV-MSCs (2×106细胞／只)。梗死后4周通过心脏超声和左心导管检测心功能；通过病理组织学检测归巢至梗死心肌的MSCs、炎症细胞浸润及纤维化,TUNEL法检测凋亡；通过蛋白芯片检测梗死周边心肌组织中促炎和抗炎因子的水平；通过免疫荧光法检测血管新生、内源性c-Kit+干细胞的数量以及MSCs向心肌分化情况。结果：与AMI组相比,口服强化ATV可显著提高SDF-1的mRNA和蛋白表达,在1周时达高峰,选择1周作为MSCs移植时间点。与MSCs组相比,ATV-MSCs或ATV+MSCs组能够显著提高左室射血分数(LVEF)、左室短轴缩短率(LVFS)和等容收缩期左室内压力上升的最大速率(dp/dt),显著降低左室舒张末期直径(LVEDd)、左室收缩末期直径(LVESd)和左室舒张压力(LVEDP); ATV+ATV-MSCs组的LVEF和LVFS与ATV-MSCs组相比进一步提高,LVEDP和dp/dt与ATV-MSCs组和ATV+MSCs组相比均有显著改善；而加入AMD3100阻断SDF-1与CXCR4的结合后,ATV+ATV-MSCs对左心收缩和舒张功能的改善作用均被明显抑制。组织学分析显示,与MSCs组相比,ATV-MSCs和ATV+MSCs组的心肌纤维化面积显著减小,炎细胞浸润明显减轻;ATV+ATV-MSCs组炎细胞浸润进一步减少,与ATV-MSCs组相比,纤维化面积显著减小,上述作用可被AMD3100部分阻断。蛋白芯片结果显示,在梗死周边心肌组织中,与AMI组相比,ATV+ATV-MSCs组的炎症因子IL-1α、IL-1β、IL-6、TNF-α均下降最为显著,抑炎因子IL-10水平明显升高。ATV-MSCs和ATV+MSCs组归巢到梗死周边区心肌组织的MSCs数、新生动脉数和毛细血管数均明显高于MSCs组,心肌细胞的凋亡显著少于MSCs组；ATV+ATV-MSCs组的归巢MSCs数和新生毛细血管数与ATV-MSCs和ATV+MSCs组相比进一步提高,心肌细胞凋亡数继续显著减少；上述指标在给予AMD3100后均受到显著抑制。与AMI组相比,移植MSCs组的心肌组织中内源性c-Kit+干细胞数显著增多,ATV+ATV-MSCs组进一步提高了c-Kit+干细胞的数量,但并未显著增加MSCs向心肌细胞的分化。结论：ATV预处理或口服强化ATV均可以增强MSCs向梗死心肌的归巢,二者联合可通过SDF-1/CXCR4轴进一步增加MSCs的归巢和内源性c-Kit+干细胞数量,促进血管新生,抑制心肌细胞凋亡和炎症反应,减少纤维化面积,提高心肌梗死后心功能。口服强化ATV联合移植ATV预处理的MSCs可能成为提高干细胞疗效的一有效方法。球形脂联素降低缺氧无血清诱导骨髓间充质干细胞凋亡的实验研究目的：骨髓来源的间充质干细胞(mesenchymal stem cells, MSCs)在恶劣的梗死微环境中的低存活率极大限制了其治疗急性心肌梗死的疗效。脂联素(adiponectin,APN)是一种脂肪细胞分泌的细胞因子,球形脂联素(globular adiponectin, gAPN)是其C端球形结构域,可以抗多种细胞凋亡,并具有干细胞调控特性。脂联素主要通过与细胞表面的脂联素受体1(AdipoR1)或受体2(AdipoR2)结合发挥生物学作用。单磷酸腺苷活化蛋白激酶(AMP-activated protein kinase, AMPK)是调控细胞能量代谢的核心分子,在调节细胞凋亡中发挥重要作用。因此我们在体外建立缺氧无血清(hypoxia and serum deprivation, H/SD)模型来模拟心肌梗死后体内缺血缺氧的微环境,探讨脂联素能否减少MSCs的凋亡,并明确发挥作用的受体和下游的信号通路。方法：分离并培养3-4周龄雄性Sprague-Dawley大鼠骨髓MSCs,将第3代细胞随机分为正常对照组、H/SD对照组、不同浓度梯度gAPN组(0.01 μg/mL,0.1 μg/mL、 1μg/mL)、通过小干扰RAN (small interfering RNA, siRNA)干扰AdipoR1组、干扰AdipoR2组、干扰AdipoRl+AdipoR2组、AMPK通路抑制剂Compound C组(10μM)。在荧光显微镜下观察Hocchst33342染色阳性细胞,Annexin V/PI流式细胞术检测各组细胞的凋亡比例,Caspase-3活性试剂盒检测Caspase-3活性；荧光探针JC-1染色检测线粒体膜电位(mitochondrial membrane potential, MMP)的变化,并进一步采用Western blot方法检测凋亡相关蛋白Caspase-3、Bax、Bcl-2水平及AMPK及其磷酸化蛋白的水平。结果：gAPN剂量依赖性的降低H/SD诱导的MSCs凋亡,表现为核皱缩和变性(Hocchst33342染色阳性)细胞减少,早期凋亡(Annexin V+/PI-)细胞和晚期凋亡(Annexin V+/PI+)细胞均呈剂量依赖性减少,Caspase-3活性降低,在1μg/mL浓度时最为明显。干扰AdipoR1的表达可明显抑制gAPN的抗凋亡作用,而干扰AdipoR2的表达对gAPN的抗凋亡作用无显著影响。gAPN各组AMPK的磷酸化水平升高,加入Compound C抑制AMPK的磷酸化后,gAPN的抗凋亡作用被显著抑制。此外,gAPN升高抗凋亡蛋白Bcl-2、抑制促凋亡蛋白Bax的表达,抑制了线粒体膜电位的丧失,而干扰AdipoR1表达或加入Compound C后gAPN的上述作用被部分抑制。结论：gAPN可以剂量依赖性抑制H/SD培养条件诱导的MSCs凋亡,其机制可能是通过与AdipoR1结合后激活AMPK通路,进而抑制线粒体途径的凋亡。
[Abstract]:Oral enhancement of Atorvastatin Combined with atorvastatin to improve the efficacy of bone marrow mesenchymal stem cells in the treatment of acute myocardial infarction: bone marrow derived mesenchymal stem cells (mesenchymal stem cells, MSCs) is an effective myocardial repair after acute myocardial infarction (acute myocardial infarction, AMI). Stem cell sources, but the effect is limited, one of the reasons is that homing and surviving in infarcted myocardium have fewer MSCs numbers. The SDF-1/CXCR4 axis of matrix cell derived factor 1 (stromal cell-derived factor-1, SDF-1) and its specific receptor chemokine receptor 4 (CXCchemokine receptor 4, CXCR4) is homing in MSCs and is colonized to the site of injury. Our previous study showed that oral enhanced atorvastatin (ATV) could improve the survival of MSCs in infarct microenvironment, and ATV preconditioning could increase the expression of CXCR4 on the MSCs surface and improve the homing ability of MSCs. The purpose of this study was to explore MSCs with oral enhanced ATV combined with ATV preconditioning. ATV-MSCs) is it possible to further improve the cardiac function of the myocardial infarction and determine whether its effect is performed on the SDF-1/CXCR4 axis. Method: the first part of the 6-8 week old female Sprague-Dawley rats was randomly divided into the sham operation group (Sham), the myocardial infarction control group (AMI), and the oral strong ATV group after AMI, and were harvested at 1 days after AMI, 1 and 2 weeks respectively, through immunization. The dynamic changes of SDF-1 in the peri infarct myocardium were measured by RT-PCR and ELISA, and the peak of SDF-1 expression was selected as the time point of the second part of the transplantation of MSCs or ATV-MSCs. The rats were randomly divided into Sham group, AMI control group, transplant MSCs group, oral enhanced ATV group, MSCs group transplantation ATV pretreatment, oral strengthening combined migration. In the group of MSCs (ATV+MSCs), group of MSCs (ATV+ATV-MSCs) and ATV+ATV-MSCs+AMD3100 (SDF-1/CXCR4 blocker) group with enhanced ATV combined with ATV preconditioning, acute myocardial infarction model was made by ligating the anterior descending coronary artery. 1 weeks after the infarction, the MSCs or ATV-MSCs (2 x 106 cells / only) were injected into the cervical vein. 4 weeks after death, cardiac function was detected by echocardiography and left cardiac catheterization. MSCs, inflammatory cell infiltration and fibrosis were detected by histopathology, inflammatory cell infiltration and fibrosis, apoptosis was detected by TUNEL method. The level of proinflammatory and anti-inflammatory factors in peripheral myocardial tissue was detected by protein chip, and angiogenesis was detected by immunofluorescence. The number of source c-Kit+ stem cells and the differentiation of MSCs to the myocardium. Results: compared with the AMI group, oral enhanced ATV significantly increased the mRNA and protein expression of SDF-1, reached the peak at 1 weeks, and selected 1 weeks as the time point of MSCs transplantation. Compared with the MSCs group, the ATV-MSCs or ATV+MSCs group could significantly increase the left ventricular ejection fraction (LVEF) and the short axis contraction of the left ventricle. The maximum rate (dp/dt) of the short rate (LVFS) and the left ventricular pressure rise in the ISO systole significantly reduced the left ventricular end diastolic diameter (LVEDd), the left ventricular end systolic diameter (LVESd) and left ventricular diastolic pressure (LVEDP). The LVEF and LVFS of the ATV+ATV-MSCs group were further improved than the ATV-MSCs group, and LVEDP and dp/dt were compared with those of the ATV-MSCs group and the group. The effect of ATV+ATV-MSCs on the systolic and diastolic function of left heart was significantly inhibited by the combination of AMD3100 blocking SDF-1 and CXCR4. Histologic analysis showed that compared with the MSCs group, the area of myocardial fibrosis in ATV-MSCs and ATV+MSCs groups decreased significantly and the infiltration of inflammatory cells significantly decreased; ATV+ATV-MSCs group inflammatory cells infiltrated into the MSCs group. One step decreased, compared with the ATV-MSCs group, the area of fibrosis decreased significantly, and the above effect could be blocked by AMD3100. The results of protein chip showed that in the peripheral myocardial tissue of the infarct, the inflammatory factors of IL-1 a, IL-1 beta, IL-6, TNF- alpha in the ATV+ATV-MSCs group decreased most significantly, and the IL-10 levels of the anti inflammatory factors significantly increased.ATV-MSCs and ATV. The number of MSCs, the number of new arteries and the number of capillaries in the +MSCs group were significantly higher than that of the MSCs group. The apoptosis of myocardial cells was significantly less than that of the group MSCs, and the number of MSCs and the newborn capillaries in the ATV+ATV-MSCs group were further improved than those of the ATV-MSCs and ATV+MSCs groups, and the number of cardiomyocytes apoptosis continued to decrease significantly. Compared with the AMI group, the number of endogenous c-Kit+ stem cells in the myocardium of the transplanted MSCs group increased significantly, and the number of c-Kit+ stem cells increased in ATV+ATV-MSCs group, but did not significantly increase the differentiation of MSCs into the cardiomyocytes. Conclusion: ATV preconditioning or oral enhanced ATV can increase the number of cardiac myocytes in MSCs group. When strong MSCs is homing to the infarcted myocardium, the two combination can further increase the number of MSCs homing and endogenous c-Kit+ stem cells through the SDF-1/CXCR4 axis, promote angiogenesis, inhibit myocardial apoptosis and inflammation, reduce the area of fibrosis, and improve cardiac function after myocardial infarction. MSCs may become an enhanced ATV combined with ATV preconditioning for MSCs. An effective method to improve the efficacy of stem cells. Objective to study the objective of spherical adiponectin to reduce the apoptosis of bone marrow mesenchymal stem cells (MSCs) induced by hypoxia. Objective: the low survival rate of bone marrow derived mesenchymal stem cells (mesenchymal stem cells, MSCs) in severe infarct microenvironment restricts the curative effect of the treatment of acute myocardial infarction. Adiponectin (APN) is a cytokine secreted by adipocytes. Globular adiponectin (gAPN) is a spherical domain of its C terminal. It can resist many kinds of apoptosis and have the characteristics of stem cell regulation. Adiponectin plays biology mainly by binding to the lipoprotein receptor 1 (AdipoR1) or receptor 2 (AdipoR2) on the surface of the cell. AMP-activated protein kinase (AMPK), a core molecule that regulates cell energy metabolism, plays an important role in regulating cell apoptosis. Therefore, we establish an anoxic serum-free (hypoxia and serum deprivation, H/SD) model in vitro to simulate the microring of ischemic anoxia after myocardial infarction. To explore whether adiponectin can reduce the apoptosis of MSCs and clearly play a role in the receptor and the downstream signal pathway. Methods: the bone marrow MSCs of 3-4 weeks male Sprague-Dawley rats was separated and cultured, and the third generation cells were randomly divided into normal control group, H/SD control group, different concentration ladder gAPN group (0.01 mu g/mL, 0.1 g/mL, 1 u g/mL), through small dry. Disturbance RAN (small interfering RNA, siRNA) interfered with AdipoR1 group, interfered with AdipoR2 group, interfered with AdipoRl+AdipoR2 group, AMPK pathway inhibitor Compound C group (10 micron). The changes of mitochondrial membrane potential (mitochondrial membrane potential, MMP) were detected by fluorescent probe JC-1 staining, and the Western blot method was used to detect the apoptosis related protein Caspase-3, Bax, Bcl-2 level, AMPK and phosphorylated protein. Shrinkage and denaturation (Hocchst33342 staining positive) cells decreased, early apoptosis (Annexin V+/PI-) cells and late apoptosis (Annexin V+/PI+) cells were reduced in a dose-dependent manner, and the activity of Caspase-3 decreased, the most obvious at the concentration of 1 mu g/mL. The expression of interference AdipoR1 inhibited the anti apoptotic effect of gAPN, while the expression of AdipoR2 was interfered with gAPN. The anti apoptosis effect did not significantly affect the level of phosphorylation of AMPK in.GAPN. After adding Compound C to inhibit the phosphorylation of AMPK, the anti apoptosis effect of gAPN was significantly inhibited. In addition, gAPN increased the anti apoptotic protein Bcl-2, inhibited the expression of apoptotic protein Bax, inhibited the loss of mitochondrial membrane potential, and interfered with AdipoR1 expression or Compou. The above effects of gAPN after Nd C are partially suppressed. Conclusion: gAPN can inhibit the apoptosis of MSCs induced by H/SD culture in a dose dependent manner. The mechanism may be to activate AMPK pathway by binding to AdipoR1 and inhibit the apoptosis of mitochondrial pathway.

【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】：2016
【分类号】：R542.22
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