本文選題：急性心肌梗死 + 阿托伐他汀�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：口服強(qiáng)化阿托伐他汀-聯(lián)合阿托伐他汀預(yù)處理提高骨髓間充質(zhì)干細(xì)胞治療急性心肌梗死療效的實(shí)驗(yàn)研究目的：骨髓來源的間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是進(jìn)行急性心肌梗死(acute myocardial infarction, AMI)后心肌修復(fù)的一種有效的干細(xì)胞來源,但療效有限,原因之一是歸巢并存活于梗死心肌的MSCs數(shù)量較少。基質(zhì)細(xì)胞衍生因子1(stromal cell-derived factor-1, SDF-1)與其特異性受體趨化因子受體4 (CXCchemokine receptor 4, CXCR4)構(gòu)成的SDF-1/CXCR4軸在MSCs歸巢、定植到損傷部位參與修復(fù)的過程中發(fā)揮重要作用。我們的前期研究表明,口服強(qiáng)化阿托伐他汀(atorvastatin, ATV)能改善梗死微環(huán)境促進(jìn)MSCs存活,而且ATV預(yù)處理能增加MSCs表面CXCR4的表達(dá),提高M(jìn)SCs的歸巢能力。本研究旨在探討口服強(qiáng)化ATV聯(lián)合移植ATV預(yù)處理的MSCs (ATV-MSCs)是否可以進(jìn)一步改善心梗后心功能,并明確其作用是否是通過SDF-1/CXCR4軸發(fā)揮的。方法：第一部分將6-8周齡雌性Sprague-Dawley大鼠隨機(jī)分為假手術(shù)組(Sham)、心梗對照組(AMI)、 AMI后口服強(qiáng)化ATV組,分別于AMI后1天、1周和2周取材,通過免疫組化、RT-PCR和ELISA測定梗死周邊心肌組織SDF-1的動態(tài)變化,選擇SDF-1表達(dá)高峰作為第二部分移植MSCs或ATV-MSCs的時間點(diǎn)。第二部分將大鼠隨機(jī)分為Sham組,AMI對照組,移植MSCs組,口服強(qiáng)化ATV組,移植ATV預(yù)處理的MSCs (ATV-MSCs)組,口服強(qiáng)化ATV聯(lián)合移植MSCs (ATV+MSCs)組,同時口服強(qiáng)化ATV聯(lián)合移植ATV預(yù)處理的MSCs (ATV+ATV-MSCs)組,ATV+ATV-MSCs+AMD3100 (SDF-1/CXCR4阻滯劑)組。采用結(jié)扎冠狀動脈前降支的方法制作急性心肌梗死模型,梗死后1周經(jīng)頸靜脈注射CM-Dil標(biāo)記的MSCs或ATV-MSCs (2×106細(xì)胞／只)。梗死后4周通過心臟超聲和左心導(dǎo)管檢測心功能；通過病理組織學(xué)檢測歸巢至梗死心肌的MSCs、炎癥細(xì)胞浸潤及纖維化,TUNEL法檢測凋亡；通過蛋白芯片檢測梗死周邊心肌組織中促炎和抗炎因子的水平；通過免疫熒光法檢測血管新生、內(nèi)源性c-Kit+干細(xì)胞的數(shù)量以及MSCs向心肌分化情況。結(jié)果：與AMI組相比,口服強(qiáng)化ATV可顯著提高SDF-1的mRNA和蛋白表達(dá),在1周時達(dá)高峰,選擇1周作為MSCs移植時間點(diǎn)。與MSCs組相比,ATV-MSCs或ATV+MSCs組能夠顯著提高左室射血分?jǐn)?shù)(LVEF)、左室短軸縮短率(LVFS)和等容收縮期左室內(nèi)壓力上升的最大速率(dp/dt),顯著降低左室舒張末期直徑(LVEDd)、左室收縮末期直徑(LVESd)和左室舒張壓力(LVEDP); ATV+ATV-MSCs組的LVEF和LVFS與ATV-MSCs組相比進(jìn)一步提高,LVEDP和dp/dt與ATV-MSCs組和ATV+MSCs組相比均有顯著改善；而加入AMD3100阻斷SDF-1與CXCR4的結(jié)合后,ATV+ATV-MSCs對左心收縮和舒張功能的改善作用均被明顯抑制。組織學(xué)分析顯示,與MSCs組相比,ATV-MSCs和ATV+MSCs組的心肌纖維化面積顯著減小,炎細(xì)胞浸潤明顯減輕;ATV+ATV-MSCs組炎細(xì)胞浸潤進(jìn)一步減少,與ATV-MSCs組相比,纖維化面積顯著減小,上述作用可被AMD3100部分阻斷。蛋白芯片結(jié)果顯示,在梗死周邊心肌組織中,與AMI組相比,ATV+ATV-MSCs組的炎癥因子IL-1α、IL-1β、IL-6、TNF-α均下降最為顯著,抑炎因子IL-10水平明顯升高。ATV-MSCs和ATV+MSCs組歸巢到梗死周邊區(qū)心肌組織的MSCs數(shù)、新生動脈數(shù)和毛細(xì)血管數(shù)均明顯高于MSCs組,心肌細(xì)胞的凋亡顯著少于MSCs組；ATV+ATV-MSCs組的歸巢MSCs數(shù)和新生毛細(xì)血管數(shù)與ATV-MSCs和ATV+MSCs組相比進(jìn)一步提高,心肌細(xì)胞凋亡數(shù)繼續(xù)顯著減少；上述指標(biāo)在給予AMD3100后均受到顯著抑制。與AMI組相比,移植MSCs組的心肌組織中內(nèi)源性c-Kit+干細(xì)胞數(shù)顯著增多,ATV+ATV-MSCs組進(jìn)一步提高了c-Kit+干細(xì)胞的數(shù)量,但并未顯著增加MSCs向心肌細(xì)胞的分化。結(jié)論：ATV預(yù)處理或口服強(qiáng)化ATV均可以增強(qiáng)MSCs向梗死心肌的歸巢,二者聯(lián)合可通過SDF-1/CXCR4軸進(jìn)一步增加MSCs的歸巢和內(nèi)源性c-Kit+干細(xì)胞數(shù)量,促進(jìn)血管新生,抑制心肌細(xì)胞凋亡和炎癥反應(yīng),減少纖維化面積,提高心肌梗死后心功能。口服強(qiáng)化ATV聯(lián)合移植ATV預(yù)處理的MSCs可能成為提高干細(xì)胞療效的一有效方法。球形脂聯(lián)素降低缺氧無血清誘導(dǎo)骨髓間充質(zhì)干細(xì)胞凋亡的實(shí)驗(yàn)研究目的：骨髓來源的間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)在惡劣的梗死微環(huán)境中的低存活率極大限制了其治療急性心肌梗死的療效。脂聯(lián)素(adiponectin,APN)是一種脂肪細(xì)胞分泌的細(xì)胞因子,球形脂聯(lián)素(globular adiponectin, gAPN)是其C端球形結(jié)構(gòu)域,可以抗多種細(xì)胞凋亡,并具有干細(xì)胞調(diào)控特性。脂聯(lián)素主要通過與細(xì)胞表面的脂聯(lián)素受體1(AdipoR1)或受體2(AdipoR2)結(jié)合發(fā)揮生物學(xué)作用。單磷酸腺苷活化蛋白激酶(AMP-activated protein kinase, AMPK)是調(diào)控細(xì)胞能量代謝的核心分子,在調(diào)節(jié)細(xì)胞凋亡中發(fā)揮重要作用。因此我們在體外建立缺氧無血清(hypoxia and serum deprivation, H/SD)模型來模擬心肌梗死后體內(nèi)缺血缺氧的微環(huán)境,探討脂聯(lián)素能否減少M(fèi)SCs的凋亡,并明確發(fā)揮作用的受體和下游的信號通路。方法：分離并培養(yǎng)3-4周齡雄性Sprague-Dawley大鼠骨髓MSCs,將第3代細(xì)胞隨機(jī)分為正常對照組、H/SD對照組、不同濃度梯度gAPN組(0.01 μg/mL,0.1 μg/mL、 1μg/mL)、通過小干擾RAN (small interfering RNA, siRNA)干擾AdipoR1組、干擾AdipoR2組、干擾AdipoRl+AdipoR2組、AMPK通路抑制劑Compound C組(10μM)。在熒光顯微鏡下觀察Hocchst33342染色陽性細(xì)胞,Annexin V/PI流式細(xì)胞術(shù)檢測各組細(xì)胞的凋亡比例,Caspase-3活性試劑盒檢測Caspase-3活性；熒光探針JC-1染色檢測線粒體膜電位(mitochondrial membrane potential, MMP)的變化,并進(jìn)一步采用Western blot方法檢測凋亡相關(guān)蛋白Caspase-3、Bax、Bcl-2水平及AMPK及其磷酸化蛋白的水平。結(jié)果：gAPN劑量依賴性的降低H/SD誘導(dǎo)的MSCs凋亡,表現(xiàn)為核皺縮和變性(Hocchst33342染色陽性)細(xì)胞減少,早期凋亡(Annexin V+/PI-)細(xì)胞和晚期凋亡(Annexin V+/PI+)細(xì)胞均呈劑量依賴性減少,Caspase-3活性降低,在1μg/mL濃度時最為明顯。干擾AdipoR1的表達(dá)可明顯抑制gAPN的抗凋亡作用,而干擾AdipoR2的表達(dá)對gAPN的抗凋亡作用無顯著影響。gAPN各組AMPK的磷酸化水平升高,加入Compound C抑制AMPK的磷酸化后,gAPN的抗凋亡作用被顯著抑制。此外,gAPN升高抗凋亡蛋白Bcl-2、抑制促凋亡蛋白Bax的表達(dá),抑制了線粒體膜電位的喪失,而干擾AdipoR1表達(dá)或加入Compound C后gAPN的上述作用被部分抑制。結(jié)論：gAPN可以劑量依賴性抑制H/SD培養(yǎng)條件誘導(dǎo)的MSCs凋亡,其機(jī)制可能是通過與AdipoR1結(jié)合后激活A(yù)MPK通路,進(jìn)而抑制線粒體途徑的凋亡。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R542.22
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本文編號：1773471