药物提高骨髓间充质干细胞治疗急性心肌梗死效果的实验研究

发布时间：2018-07-06 10:00

本文选题：急性心肌梗死 + 阿托伐他汀　；参考：《北京协和医学院》2014年博士论文

【摘要】：目的：随着干细胞再生医学不断发展,应用骨髓来源的间充质干细胞(mesenchymal stem cells, MSCs)进行急性心肌梗死(acute myocardial infarction, AMI)后心肌修复成为一种有前景的方法。然而干细胞移植进行梗死心肌修复虽然有效,但是疗效有限。其疗效底下的原因之一是干细胞归巢至梗死心肌的数量较少。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(CXC chemokine receptor4, CXCIR4)构成的SDF-1/CXCR4生物学轴在MSCs归巢、定植到损伤部位参与修复的过程中发挥重要作用。既往研究表明,他汀不但能改善梗死微环境促进MSCs存活,而且能增强MSCs的自身抗凋亡能力。然而,他汀能否促进MSCs的迁移和归巢能力尚无相关研究。因此,我们进行此项研究,拟明确阿托伐他汀(atorvastatin, ATV)预处理能否增加MSCs表面CXCR4的表达,并提高MSCs的归巢能力,进而改善心梗后心功能。方法：实验分为体外实验和体内实验两部分。体外实验部分：分离并培养Sprague-Dawley大鼠MSCs,将第3代细胞随机分为正常对照组、ATV不同浓度梯度组(0.01μM、0.1μM、1μM、10μM)、ATV不同时间梯度组(1h、3h、6h、12h、24h、36h、48h)；机制探讨部分设MSCs组、最适ATV预处理组(ATV-MSCs). MSCs+CXCR4中和抗体组(MSCs-NA)、ATV-MSCs+CXCR4中和抗体组(ATV-MSCs-NA)。流式细胞术和RT-PCR检测各组CXCR4的表达,Transwell小室评估MSCs的迁移能力。体内实验部分：共108只雌性SD大鼠(6-8周龄),随机分为SDF-1动态测定组(AMI30min、1h、12h、24h、48h、72h、5d、7d)、假手术组(Sham)、心梗对照组AMI)、MSCs移植组(MSCs)、ATV预处理MSCs移植组(ATV-MSCs).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24小时经尾静脉注射MSCs或ATV-MSCs(2x106细胞/只),对照组动物注射等体积PBS。在干细胞移植后3天(基线)及30天(终点)分别行心脏超声检测、病理组织学及分子生物学检测。结果：体外实验表明,与正常对照组相比,ATV剂量依赖性的增加细胞表面CXCR4表达,以1μM ATV处理组最为明显(14.76±3.05%vs.1.98±0.40%,P0.001)；与正常对照组相比,ATV处理组CXCR4的表达呈一定的时间依赖性,其中ATV处理12小时CXCR4的表达至峰值(22.77±2.03%vs.2.20±0.18%,P0.001),24小时仍保持在较高水平(20.34±4.13%vs.2.20±0.18%,P0.001)。CXCR4mRNA的表达趋势与细胞表面CXCR4的表达趋势相似。‘Transwell迁移结果显示,ATV预处理组MSCs向SDF-1迁移的数量是未处理组的2倍左右(24.65±5.57vs.12.70±2.40,P0.001),然而加入CXCR4的中和抗体后MSCs的迁移能力被抑制。体内试验发现,干细胞移植后3天,冰冻切片激光共聚焦显微镜下观察到ATV-MSCs组归巢到梗死心肌区域的细胞数明显高于MSCs组(41.68±10.80vs.65.30±13.37,P0.05),在非梗死区域未发现移植的干细胞；在细胞移植后30天,ATV-MSCs组和MSCs组的存活率都很低,MSCs组的存活率更低。与AMI组相比,MSCs组的左室收缩末期内径和左室射血分数有适度改善。与MSCs组相比,ATV-MSCs组的左室舒张末期内径和左室收缩末期内径进一步减小,左室射血分数(62.28±3.27%vs.52.77±7.05%,P=0.014)和左室短轴缩短率(27.80±2.16%vs.22.27±3.84%,P=0.015)显著改善。组织学分析显示,与AMI组和MSCs组相比,ATV-MSCs组的心肌纤维化面积显著减小,炎细胞浸润明显减轻。Western blot分析显示,细胞移植后3天,ATV-MSCs组IL-6和TNF-α的表达水平明显降低；细胞移植后30天,其表达水平进一步降低。结论：ATV预处理可以提高MSCs表面CXCR4的表达,进而增强MSCs的迁移和归巢能力,并通过改善梗死心肌中炎性因子的表达,进一步改善心肌梗死后心功能,ATV预处理MSCs可能成为提高干细胞移植疗效的一有效方法。目的：骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)是移植治疗急性心肌梗死的理想种子。目前移植的瓶颈问题是移植后的干细胞在心梗后恶劣的微环境大量凋亡,导致其疗效受限。通心络是一种传统中药,在我国广泛应用于心血管疾病的治疗。我们既往的体内研究表明,中药通心络可以增加MSCs的存活,提高干细胞移植疗效,但是通心络在能否减少MSCs的凋亡及其具体机制尚未明确。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,内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)是AMPK下游的重要分子,AMPK/eNOS通路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环境,探讨通心络(tongxinluo,TXL)能否减少MSCs的凋亡,并明确AMPK/eNOS通路在其中发挥的作用。方法：分离并培养Sprague-Dawley大鼠MSCs,将第3代细胞随机分为正常对照组、H/SD对照组、通心络不同浓度梯度组(50μg/mL、100μg/mL、200μg/mL、400μg/mL)、 AMPK通路抑制剂Compound C组(10μM)。在荧光显微镜下观察Hocchst33342染色阳性细胞以及TUNEL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,荧光探针JC-1流式细胞术检测线粒体膜电位的变化,并进一步采用westernblot方法检测凋亡相关蛋白细胞色素C、bax、bcl-2水平及AMPK、eNOS及其磷酸化蛋白的水平。结果：与正常组相比,H/SD条件下细胞凋亡明显增加Annexin V+/PI-细胞：21.91±3.28%vs.2.13±0.33%,P0.001；JC-1红/绿色荧光比值：5.40±0.43vs.2.34±0.25,P0.001)。通心络剂量依赖性的降低MSCs凋亡,在400μg/mL浓度时最为明显,表现为nnexin V+/PI-细胞减少(3.47±0.69%vs.21.91±3.28%, P0.001), JC-1红/绿色荧光比值增高(4.78±0.37vs.2.34±0.25,P0.001),促凋亡蛋白bax表达降低(1.06±0.24vs.2.92±0.29,P0.001),抗凋亡蛋白bcl-2表达增高(2.59±0.15vs.1.14±0.09,P0.001)。而且,通心络各组AMPK和eNOS的磷酸化水平显著升高。加入AMPK抑制剂Compound C后通心络组AMPK和eNOS的磷酸化水平下降,同时减弱了通心络的抗凋亡作用。结论：通心络可以抑制缺氧无血清培养条件下诱导的MSCs凋亡,其中AMPK/eNOS通路在其中发挥重要作用。这也为通心络在心血管系统中的多效性提供了新的解释。通心络可能成为提高心肌梗死患者MSCs存活的又一有效方法。
[Abstract]:Objective: with the continuous development of stem cell regenerative medicine, myocardial repair after acute myocardial infarction (acute myocardial infarction, AMI) has become a promising method using bone marrow derived mesenchymal stem cells (mesenchymal stem cells, MSCs). However, the repair of infarcted myocardium by stem cell transplantation is effective, but the curative effect is limited. One of the reasons for its effect is that the number of stem cells homing to the infarcted myocardium is small. The SDF-1/CXCR4 biologic axis of the matrix derived factor 1 (stromal cell-derived factor-1, SDF-1) and its specific receptor chemokine receptor 4 (CXC chemokine receptor4, CXCIR4) is homing in MSCs and is fixed to the injured site for repair. Previous studies have shown that statins not only improve the survival of the infarct microenvironment to promote MSCs survival, but also enhance the ability of MSCs to resist apoptosis. However, there is no related research on whether statins promote the migration and homing of MSCs. Therefore, we do this study to identify the preposition of atorvastatin (atorvastatin, ATV). Can it increase the expression of CXCR4 on the surface of MSCs and improve the homing ability of MSCs, thereby improving the cardiac function after myocardial infarction.
Methods: the experiment was divided into two parts: in vitro experiment and in vivo experiment. In vitro experiment part: isolation and cultivation of Sprague-Dawley rat MSCs. The third generation cells were randomly divided into normal control group, ATV concentration gradient group (0.01 mu M, 0.1 mu M, 1 micron M, 10 M), ATV in different time gradient groups (1H, 3h, 6h, 12h, wasting,) The best ATV preconditioning group (ATV-MSCs). MSCs+CXCR4 neutralization antibody group (MSCs-NA), ATV-MSCs+CXCR4 neutralization antibody group (ATV-MSCs-NA). Flow cytometry and RT-PCR detection of CXCR4 expression, Transwell compartment evaluation of MSCs migration ability. In vivo experimental part: a total of 108 female SD rats (6-8 weeks of age), randomly divided into SDF-1 dynamic determination group N, 1H, 12h, 24h, 48h, 72h, 5D, 7D), the sham operation group (Sham), the myocardial infarction control group AMI), the MSCs transplantation group (MSCs), and the pretreated anterior descending branch of the coronary artery to make the acute myocardial infarction model. 24 hours after the infarction were injected into the tail vein, and the control group was injected with the same volume. Echocardiography, histopathology and molecular biology were performed on 3 days (baseline) and 30 days (end point) after stem cell transplantation.
Results: in vitro experiments showed that, compared with the normal control group, ATV dose dependent increase the expression of CXCR4 on the cell surface, and the most obvious (14.76 + 3.05%vs.1.98 + 0.40%, P0.001) in the 1 u M ATV treatment group. Compared with the normal control group, the expression of CXCR4 in the ATV treatment group showed a certain time dependence, and the expression of ATV processing 12 hours CXCR4 to the peak value was the peak value. (22.77 + 2.03%vs.2.20 + 0.18%, P0.001), the tendency to remain at a higher level (20.34 + 4.13%vs.2.20 + 0.18%, P0.001).CXCR4mRNA was similar to the expression trend of CXCR4 on the cell surface. 'Transwell migration results showed that the number of MSCs to SDF-1 in ATV preconditioning group was 2 times as much as that of the untreated group (24.65 +. 2.40, P). 0.001) however, the migration ability of MSCs was inhibited after neutralization antibody was added to CXCR4.
In vivo test, 3 days after the stem cell transplantation, the number of cells returned to the infarcted myocardium in the ATV-MSCs group was significantly higher than that in the MSCs group (41.68 + 10.80vs.65.30 + 13.37, P0.05), and the stem cells were not found in the non infarct area, and the survival of the ATV-MSCs and MSCs groups after the transplantation. The survival rate of the MSCs group was lower. Compared with the AMI group, the left ventricular end systolic diameter and left ventricular ejection fraction in the MSCs group improved moderately. Compared with the group MSCs, the left ventricular end diastolic diameter and left ventricular end systolic diameter of the ATV-MSCs group were further reduced, and the left ventricular ejection fraction (62.28 + 3.27%vs.52.77 + 7.05%, P=0.014) and the left ventricular short axis contraction were reduced. The short rate (27.80 + 2.16%vs.22.27 + 3.84%, P=0.015) significantly improved. Histological analysis showed that compared with group AMI and MSCs, the area of myocardial fibrosis in ATV-MSCs group decreased significantly, and the infiltration of inflammatory cells significantly reduced.Western blot analysis, and the expression level of IL-6 and TNF- a in ATV-MSCs group decreased obviously at 3 days after transplantation; 30 days after cell transplantation. The level of expression is further reduced.
Conclusion: ATV preconditioning can improve the expression of CXCR4 on the surface of MSCs, enhance the migration and homing ability of MSCs, and further improve the cardiac function of myocardial infarction by improving the expression of inflammatory factors in the infarcted myocardium. ATV preconditioning MSCs may be an effective method to improve the effect of stem cell transplantation.
Objective: bone marrow derived mesenchymal stem cells (mesenchymal stem cells, MSCs) are ideal seeds for transplantation in the treatment of acute myocardial infarction. The bottleneck of transplantation is a large number of apoptotic stem cells after transplantation in bad microenvironment after myocardial infarction, resulting in limited effect. Tongxinluo is a traditional Chinese medicine and is widely used in our country. Our previous research in vivo shows that Tongxinluo can increase the survival of MSCs and improve the effect of stem cell transplantation, but it is not clear whether Tongxinluo can reduce the apoptosis of MSCs and its specific mechanism. AMP-activated protein kinase (AMPK) is the nuclear regulation of cell energy metabolism. Endothelial nitric oxide synthase (eNOS) is an important molecule in the downstream of AMPK. The AMPK/eNOS pathway plays an important role in the regulation of apoptosis. Therefore, we establish a hypoxic serum-free (hypoxia and serum deprivation H/SD) model in vitro to simulate the environment of ischemia and hypoxia after myocardial infarction. To investigate whether Tongxinluo (TXL) can reduce the apoptosis of MSCs and clarify the role of AMPK/eNOS pathway in it.
Methods: Sprague-Dawley rat MSCs was isolated and cultured. The third generation cells were randomly divided into normal control group, H/SD control group, different concentration gradient group of Tongxinluo (50 g/mL, 100 mu g/mL, 200 mu g/mL, 400 mu g/mL), Compound C group of AMPK pathway inhibitor (10 mu M). The positive cells of Hocchst33342 staining and positive staining were observed under the fluorescent microscope. The cell apoptosis ratio was detected by Annexin V/PI flow cytometry. The changes of mitochondrial membrane potential were detected by fluorescence probe JC-1 flow cytometry, and Westernblot method was used to detect the levels of apoptosis related protein cytochrome C, Bax, bcl-2 level and AMPK, eNOS and phosphorylated protein.
Results: compared with the normal group, the apoptosis of Annexin V+/PI- cells increased significantly under H/SD conditions: 21.91 + 3.28%vs.2.13 + 0.33%, P0.001, JC-1 red / green fluorescence ratio: 5.40 + 0.43vs.2.34 + 0.25, P0.001). The dose dependence of Tongxinluo on MSCs apoptosis was the most obvious at 400 mu g/mL concentration, which was 3 of nnexin V+/PI- cells (3 .47 + 0.69%vs.21.91 + 3.28%, P0.001), the ratio of JC-1 red / green fluorescence increased (4.78 + 0.37vs.2.34 + 0.25, P0.001), the expression of apoptotic protein Bax decreased (1.06 + 0.24vs.2.92 + 0.29, P0.001), and the expression of anti apoptotic protein Bcl-2 increased (2.59 + 0.15vs.1.14 + 0.09, P0.001). After AMPK inhibitor Compound C, the phosphorylation level of AMPK and eNOS in Tongxinluo group decreased, while the anti apoptotic effect of Tongxinluo was weakened.
Conclusion: Tongxinluo can inhibit the apoptosis of MSCs induced by hypoxia and serum-free culture, and the AMPK/eNOS pathway plays an important role in it. This also provides a new explanation for the pleiotropic effect of Tongxinluo on the cardiovascular system. Tongxinluo may be another effective method to improve the survival of MSCs in patients with myocardial infarction.
【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】：2014
【分类号】：R542.22

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