本文選題：急性心肌梗死 + 阿托伐他汀�。� 參考：《北京協(xié)和醫(yī)學院》2014年博士論文

【摘要】：目的：隨著干細胞再生醫(yī)學不斷發(fā)展,應用骨髓來源的間充質(zhì)干細胞(mesenchymal stem cells, MSCs)進行急性心肌梗死(acute myocardial infarction, AMI)后心肌修復成為一種有前景的方法。然而干細胞移植進行梗死心肌修復雖然有效,但是療效有限。其療效底下的原因之一是干細胞歸巢至梗死心肌的數(shù)量較少。基質(zhì)細胞衍生因子1(stromal cell-derived factor-1, SDF-1)與其特異性受體趨化因子受體4(CXC chemokine receptor4, CXCIR4)構成的SDF-1/CXCR4生物學軸在MSCs歸巢、定植到損傷部位參與修復的過程中發(fā)揮重要作用。既往研究表明,他汀不但能改善梗死微環(huán)境促進MSCs存活,而且能增強MSCs的自身抗凋亡能力。然而,他汀能否促進MSCs的遷移和歸巢能力尚無相關研究。因此,我們進行此項研究,擬明確阿托伐他汀(atorvastatin, ATV)預處理能否增加MSCs表面CXCR4的表達,并提高MSCs的歸巢能力,進而改善心梗后心功能。 方法：實驗分為體外實驗和體內(nèi)實驗兩部分。體外實驗部分：分離并培養(yǎng)Sprague-Dawley大鼠MSCs,將第3代細胞隨機分為正常對照組、ATV不同濃度梯度組(0.01μM、0.1μM、1μM、10μM)、ATV不同時間梯度組(1h、3h、6h、12h、24h、36h、48h)；機制探討部分設MSCs組、最適ATV預處理組(ATV-MSCs). MSCs+CXCR4中和抗體組(MSCs-NA)、ATV-MSCs+CXCR4中和抗體組(ATV-MSCs-NA)。流式細胞術和RT-PCR檢測各組CXCR4的表達,Transwell小室評估MSCs的遷移能力。體內(nèi)實驗部分：共108只雌性SD大鼠(6-8周齡),隨機分為SDF-1動態(tài)測定組(AMI30min、1h、12h、24h、48h、72h、5d、7d)、假手術組(Sham)、心梗對照組AMI)、MSCs移植組(MSCs)、ATV預處理MSCs移植組(ATV-MSCs).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24小時經(jīng)尾靜脈注射MSCs或ATV-MSCs(2x106細胞/只),對照組動物注射等體積PBS。在干細胞移植后3天(基線)及30天(終點)分別行心臟超聲檢測、病理組織學及分子生物學檢測。 結(jié)果：體外實驗表明,與正常對照組相比,ATV劑量依賴性的增加細胞表面CXCR4表達,以1μM ATV處理組最為明顯(14.76±3.05%vs.1.98±0.40%,P0.001)；與正常對照組相比,ATV處理組CXCR4的表達呈一定的時間依賴性,其中ATV處理12小時CXCR4的表達至峰值(22.77±2.03%vs.2.20±0.18%,P0.001),24小時仍保持在較高水平(20.34±4.13%vs.2.20±0.18%,P0.001)。CXCR4mRNA的表達趨勢與細胞表面CXCR4的表達趨勢相似。‘Transwell遷移結(jié)果顯示,ATV預處理組MSCs向SDF-1遷移的數(shù)量是未處理組的2倍左右(24.65±5.57vs.12.70±2.40,P0.001),然而加入CXCR4的中和抗體后MSCs的遷移能力被抑制。 體內(nèi)試驗發(fā)現(xiàn),干細胞移植后3天,冰凍切片激光共聚焦顯微鏡下觀察到ATV-MSCs組歸巢到梗死心肌區(qū)域的細胞數(shù)明顯高于MSCs組(41.68±10.80vs.65.30±13.37,P0.05),在非梗死區(qū)域未發(fā)現(xiàn)移植的干細胞；在細胞移植后30天,ATV-MSCs組和MSCs組的存活率都很低,MSCs組的存活率更低。與AMI組相比,MSCs組的左室收縮末期內(nèi)徑和左室射血分數(shù)有適度改善。與MSCs組相比,ATV-MSCs組的左室舒張末期內(nèi)徑和左室收縮末期內(nèi)徑進一步減小,左室射血分數(shù)(62.28±3.27%vs.52.77±7.05%,P=0.014)和左室短軸縮短率(27.80±2.16%vs.22.27±3.84%,P=0.015)顯著改善。組織學分析顯示,與AMI組和MSCs組相比,ATV-MSCs組的心肌纖維化面積顯著減小,炎細胞浸潤明顯減輕。Western blot分析顯示,細胞移植后3天,ATV-MSCs組IL-6和TNF-α的表達水平明顯降低；細胞移植后30天,其表達水平進一步降低。 結(jié)論：ATV預處理可以提高MSCs表面CXCR4的表達,進而增強MSCs的遷移和歸巢能力,并通過改善梗死心肌中炎性因子的表達,進一步改善心肌梗死后心功能,ATV預處理MSCs可能成為提高干細胞移植療效的一有效方法。 目的：骨髓來源的間充質(zhì)干細胞(mesenchymal stem cells,MSCs)是移植治療急性心肌梗死的理想種子。目前移植的瓶頸問題是移植后的干細胞在心梗后惡劣的微環(huán)境大量凋亡,導致其療效受限。通心絡是一種傳統(tǒng)中藥,在我國廣泛應用于心血管疾病的治療。我們既往的體內(nèi)研究表明,中藥通心絡可以增加MSCs的存活,提高干細胞移植療效,但是通心絡在能否減少MSCs的凋亡及其具體機制尚未明確。單磷酸腺苷活化蛋白激酶(AMP-activated protein kinase,AMPK)是調(diào)控細胞能量代謝的核心分子,內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)是AMPK下游的重要分子,AMPK/eNOS通路在調(diào)節(jié)細胞凋亡中發(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)缺血缺氧環(huán)境,探討通心絡(tongxinluo,TXL)能否減少MSCs的凋亡,并明確AMPK/eNOS通路在其中發(fā)揮的作用。 方法：分離并培養(yǎng)Sprague-Dawley大鼠MSCs,將第3代細胞隨機分為正常對照組、H/SD對照組、通心絡不同濃度梯度組(50μg/mL、100μg/mL、200μg/mL、400μg/mL)、 AMPK通路抑制劑Compound C組(10μM)。在熒光顯微鏡下觀察Hocchst33342染色陽性細胞以及TUNEL染色陽性細胞,Annexin V/PI流式細胞術檢測各組細胞的凋亡比例,熒光探針JC-1流式細胞術檢測線粒體膜電位的變化,并進一步采用westernblot方法檢測凋亡相關蛋白細胞色素C、bax、bcl-2水平及AMPK、eNOS及其磷酸化蛋白的水平。 結(jié)果：與正常組相比,H/SD條件下細胞凋亡明顯增加Annexin V+/PI-細胞：21.91±3.28%vs.2.13±0.33%,P0.001；JC-1紅/綠色熒光比值：5.40±0.43vs.2.34±0.25,P0.001)。通心絡劑量依賴性的降低MSCs凋亡,在400μg/mL濃度時最為明顯,表現(xiàn)為nnexin V+/PI-細胞減少(3.47±0.69%vs.21.91±3.28%, P0.001), JC-1紅/綠色熒光比值增高(4.78±0.37vs.2.34±0.25,P0.001),促凋亡蛋白bax表達降低(1.06±0.24vs.2.92±0.29,P0.001),抗凋亡蛋白bcl-2表達增高(2.59±0.15vs.1.14±0.09,P0.001)。而且,通心絡各組AMPK和eNOS的磷酸化水平顯著升高。加入AMPK抑制劑Compound C后通心絡組AMPK和eNOS的磷酸化水平下降,同時減弱了通心絡的抗凋亡作用。 結(jié)論：通心絡可以抑制缺氧無血清培養(yǎng)條件下誘導的MSCs凋亡,其中AMPK/eNOS通路在其中發(fā)揮重要作用。這也為通心絡在心血管系統(tǒng)中的多效性提供了新的解釋。通心絡可能成為提高心肌梗死患者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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R542.22

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