【作者】 张力；

【导师】 陈莹莹；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2016， 硕士

【摘要】 背景传统的观念认为,成年哺乳动物的心脏是终末分化的组织,没有自我更新的能力。而近年来越来越多的证据表明,在成年人类及其他动物的心脏中,存在干细胞表面标志物(如C-Kit, MDR-1, Sca-1)阳性的心肌源性干细胞,即心肌干细胞(cardiac stem cells, CSCs)。研究报道,在心肌缺血等病理性心肌损伤后,心肌组织中CSCs增殖／迁移能力的改变是决定心肌功能是否恢复或恢复程度好坏的关键。炎症是不同心血管疾病,比如心衰、冠心病、高血压等发生发展机制中一个重要的病理过程。脂多糖(Lipopolysaccharide, LPS)是革兰氏阴性细菌细胞外膜上主要的分子组分,也是引起脓毒症休克的主要致病因素,可导致包括心功能损伤在内的多器官功能障碍。然而,近来有研究表明, LPS预处理可缩小梗死面积,促进缺血复灌心脏心功能的恢复。可见,炎性介质在心肌损伤及修复中起着双重作用,但目前其机制尚不清楚。我们推测可能与炎性介质促进或抑制CSCs的增殖／迁移有关。Wnt/β-catenin信号通路是一条在生物进化中极为保守的通路,控制着细胞增殖、干细胞维持、以及细胞迁移等重要的生命过程。在小鼠急性肺损伤模型的研究上发现,激活Wnt/p-catenin通路可促进间充质干细胞向损伤肺组织迁移,从而对受损组织给予修复。β-catenin是否参与了LPS引起的CSCs增殖、迁移的改变还尚不清楚。目的本研究目的是观察不同浓度LPS对CSCs增殖和迁移的影响；探讨β-catenin是否参与了其潜在机制。同时,在大鼠缺血复灌心肌损伤模型上明确移植LPS预处理的CSCs是否可通过增强其增殖和／或迁移能力,对抗心肌缺血损伤。方法从新生SD大鼠心脏中分离CSCs,用不同浓度LPS (0.0.01,0.1, 1μg/ml)刺激后,用CCK-8法检测细胞生存情况,Transwell小室法测定心肌干细胞体外迁移能力。结扎左冠脉前降支法建立成年大鼠心肌缺血模型。将LPS (0.01 μg/ml)预处理24h后的CSCs注射入大鼠缺血危险区边缘,CFDA荧光标记法观察CSCs向危险区中心迁移情况,TTC染色法评价心肌梗死面积。Western blotting法检测心肌干细胞Toll样受体4 (toll like receptor 4, TLR4),连接蛋白43(connexin 43, CX43)、β-catenin、热休克蛋白90 (heat shock protein 90, HSP90)的表达。结果(1) 与空白对照组相比,低、中浓度LPS (0.01、0.1μg/ml)孵育CSCs Id、3d、7d后,细胞生存率无明显改变。低浓度LPS (0.01μg/ml)处理CSCs 24 h后,细胞迁移数目明显增多。虽然0.1 μg/ml LPS作用于心肌干细胞也能促进CSCs迁移,但是效果不如低浓度处理组明显。高浓度LPS (1μg/ml)孵育CSCs 1d、3d后,虽然细胞生存率无明显改变,但第7d的细胞生存率与空白对照组相比,明显下降。且高浓度LPS处理CSCs 24小时后,细胞迁移数目明显下降。(2) 将低浓度LPS (0.01 μg/ml)预处理过的CSCs(事先用CFDA荧光标记)注射入缺血30 min/复灌120 min的大鼠心脏。免疫组化结果显示,与未经LPS预处理的CSCs相比,经LPS预处理后CSCs缺血危险区中心迁移的数量明显增多。同时,TTC染色结果显示,移植了LPS预处理后的CSCs,可使缺血后心肌的梗死面积明显缩小。(3) 体外培养的CSCs低浓度LPS刺激24h后,对CSCs的CX43、TLR4、总β-catenin蛋白表达无明显影响。但细胞内活化β-catenin(即未磷酸化的β-catenin)和细胞核内β-catenin水平却明显增高。β-catenin的抑制剂FH535可抑制低浓度LPS诱导的细胞迁移增加。(4) 与空白对照组相比,体外培养的CSCs用低浓度LPS (0.01μg/ml)刺激后可显著提高其HSP90的表达。与单纯低浓度LPS组相比,采用HSP90抑制剂17-AAG与LPS共同孵育心肌干细胞后,β-catenin活化及核转位水平明显下降；同时LPS的促细胞迁移作用明显受抑制。结论本实验结果表明,低浓度LPS可促进CSCs迁移,缩小缺血复灌心肌的梗死面积。其作用机制可能与HSP90依赖性β-catenin活化及核转位有关。该实验结果将为临床使用CSCs修复损伤的心肌组织提供新实验证据和理论依据。更多还原

【Abstract】 BackgroundAdult mammalian heart tissue is traditionally thought to be terminally differentiated cells without self-renewal potential. This classic concept leads to a consensus that the damaged heart tissue cannot be self-repaired, scar tissue replaces damaged myocardial tissue resulting in the impairment of cardiac function. It has been reported that cardiac stem cells (CSCs) can repair the damage to the muscle, which occurs during many pathological conditionings such as heart attack, due to its ability of regeneration and migration. The fate of CSCs under various heart disorders is crucial for the cardiac function preservation or deterioration. Lipopolysaccharide (LPS), the major molecular component of the outer membrane of gram-negative bacteria, is a main causative agent of septic shock which could result in cardiac dysfunction. However, LPS-preconditioning has recently been reported to induce the reduction of infarct size and promote the recovery of post-ischemic cardiac function in the ischemia-reperfusion heart. The mechanism that underlies the paradoxical role of inflammation in cardiac injury and repair is unclear. We hypothesized that it might be due to activation or inactivation of CSCs proliferation/migration. The Wnt/β-catenin signaling pathway plays a crucial role during embryonic development by controlling cell proliferation, stem cell maintenance and cell fate decisions, and migration. Whether activation of P-catenin is also involved in the LPS-pretreatment induced changes in proliferation and migration of CSCs is unclear.ObjectivesThe aims of this study were to investigate the effect of LPS on the proliferation and migration of CSCs, and to explore whether β-catenin is involved in its underlying mechanism, and finally to observe whether LPS pretreatment-induced CSCs proliferation or migration play an important role in the cardiac repair in the rat model of ischemia-reperfusion injury.MethodsCSCs were isolated from the hearts of neonatal Sprague-Dawley rats. CSCs were exposed to different dose of LPS (0,0.01,0.1,1 μg/ml) with or without pretreatment of 17-AAG or FH535. CCK-8 assay kit was used to evaluate the cell proliferation of CSCs. Migration was detected using 24-well transwell system in vitro cultured CSCs. Male adult SD rat hearts were suffered 30 min of ischemia and 120 min of reperfusion. At the end of ischemia, cultured CSCs pretreated with or without LPS (0.01 μg/ml) were injected into four sites along the border zone of the ischemia area (area at risk). Migration of CSCs was detected using carboxyfluorescein diacetate (CFDA)-labelled method in myocardial infarction rat model. The expression of toll like receptor 4 (TLR4), connexin 43 (CX43), β-catenin, active β-catenin, heat shock protein 90 (HSP90) were analyzed using western blotting.Results(1) Compared with control group, treatment of CSCs with LPS (0.01 or 0.1 μg/mL) for 1,3, or 7 d had no effect on cell proliferation. A transwell-based migration assay was performed after 24 h treatment of LPS. Compared with control group, lower and medium doses of LPS significantly increased the number of migrated CSCs, which reached a peak at 0.01 μg/mL.(2) The effect of LPS (0.01 μg/mL) on CSCs migration in the isolated rat hearts were assessed by using CFDA-labeled method. At the end of reperfusion, the number of LPS-pretreated CSCs migrated into the risk area was higher than the LPS-untreated CSCs. Injection of LPS-pretreated CSCs also caused a significant decrease in infarct size than that of LPS-untreated CSCs group.(3) Compared with control group, lower dose of LPS did not influence the expression of CX43, TLR4, and total amount of β-catenin protein in CSCs. However, it enhanced the levels of active β-catenin and nuclear β-catenin protein. Meanwhile, β-catenin inhibitor FH535 inhibited the enhancement of lower dose of LPS-induced migration.(4) HSP90 protein expression markedly increased in CSCs treated with LPS (0.01μg/mL). Compared with LPS group, after preincubated with HSP90 inhibitor 17-AAG, the LPS-induced enhancement of active β-catenin protein and nuclear P-catenin protein was abolished. And 17-AAG also prevented the lower dose of LPS-induced cell migration.ConclusionIn summary, the present study showed that low dose of LPS induced increase in migration of CSCs, reduced the infarct size in ischemic-reperfusion heart. The mechanism might be due to the activation and translocation of β-catenin via HSP90-dependent manner. These results suggested the LPS-pretreated CSCs as potential treatment for the repair of damaged heart tissue.更多还原