糖皮质激素在主动脉夹层血管重构中的调控机制研究

发布时间：2018-08-16 08:30

【摘要】：研究背景随着社会老龄化的到来和人民生活水平的提高,血管系统疾病的发病率逐年上升,而以主动脉夹层(aortic dissection,AD)为代表的主动脉扩张病是其中最凶险的一类。尽管AD的治疗方式从巨创向微创发生了革命性变化,夹层的并发症率和死亡率有了明显降低,但仍有很多患者在明确诊断前或开始救治前即因夹层破裂而死亡。因此,阐明AD发病和转归的机制,对改善夹层预后至关重要。有证据表明血管炎症反应在主动脉血管重构过程中起着重要作用,而糖皮质激素因其强大的抗炎作用被广泛用于临床。最新的随机对照临床研究表明术前单次应用大剂量的糖皮质激素,在没有增加不良事件发生率的基础上能明显降低腹主动脉瘤患者腔内隔绝术后全身炎症反应综合征的发生率,但糖皮质激素与AD的关系仍模糊不清。研究目的探讨糖皮质激素在AD的发生、发展和转归中的作用,并进一步阐明糖皮质激素参与主动脉血管重构的细胞分子机制。研究方法1临床样本研究:在符合伦理审查和患者知情同意情况下,收集AD、非破裂主动脉瘤(non-ruptured aortic aneurysm,n AA)和健康对照者的血液标本,放射免疫法检测血清皮质醇、血浆促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)的含量;同时收集行开放手术的AD、n AA患者和遗体捐献者的主动脉,免疫组化法检测主动脉中糖皮质激素受体(glucocorticoid receptor,GCR)表达水平;收集夹层患者的临床信息进行多元线性回归分析,探讨血清皮质醇含量的影响因素。2动物实验研究:在获得第二军医大学动物保护和使用协会的批准后,切除80只C57BL/6小鼠的双侧肾上腺,按2:2:1完全随机分成三组,用血管紧张素II建立AD模型,外加糖皮质激素或溶剂对照干预,三组分别为糖皮质激素干预组、模型组、溶剂对照组:记录小鼠的死亡时间,Kaplan-Meier曲线计算小鼠累积生存率;血管紧张素II干预前后每周尾动脉测压,验证血管紧张素II是否起效;解剖获取小鼠主动脉,借助Image-Pro Plus软件测量小鼠主动脉外径;苏木素-伊红染色,测量主动脉中膜厚度及观察夹层发生率;Masson染色,检测主动脉胶原容积分数;免疫组化法检测小鼠主动脉GCR表达及巨噬细胞的含量。3细胞分子研究:体外培养人主动脉平滑肌细胞(human aortic smooth muscle cell,HA-SMC)和巨噬细胞,酶联免疫吸附实验检测糖皮质激素对巨噬细胞分泌基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-2抑制剂(tissue inhibitor of metalloproteinase-2,TIMP-2)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的影响;划痕实验检测糖皮质激素对HA-SMC迁移的影响,细胞免疫荧光检测糖皮质激素对HA-SMC表型转换的影响;建立HA-SMC和巨噬细胞间接共培养体系,细胞流式实验检测细胞共培养及糖皮质激素对HA-SMC凋亡的影响,细胞免疫荧光检测细胞共培养及糖皮质激素对HA-SMC迁移的影响;高通量蛋白微阵列芯片检测细胞间相互作用的关键因子,并用相应因子的中和抗体验证其作用;应激和凋亡通路试剂盒检测关键因子参与血管重构的信号分子通路,western blot验证该信号分子的变化。结果1临床样本研究:2012年10月至2013年12月,共收集在我血管外科中心治疗经可靠影像学方法诊断的82例AD,68例n AA及在我院体检中心体检的76例健康者的血液样本,三组人群血清皮质醇含量分别为173.98±23.44 ng/m L,136.60±21.83 ng/m L和129.00±27.56 ng/m L,AD组明显高于n AA组(P0.001)和健康体检组(P0.001);三组人群血浆ACTH含量分别为26.76±7.41 pg/m L,24.75±8.21pg/m L和25.64±7.38 pg/m L,三组间未见统计学差异;同期收集行开放手术的8例AD,8例n AA和8例遗体捐献者的主动脉,三组GCR含量分别为14.25±1.31%,13.39±1.42%和12.55±2.12%,未见明显统计学差异;收集夹层患者的临床信息行多元回归分析发现夹层裂口数目是血清皮质醇含量的影响因素(偏回归系数0.920,P=0.029)。2动物实验研究:最终纳入糖皮质激素干预组、模型组和溶剂对照组分析的小鼠数量分别是31、27和13;模型组与糖皮质激素干预组(P=0.139)或溶剂对照组(P=0.107)的累积生存率无统计学差异(Log-rank检验);模型组的小鼠收缩压在血管紧张素II干预后明显高于溶剂对照组,在血管紧张素II干预后7天、14天、21天明显低于糖皮质激素干预组;模型组的小鼠主动脉外径明显大于溶剂对照组(升主动脉:1458.49±257.96μm vs.1094.61±154.57μm,P0.001;主动脉弓:1449.48±290.34μm vs.986.38±151.16μm,P0.001;降主动脉:1343.47±238.48μm vs.980.36±98.48μm,P0.001),模型组小鼠的升主动脉外径明显大于糖皮质激素干预组(1458.49±257.96μm vs.1279.17±204.24μm,P=0.005),主动脉弓和降主动脉外径无明显差异;模型组(29.6%)的小鼠AD发生率明显高于糖皮质激素干预组(6.5%,P=0.034)和溶剂对照组(0%,P=0.037);模型组小鼠的主动脉中膜厚度明显高于溶剂对照组(89.6±14.7μm vs.68.4±11.6μm,P0.001),但糖皮质激素干预组与模型组之间无统计学差异(89.8±13.5μm vs.89.6±14.7μm,P=0.937);模型组(24.2±6.0%)小鼠的主动脉胶原容积分数明显低于溶剂对照组(31.7±6.3%,P=0.001)和糖皮质激素干预组(30.1±8.2%,P=0.003);模型组(9.3±3.0%)小鼠的主动脉GCR水平与糖皮质激素干预组(8.5±2.1%,P=0.235)或溶剂对照组(7.6±1.1%,P=0.115)无明显不同;但模型组(9.3±3.5%)小鼠的主动脉巨噬细胞含量明显高于溶剂对照组(2.2±1.0%,P0.001)或糖皮质激素干预组(7.4±3.2%,P=0.041)。3细胞分子研究:结果表明糖皮质激素能明显抑制巨噬细胞分泌MMP-2,对TIMP-2的分泌无明显影响;另外,高浓度糖皮质激素抑制巨噬细胞分泌TNF-α,低浓度糖皮质激素促进TNF-α的分泌;糖皮质激素减少HA-SMC的迁移,抑制HA-SMC向分泌型表型转换;HA-SMC与巨噬细胞共培养抑制了HA-SMC的凋亡,糖皮质激素加强这一效应,但对细胞迁移无影响;高通量蛋白微阵列鉴定出白介素-6(interleuin-6,IL-6)和可溶性肿瘤坏死因子II型受体(soluble tumor necrosis factor receptor II,TNF-s RII)可能是细胞间相互作用的关键因子,但抗体中和实验证实了TNF-s RII是关键因子,IL-6的作用未得到验证。细胞共培养或糖皮质激素干预时,应激和凋亡通路试剂盒检测到bad、HSP27、p38MAPK和Ik Ba信号分子磷酸化水平的变化,western blot结果表明细胞共培养和糖皮质激素干预影响了p38MAPK和HSP27的磷酸化通路,参与血管重构。结论糖皮质激素正性调控主动脉血管重构,对AD的发生起着保护性作用。AD患者的血清皮质醇含量升高是机体为应对病理性血管重构作出的积极反应。糖皮质激素降低巨噬细胞分泌MMP-2减少主动脉胶原的降解,抑制巨噬细胞分泌TNF-α减轻管壁炎症反应,抑制HA-SMC迁移维持管壁SMC数量稳定,抑制HA-SMC向分泌型转变维持管壁功能稳态;HA-SMC与巨噬细胞共培养增加了游离TNF-s RII的含量,抑制p38MAPK-HSP27信号分子磷酸化,糖皮质激素干预进一步降低了TNF-α/TNF-s RII的比值,积极参与主动脉血管重构。也许在将来,糖皮质激素或TNF-s RII能成为主动脉血管重构的可干预靶标。
[Abstract]:Background With the advent of social aging and the improvement of people's living standards, the incidence of vascular diseases is increasing year by year, and aortic dissection (AD) represented by aortic dissection is one of the most dangerous. The morbidity and mortality have been significantly reduced, but many patients die of dissection rupture before diagnosis or treatment. Therefore, it is important to clarify the pathogenesis and prognosis of AD to improve the prognosis of dissection. Latest randomized controlled clinical studies have shown that a single dose of glucocorticoids before surgery can significantly reduce the incidence of systemic inflammatory response syndrome in patients with abdominal aortic aneurysms after endovascular exclusion without increasing the incidence of adverse events, but glucocorticoids and AD Objective To investigate the role of glucocorticoids in the occurrence, development and prognosis of AD, and further elucidate the cellular and molecular mechanisms of glucocorticoids involved in aortic remodeling. Methods 1 Clinical sample study: AD, non-ruptured aortic aneurysm (n) was collected with ethical review and informed consent of patients. Blood samples from on-ruptured aortic aneurysm (n AA) and healthy controls were examined for serum cortisol and plasma adrenocorticotropic hormone (ACTH) levels by radioimmunoassay, and aortas from patients with AD, n AA and cadaver donors undergoing open surgery were collected and glucocorticoid stimulation was detected by immunohistochemistry. Glucocorticoid receptor (GCR) expression level; clinical information of dissection patients was collected for multiple linear regression analysis to explore the influencing factors of serum cortisol content. 2 Animal experimental study: 80 C57BL/6 mice were excised bilateral adrenal glands after obtaining the approval of the Animal Protection and Use Association of the Second Military Medical University, and completed by 2:2:1. All the mice were randomly divided into three groups: the AD model was established with angiotensin II, and the AD model was treated with glucocorticoid or solvent. The three groups were divided into glucocorticoid intervention group, model group and solvent control group. The time of death was recorded, and the cumulative survival rate was calculated by Kaplan-Meier curve. Angiotensin II was effective; aorta of mice was dissected and the diameter of aorta was measured by image-Pro Plus software; aortic media thickness and dissection rate were measured by hematoxylin-eosin staining; aortic collagen volume fraction was detected by Masson staining; GCR expression and macrophages were detected by immunohistochemistry method. Content. 3 Cell Molecular Study: Human aortic smooth muscle cell (HA-SMC) and macrophages were cultured in vitro. Enzyme-linked immunosorbent assay was used to detect the secretion of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-2 inhibitor of macrophages by glucocorticoids. Metaloproteinase-2, TIMP-2, tumor necrosis factor-alpha (TNF-alpha) effect; scratch test to detect the effect of glucocorticoids on the migration of HA-SMC, cell immunofluorescence to detect the effect of glucocorticoids on the phenotype conversion of HA-SMC; establish HA-SMC and macrophage indirect co-culture system, cell flow cytometry to detect the cell. The effects of co-culture and glucocorticoids on apoptosis of HA-SMC, the effects of co-culture and glucocorticoids on migration of HA-SMC, the key factors of cell-to-cell interaction were detected by high-throughput protein microarray chip, and the neutralizing experience of the corresponding factors was used to verify the role of co-culture and glucocorticoids on apoptosis of HA-SMC. Results 1 Clinical sample study: From October 2012 to December 2013, blood samples from 82 AD patients, 68 n AA patients and 76 healthy volunteers who underwent physical examination in our vascular surgery center were collected. The levels of serum cortisol in AD group were significantly higher than those in n AA group (P The GCR levels of 8 AD patients, 8 NAA patients and 8 cadaver donors were 14.25 (+ 1.31%), 13.39 (+ 1.42%) and 12.55 (+ 2.12%) respectively, and there was no significant difference among the three groups. 920, P = 0.029).2 Animal experiment: The number of mice in the model group and the solvent control group were 31, 27 and 13, respectively. The cumulative survival rates of the model group and the glucocorticoid intervention group (P = 0.139) or the solvent control group (P = 0.107) were not significantly different (Log-rank test). The diameter of aorta in the model group was significantly larger than that in the solvent group (ascending aorta: 1458.49 + 257.96 microns vs. 1094.61 + 154.57 microns, P 0.001; aortic arch: 1449.48 + 290.34 microns vs. 986.38 + 151.38 microns). The diameter of ascending aorta in the model group was significantly larger than that in the glucocorticoid intervention group (1458.49 257.966550 The thickness of aortic media in the model group was significantly higher than that in the solvent group (89.6 65507 The volume fraction of collagen in aorta of rats was significantly lower than that of solvent control group (31.7 65507 The content of macrophages in the aorta of mice was significantly higher than that of the solvent control group (2.2+1.0%, P 0.001) or the glucocorticoid intervention group (7.4+3.2%, P=0.041). The results showed that glucocorticoid could significantly inhibit the secretion of MMP-2 by macrophages, but had no significant effect on the secretion of TIMP-2. TNF-a secretion was promoted by low concentration of glucocorticoids; glucocorticoids reduced the migration of HA-SMC and inhibited the transformation of HA-SMC to secretory phenotype; HA-SMC co-cultured with macrophages inhibited the apoptosis of HA-SMC, glucocorticoids enhanced the effect, but had no effect on cell migration; high-throughput protein microarray identified interleukin-6 (inte-6). Rleuin-6, IL-6 and soluble tumor necrosis factor receptor II (TNF-s RII) may be the key factor s in cell-cell interaction, but antibody neutralization confirmed that TNF-s RII is the key factor, and the role of IL-6 has not been verified. Stress and apoptosis are mediated by cell co-culture or glucocorticoid intervention. The phosphorylation levels of bad, HSP27, p38 MAPK and Ik Ba signaling molecules were detected by the kit. Western blot analysis showed that co-culture and glucocorticoid intervention affected the phosphorylation pathway of p38 MAPK and HSP27 and involved in vascular remodeling. Glucocorticoid reduces the secretion of MMP-2 by macrophages, decreases the degradation of collagen in aorta, inhibits the secretion of TNF-alpha by macrophages, reduces inflammation in vascular wall, inhibits the migration of HA-SMC and maintains the stability of SMC in vascular wall, and inhibits the secretion of HA-SMC. HA-SMC co-cultured with macrophages increased the content of free TNF-s RII, inhibited the phosphorylation of p38MAPK-HSP27 signal molecule, and glucocorticoid intervention further reduced the ratio of TNF-a to TNF-s RII and actively participated in aortic vascular remodeling. Perhaps in the future, glucocorticoid or TNF-s RII could become aortic blood. The reconfigurable target of tube reconstruction.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.1

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