【作者】 刘晓红；

【导师】 徐志云； 黄盛东； 韩林； 陆方林； 唐昊；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 目的:近年来,特发性和心脏损伤后缩窄性心包炎(Idiopathic and post–cardiac-injury constrictive pericarditis, IAPCP)的发病率逐年上升,已成为心包疾病防治领域中一个亟待解决的重要问题,也是影响心脏手术病人生存质量的一个关键因素。最新研究进展表明心包纤维钙化是IAPCP产生心包缩窄症状的主要原因,但其具体的发病机制尚不清楚,临床上无有效药物和非手术干预方法。因而,我们以此为切入点,从基因、细胞及组织三个不同的层面深入探索IAPCP心包纤维钙化的发病机制、初步尝试针对性的干预实验,旨在为今后IAPCP的临床防治提供理论基础和实验数据。方法:为以上目的,本研究分为以下六部分。1. IAPCP心包的分子病理研究:收集上海长海医院资料完整、临床病理诊断明确的IAPCP心包标本共45例,其中特发性心包炎(Idiopathic constrictive pericarditis, ICP)43例,心脏损伤后心包炎(Post–cardiac-injury constrictive pericarditis, PCP)2例。所有IAPCP病例均有石蜡包埋组织,其中18例ICP有新鲜标本、液氮保存。正常对照心包12例,均为液氮保存的新鲜标本。采用苏木素-伊红染色(Hematoxylin and Eosin, HE)观察IAPCP心包的组织学特征、维多利亚蓝-苦味酸酸性复红染色(Victoria blue-van Gieson, VG)确定纤维化程度、Von Kossa染色检测心包内钙盐沉积情况。采用免疫组织化学和脱氧核糖核苷酸末端转移酶介导的缺口末端标记( Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, TUNEL)染色确定IAPCP的心包间质细胞(Pericardial interstitial cells, PICs)表型的改变和凋亡的发生。抽提新鲜组织标本的总RNA,qRT-PCR分析细胞外基质相关基因表达的改变。2. TGF-β1在IAPCP心包中的表达及对PICs的作用:采用western blot和免疫组织化学方法检测转化生长因子-β1(Transforming growth factor-β1, TGF-β1)在IAPCP心包中的表达丰度及细胞定位。分离培养正常对照心包的PICs,并给予TGF-β1因子;采用qRT-PCR分析TGF-β1处理前后PICs细胞外基质相关基因表达的改变;明胶酶酶谱法检测明胶酶活性的改变;动态观察TGF-β1诱导融合的PICs形成钙化结节的过程;免疫细胞化学染色、TUNEL染色、Von Kossa染色确定PICs表型的改变、凋亡及钙盐沉积情况;流式细胞仪和比色法定量凋亡率和钙沉积程度。3. FGF-2、PDGF、BMP-2在IAPCP心包中的表达及对PICs的作用:采用免疫组织化学方法检测成纤维生长因子-2(Fibroblast growth factor-2, FGF-2)、血小板源性生长因子(Platelet-derived growth factor, PDGF)和骨形成蛋白-2(Bone morphogenetic protein-2, BMP-2)在IAPCP心包中的表达;分离培养PICs,分别给予FGF-2、PDGF-AA、BMP-2因子后,采用免疫细胞化学方法分析PICs表型的改变。4.自体心包瓣置换主动脉瓣:再次手术心包瓣的病理分析:对本科室所完成的15例自体心包瓣置换主动脉瓣手术病人行长期随访;收集再次手术标本,采用HE、VG和免疫组织化学染色观察自体心包瓣的病理组织学改变、检测PICs的表型。5. PICs的生物学特征研究:分离培养PICs和人骨髓间充质干细胞(Mesenchymal stem cells, MSCs)。观察PICs的形态和生长特征,β-半乳糖苷酶染色检测各代细胞中的衰老细胞。流式细胞仪分析PICs的免疫表型,特定培养基诱导PICs向骨母、软骨母细胞及脂肪细胞分化,通过免疫组织化学、免疫细胞化学及油红O染色分析PICs的分化能力。MTT法测定PICs和MSCs的细胞增殖活性,流式细胞仪分析两种细胞的细胞周期分布。通过观察成纤维细胞集落形成单位(Fibroblastic colony-forming unit, CFU-f)确定两种细胞的自我更新能力;采用RT-PCR方法比较两种细胞表达自我更新相关基因的差异。6. Klf4逆转心包肌纤维母细胞至PICs的研究:分离培养IAPCP的心包肌纤维母细胞,采用流式细胞仪、β-半乳糖苷酶染色、原位凋亡染色等方法观察肌纤维母细胞的生物学特征。给予Krüppel样因子4腺病毒(Adenoviral Krüppel-like factor 4, AdKlf4)和增强型绿色荧光蛋白腺病毒(Adenoviral enhanced green fluorescent protein, AdEGFP),荧光显微镜下观察和流式细胞仪检测确定细胞感染效率,免疫细胞化学检测目标蛋白的表达。通过形态学观察、β-半乳糖苷酶染色、western blot、RT-PCR确定Klf4逆转心包肌纤维母细胞表型及降低胶原合成的能力,同时确定Klf4减少细胞衰老、阻止衰老基因表达的可行性。结果:1. IAPCP心包的分子病理研究:HE和VG染色显示纤维化和钙化是IAPCP心包的主要病理改变,其中5例钙化心包见骨化区域。免疫组织化学染色表明IAPCP心包的PICs异常转分化,表达肌纤维母细胞表型标记物α-平滑肌肌动蛋白(α-Smooth muscle actin,α-SMA)和骨母细胞表型标记物碱性磷酸酶(Alkaline phosphatase, ALP);3例无钙化心包亦检测到ALP阳性细胞,心包钙化程度与PICs骨母细胞转分化率成正相关(rs=0.7579, P<0.05)。TUNEL染色表明随着钙化程度的升高,凋亡率逐渐增多,心包钙化程度与PICs的凋亡率成正相关(rs=0.4826, P<0.05)。qRT-PCR表明ICP心包中I型胶原和III型胶原的转录水平、金属基质蛋白酶-2(Matrix metalloproteinase-2, MMP-2)/金属基质蛋白酶抑制物-2(Tissue inhibitor of metalloproteinase-2, TIMP-2)和MMP-9/TIMP-1的比率明显高于正常对照心包,弹力蛋白的转录水平、MMP-1/TIMP-1和MMP-13/TIMP-1的比率降低(所有P<0.05)。2. TGF-β1在IAPCP心包中的表达及对PICs的作用:IAPCP心包表达TGF-β1蛋白,其蛋白丰度与胶原/弹力比率(r=0.6585, P<0.05)和心包钙化程度(rs=0.5328, P<0.05)呈正相关。细胞特异性免疫组织染色证实TGF-β1及其受体主要表达于PICs,提示TGF-β1以自-旁分泌的形式作用于PICs。体外模型证实TGF-β1促进PICs向肌纤维母细胞转分化;在诱导细胞转分化的同时,TGF-β1还上调细胞中Ⅰ型胶原、Ⅲ型胶原、MMP-2、MMP-8、MMP-9、TIMP-1和TIMP-2的转录水平,并下调MMP-1和MMP-13的表达(所有P<0.05)。明胶酶酶谱法表明TGF-β1处理组的细胞上清中存在MMP-9和中性粒细胞明胶酶相关脂质运载蛋白(Neutrophil gelatinase-associated lipocalin, NGAL)的异源二聚体。给予TGF-β1,融合的PICs能自发性收缩、聚集生长、形成结节、表达ALP蛋白,在形成的结节中部分细胞凋亡坏死。与未处理的细胞相比,TGF-β1诱导的细胞结节表达较高的ALP活性、含有更多的钙盐和凋亡细胞(所有P<0.05)。3. FGF-2、PDGF、BMP-2在IAPCP心包中的表达及对PICs的作用:免疫组织化学染色证实IAPCP心包表达FGF-2、PDGF和BMP-2蛋白,体外实验表明FGF-2、PDGF-AA和BMP-2均能诱导PICs表达α-SMA,而且BMP-2还能诱导PICs表达ALP。4.自体心包瓣置换主动脉瓣:再次手术心包瓣的病理分析:随访结束时,瓣膜交界撕裂、心内膜炎、瓣膜退行性变(纤维化和/或钙化)、再次手术的免除率分别为100%、93%、80%和67%,瓣膜相关的死亡率为0%。手术切除的自体心包瓣均行病理分析,结果表明自体心包瓣在宿主体内5个月就被覆VIII因子阳性的内皮细胞,内皮下细胞表达α-SMA,HE和VG染色表明该瓣膜无明显纤维化。因纤维钙化而衰败的心包瓣(n=3)中的PICs表达α-SMA,部分细胞表达ALP;VG染色显示心包瓣的心室面有一层弹力板,这种弹力板见于主动脉瓣,但是正常心包和早期手术的心包瓣中没有。5. PICs的生物学特征研究:体外培养的PICs呈成纤维细胞样外观,在传代过程中,细胞体积逐渐增大、变宽,衰老细胞增多,并逐渐表达α-SMA。细胞周期检测表明随着细胞代数的增加,细胞周期向G2/M～S期进展。PICs与MSCs相类似,表达粘附相关分子,同样不表达造血干细胞标记物。细胞分化实验证实PICs具有向骨母细胞、软骨母细胞以及脂肪细胞分化的能力。PICs在培养的各个时间点的增殖活性均高于MSCs(P<0.05)。细胞周期分析表明≈90%的MSCs处于细胞周期的G0/G1期;然而仅有≈64%的PICs处于细胞周期的G0/G1期。MSCs能够不断产生CFU-f,但是PICs的CFU-f形成效率低于MSCs(P<0.05)。RT-PCR证实MSCs和PICs均表达自我更新相关基因Oct3/4和Bmi-1,但是其在PICs的表达量低于MSCs。6. Klf4逆转心包肌纤维母细胞至PICs的研究:与PICs相比,肌纤维母细胞多呈聚集样生长,细胞扁平、宽大,表达α-SMA,第一代衰老、凋亡细胞就明显高于PICs。肌纤维母细胞感染Klf4腺病毒(Adenoviral Klf4, AdKlf4)后细胞逐渐失去聚集样生长特征、并在传代过程中由扁平细胞向梭形细胞转变。AdKlf4感染早期(5天),细胞周期分析表明细胞周期阻滞。RT-PCR证实Klf4促进p53基因的转录、并抑制Ⅰ型胶原和Ⅲ型胶原的mRNA表达,而Klf4对α-SMA的表达则无明显作用。腺病毒感染5周时,行western blot和RT-PCR检测均证实Klf4下调α-SMA和p53的表达。结论:1.纤维化和钙化是IAPCP心包最主要的病理学特征;纤维化表现为PICs向肌纤维母细胞转分化、细胞外胶原沉积、弹力降解、MMPs和TIMPs的平衡失调;心包钙化与PICs凋亡、骨母细胞转分化相关,而且钙化心包中有骨化区域,提示IAPCP的心包钙化是多种机制参与的、由PICs介导的主动调控过程。2.通过研究TGF-β1、FGF-2、PDGF和BMP-2在IAPCP心包中的表达及对PICs的作用,证实IAPCP心包纤维钙化是多因素参与的主动调控过程,其中心环节为PICs向肌纤维母细胞和骨母细胞转分化。3.在纤维钙化的自体心包瓣中获得PICs向肌纤维母细胞和骨母细胞转分化的证据,1例无纤维化的心包中也检测到PICs向肌纤维母细胞转分化,提示PICs的异常转分化是心包修复材料衰败的始作俑者。4.通过对比PICs和MSCs的生物学特征,证实PICs具有MSCs相似的表型可塑性(即多向分化潜力),而这种可塑性为复杂的致病信号提供底物,导致PICs异常转分化,并在转分化过程中改变其生物学特征,最终引起心包纤维化及钙化的形成。因此,维持PICs原始表型,阻断甚至是逆转异常转分化,有望成为心包纤维钙化治疗的突破口。5.初步实验证实Klf4逆转肌纤维母细胞表型是一个多步骤、有序的、高度调控的过程,其在逆转肌纤维母细胞之前就可抑制胶原纤维的表达,提示Klf4所诱导的细胞表型转分化可减缓组织纤维化进程。更多还原

【Abstract】 Objective: Idiopathic and post-cardiac-injury constrictive pericarditis (IAPCP) has become increasingly prevalent in recent years. Some studies showed that pericardial fibrosis and/or calcification played an important role in the initiation and progression of IAPCP. The objective of the present study is to examine the mechanism underlying the initiation and progression of IAPCP, and explore possible methods to prevent or delay the occurrence of IAPCP.Methods: The present study was divided into six sections. 1. Molecular pathology of IAPCP pericardia: To evaluate histological features, data were collected from 45 consecutive IAPCP patients (idiopathic constrictive pericarditis [ICP], 43 cases; post–cardiac-injury constrictive pericarditis [PCP], two cases) who had archived paraffin-embedded pericardial specimens available for study, including fresh pericardial specimens from 18 patients (all ICP). Twelve fresh pericardial specimens free of pericarditis were obtained at autopsy or during cardiac surgery as controls. The histological features and the degree of fibrosis and calcification were observed by Hematoxylin and Eosin (HE), Victoria blue-van Gieson (VG) and Von Kossa staining respectively. Cellular phonotype and apoptosis were determined by immunohistochemistry and TUNEL staining respectively. Total RNA was extracted from the fresh samples, and change of extracellular matrix (ECM)-related genes was examined by quantitative RT-PCR analysis. 2. TGF-β1 expression in IAPCP pericardia and effect on PICs: Expression and distribution of transforming growth factor-β1 (TGF-β1) in IAPCP samples were examined by western blot and immunohistochemistry. Pericardial interstitial cells (PICs) were isolated from normal pericardia and treated with TGF-β1. Changes in ECM-related genes before and after TGF-β1 treatment were compared by quantitative RT-PCR. The activity of gelatinases was determined by Gelatin zymography. The course of TGF-β1-induced nodules in confluent PICs was monitored. Cellular phonotype, apoptosis and calcium deposition were examined by immunocytochemistry, TUNEL and Von Kossa staining respectively. The degree of calcium deposition and the index of apoptosis were quantified colorimetrically and by flow cytometry respectively. 3. FGF-2, PDGF and BMP-2 expression in IAPCP pericardia and effect on PICs: Expression of fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF) and bone morphogenetic protein-2 (BMP-2) in IAPCP pericardia was examined by immunohistochemistry. PICs were isolated from normal pericardia and treated with FGF-2, PDGF-AA and BMP-2. Cellular phonotype was determined by immunocytochemistry. 4. Aortic valve replacement with autologous pericardium: pathological examination of surgically removed pericardial valves: Fifteen patients who underwent aortic valve replacement with autologous pericardium were followed up for the prevalence of reoperation. The pathological features of pericardial valves and the phenotype of PICs were analyzed by HE, VG and immunohistochemical staining. 5. Biological characteristics of PICs: PICs and mesenchymal stem cells (MSCs) were isolated and cultured. Senescent cells in each passpage were determined byβ-galactosidase staining, and the immunophenotype of PICs was analyzed by flow cytometry. PICs were induced to differentiate into osteoblasts, chondroblasts and adipocytes with appropriate inducing media. The differentiation capacity of PICs was determined by immunocytochemistry, immunohistochemistry and oil O staining. Cell proliferation, cell-cycle distribution and self-renewal were examined by MTT, flow cytometry and fibroblastic colony-forming unit (CFU-f) assay. Expression differences in self-renewal related genes between MSCs and PICs were compared by RT-PCR. 6. Transdifferentiation of pericardial myofibroblasts to PICs by Klf4: Pericardial myofibroblasts derived from IAPCP pericardia were isolated and cultured. Biological characteristics of myofibroblasts were determined by usingβ-galactosidase staining, flow cytometry and in-situ apoptosis. Infection efficiency and changes in morphology, cellular phonotype and collagen synthesis were examined by flow cytometry and western blot after infection of myofibroblasts with adenovirus-mediated Krüppel-like factor 4 (AdKlf4) and adenovirus-mediated enhanced green fluorescent protein (AdEGFP). In addition, the effect of Klf4 on cellular senescence and the expression of senescent genes were examined byβ-galactosidase staining and RT-PCR.Results: 1. Molecular pathology of IAPCP pericardia: HE and VG staining showed that the architecture of IAPCP pericardia was distorted markedly due to moderate-to-severe fibrosis and calcification (bone formation in 5 cases). Immunohistochemical staining ofα-smooth muscle actin (α-SMA) and alkaline phosphatase (ALP) showed that some PICs in IAPCP pericardia were myofibroblasts and osteoblasts. Even PICs in three cases without calcification also expressed ALP protein. The index of osteoblast transdifferentiation of PICs significantly correlated with the degree of calcification (rs=0.7579, P<0.05). Strong positive signals of apoptosis were present in PICs as shown by TUNEL staining. The apoptosis rate increased gradually with the degree of calcification increasing (rs=0.4826, P<0.05). Compared with the normal pericardia, the expression of collagenⅠand collagenⅢwas significantly increased in ICP pericardia, whereas the expression of elastin decreased. Furthermore, mRNA expression of matrix metalloproteinase-2 (MMP-2), MMP-8, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 increased significantly in ICP as compared with normal pericardia, whereas the expression of MMP-1 and MMP-13 decreased. Importantly, the MMP-1/TIMP-1 and MMP-13/TIMP-1 ratios decreased significantly in ICP compared with normal pericardia, while the MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios were significantly higher in ICP pericardia. In contrast, the MMP-8/TIMP-1 radio did not differ significantly between ICP and normal pericardia (all P<0.05). 2. TGF-β1 expression in IAPCP pericardia and effect on PICs: IAPCP pericardia expressed TGF-β1 protein, and TGF-β1 protein abundance significantly correlated with an increase in the collagen/elastin ratio (r=0.6585, P<0.05) and the degree of calcification (rs=0.5328, P<0.05). Cellular specific immunohistochemistry confirmed the expression of TGF-β1 mainly in PICs. The in vitro model showed thatα-SMA mRNA expression increased in a concentration- and time-dependent manner after treatment of PICs with TGF-β1. Meanwhile, there was an increase in the expression of collagen I and collagen III mRNA. TGF-β1 increased the expression of MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2, although MMP-1 and MMP-13 expressions were reduced. Gelatinolytic activity in the conditioned medium of the co-culture model was measured by gelatin zymography. By day 3, TGF-β1 elevated the proteolytic band around 130 kDa, which represents the heterodimer of MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL), and this gelatinolytic activity increased in a time-dependent manner. Furthermore, TGF-β1 promoted the formation of apoptotic-enriched calcified nodules in confluent PICs. These nodules shared certain properties with the bone, including increased ALP activity, the development of osteoblast phenotype, high calcium deposition and increased apoptosis (all P<0.05). 3. FGF-2, PDGF and BMP-2 expression in IAPCP pericardia and effect on PICs: Immunohistochemistry confirmed that IAPCP pericardia expressed FGF-2, PDGF and BMP-2. In vitro experiments showed that FGF-2, PDGF-AA and BMP-2 induced transdifferentiation of PICs to myofibroblasts with the expression ofα-SMA, and BMP-2 also induced transdifferentiation of PICs to osteoblasts with the expression of ALP. 4. Aortic valve replacement with autologous pericardium: pathological examination of surgically removed pericardial valves: The freedom from commissural tear, endocarditis, valve degeneration (fibrosis and calcification) and the prevalence of re-operation at the end of follow-up was 100%, 93%, 80% and 67%, respectively. Valve-related mortality was 0%. Pathological examination confirmed partial endothelialization of pericardial valves as early as five months, and some cells under the endothelium were positive forα-SMA. PICs in degenerated pericardial valves due to fibrosis and calcification (n=3) were positive forα-SMA, and some cells were positive for ALP. VG staining revealed a band of elastic tissue of the excised autologous pericardium after long term adaptation (> nine years). 5. Biological characteristics of PICs: PICs showed a fibroid spindle-shaped appearance in vitro. The cellular morphology changed dramatically during propagation, from spindle-shaped cells to large and flat cells with increased senescent cells and higherα-SMA expression. Cell cycle analysis revealed that the number of cells in G2/M to S phase of cell cycle was increased significantly. Furthermore, PICs possessed similar surface markers as did MSCs. In vitro experiments showed that PICs had the ability to differentiate into osteoblasts, chondroblasts and adipocytes. Cell cycle analysis showed that there were more than 90% of the MSCs in the G0/G1 phase of cell cycle but there were only≈64% of the PICs in G0/G1. MSCs were able to produce CFU-f continously, but the CFU-f formation efficiency of PICs was lower than that of MSCs (P<0.05). Although PICs expressed self-renewal related genes, the transcript level of Oct3/4 and Bmi-1 was lower than that of MSCs. 6. Transdifferentiation of pericardial myofibroblasts to PICs by Klf4: As compared with PICs, confluent myofibroblasts spontaneously retracted from neighboring areas and grouped into aggregates that progressed to form nodules with increased senescent cells and higher apoptotic cells. Flow cytometry showed that Klf4 resulted in the cell cycle arrest on day five. RT-PCR confirmed that Klf4 induced the expression of p53 and inhibited the expression of collagen I and collagen III. However, Klf4 had no effect on the expression ofα-SMA. Myofibroblasts infected with AdKlf4 for five weeks, both RT-PCR and western blot showed that Klf4 significantly decreased the expressed ofα-SMA and p53.Conclusion: 1. Fibrosis and calcification are the most important histological features of IAPCP pericardia, of which fibrosis is characterized by myofibroblast transdifferentiation, abnormal collagen deposition and elastin degradation accompanied by MMPs and TIMPs imbalance; pericardial calcification correlated with apoptosis and osteoblast transdifferentiation, suggesting that pericardial calcification is a PICs-mediated active process, regulated by multiple mechanisms. 2. Although TGF-β1, FGF-2, PDGF and BMP-2 are all involved in the pathogenesis of fibrocalcification, the transdifferentiation of PICs to myofibroblasts and osteoblasts is the main mechanism for pericardial fibrocalcification, suggesting that pericardial fibrosis and calcification are active processes induced by multiple signal transduction pathways. 3. Pathological examination of the autologous pericardial valves confirmed that the abnormal transdifferentiation of PICs to myofibroblasts and osteoblasts is the main mechanism for pericardial fibrocalcification. 4. Compared with MSCs, PICs have the similar immunophenotype and differentiation potential (plasticity), whereas the proliferation activity of PICs is significantly higher than that of MSCs, and the self-renewal capacity of PICs is lower than that of MSCs. Moreover, the plasticity of adult cells may provide a substrate for growth factors or cytokines that promote inappropriate differentiation of these cells. 5. Klf4 induced-transdifferentiation from myofibroblasts to PICs is a progressive, multiple-step and highly-regulated process. Klf4 can reduce collagen secretion even before cell transdifferentiation, suggesting that phenotypic reversion of pericardial myofibroblasts is expected to prevent pericardial fibrosis.更多还原