【作者】 黄维佳；

【导师】 覃家锦；

【作者基本信息】 广西医科大学 ， 心胸外科学， 2011， 博士

【摘要】 肺动脉高压(PAH)是指以肺动脉压、肺血管阻力进行性增高,终末期导致患者右心衰竭及死亡为特征的一种疾病。肺动脉高压通过复杂的病理机制,导致患者在肺组织结构、分子信号途径和基因表达等多个方面均发生了异常。目前研究发现,肺动脉高压是在炎症、异常血流剪切力、缺氧等因素的诱发下,细胞因子及血管活性物质异常产生,并作用于血管平滑肌细胞、血管内皮等靶细胞,导致肺血管异常收缩、生长和重构,细胞外基质堆积等一系列后果,最终形成肺动脉高压,其中肺血管重构是肺动脉高压主要的病理学特征。研究表明,细胞的异常增殖,凋亡减少是组织构型重建的重要原因之一。凋亡是有核细胞在某些生理或病理情况下,基因特定程序被启动,通过DNA内切酶发生有序的主动性非炎症性死亡,又称细胞程序性死亡。细胞凋亡和增殖是矛盾的两个对立面,它们之间的平衡维持着机体组织结构的正常状态和生理功能。越来越多的研究证明,肺动脉平滑肌细胞增殖增加和/或凋亡减少都可促进肺血管中膜肥厚和血管构型重建,是肺动脉高压发病的一个重要机制。正常情况下,血管壁结构是相对稳定的,由相应细胞的增殖和凋亡共同协调维持,当血管壁在受到各种内因和外因(如细胞因子、缺氧、血流剪切力等)的刺激作用下,肺动脉平滑肌细胞内部经过一系列信号转导过程,启动平滑肌细胞增殖基因和/或凋亡抑制基因的表达,导致肺动脉平滑肌细胞的增殖增多和/或凋亡减少,促使肺动脉平滑肌细胞增殖、肥大、迁移,最终引起肺血管壁增厚和肺血管重构。目前,很多学者把抗肺血管重构的目光转移到促进肺动脉平滑肌细胞凋亡这个靶点上,促进肺动脉平滑肌细胞凋亡可能成为未来治疗肺动脉高压的突破口。5-羟色胺转运体(5-HTT)是一种膜蛋白,表达于神经元、血小板、肺动脉平滑肌细胞等体细胞中,主要功能是将5-羟色胺(5-HT)转运至细胞内部发挥生物学效应。5-羟色胺可经细胞膜上的5-羟色胺转运体介导进入肺动脉平滑肌细胞,并通过多条促增殖信号转导途径引起肺动脉平滑肌细胞增殖肥大,导致肺血管构型重建。研究结果表明5-羟色胺转运体在许多类型肺动脉高压的发病机制中起重要作用,但在左向右分流型肺动脉高压中,是否也存在5-HTT的高表达,它与肺动脉平滑肌细胞凋亡状况有无关系,目前尚未见相关报导。他汀类药物是一种3-羟基,3-甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,它所具有的降脂、稳定粥样斑块、改善内皮细胞功能、抑制血小板聚集、抗炎抗氧化、免疫调节等作用使其广泛应用于心脑血管疾病等领域。近来,越来越多的学者开始关注他汀类药物的“非调脂作用”,国内外有研究发现他汀类药物可通过与肺动脉高压发生的各个环节产生作用,降低肺动脉压力,缓解肺血管过度收缩,抑制肺血管重建,他汀类药物的这些作用可能是通过诱导肺动脉平滑肌细胞或血管内皮细胞凋亡来实现的,用于治疗和预防肺动脉高压有相当的前景。瑞舒伐他汀(rosuvastatin)是一种新型羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,是他汀类药物家族的新成员,其在肺动脉高压中的作用及机制报导甚少,更未见其对于高肺血流性肺动脉高压影响的研究。在本研究中,我们用腹腔分流法建立肺高血流大鼠肺动脉高压模型,通过观察模型大鼠肺动脉平滑肌细胞5-HTT的表达情况及肺动脉平滑肌细胞凋亡情况,了解大鼠肺高血流肺动脉高压的发生是否与这些因素有关,初步探讨肺高血流肺动脉高压可能的发病机制。同时,我们通过瑞舒伐他汀来干预肺高血流大鼠肺动脉高压模型,以观察瑞舒伐他汀是否对肺高血流大鼠肺组织中5-HTT的表达及肺动脉平滑肌细胞的凋亡产生影响,初步探讨瑞舒伐他汀在肺高血流模型大鼠肺血管重构中的作用机制。1.将40只SD大鼠随机分为4组:①分流11周组、②分流8周组、③分流4周组、④对照组,每组各10只。分流11周组、分流8周组和分流4周组采用腹主动脉-下腔静脉分流法建立左向右分流大鼠模型,而对照组开腹但不建立分流。2.在相应时间内处死大鼠,处死前测量大鼠肺动脉平均压(mPAP),处死后对大鼠右心室和左心室+室间隔进行称重,计算右心室肥厚指数(RVI)。3.常规石蜡包埋大鼠肺组织,切片并HE染色,观察各组大鼠肺组织小动脉的形态变化,计算管壁厚度占外径的百分比(WT%)及管壁面积占血管总面积的百分比(WA%)作为反映大鼠肺组织血管重构程度的指标。4.用免疫组化SP法(链霉卵白素-过氧化物酶法)检测大鼠肺组织中5-HTT的表达情况。5.用原位末端标记法(TUNEL法)检测大鼠肺动脉平滑肌细胞凋亡情况并计算TUNEL阳性细胞百分比(AI值)。6.用Western Blot法检测各组大鼠肺组织中5-HTT,bcl-2蛋白的水平。7.用RT-PCR检测各组大鼠肺组织中5-HTTmRNA的表达情况。8.在上述实验的基础上,另将30只SD大鼠随机分为3组(每组10只):①分流组;②分流给药组:分流手术后第二天开始给予瑞舒伐他汀(10 mg/kg)灌胃,每日1次,连续11周;③对照组。观察瑞舒伐他汀对高肺血流肺动脉高压模型大鼠肺组织中5-HTT、bcl-2蛋白表达(Western Blot法)及肺动脉平滑肌细胞凋亡(TUNEL法)的影响。9.所得数据采用SPSS13.0软件进行统计学分析,所有数据以mean士SD表示,两组间比较采用成组t检验,多组间比较采用单因素方差分析,p<0.05时认为差异具有统计学意义。1.随着分流时间的延长,分流各组大鼠平均肺动脉压、右心室肥厚指数(RVI)逐步升高,第11周模型大鼠出现明显肺动脉压力升高和右心室肥厚的表现,与其他各组间上述指标的差异有统计学意义(P<0.05)。2.随着分流时间的延长,分流各组大鼠肺组织血管壁明显增厚,出现肺动脉平滑肌细胞肥大、增生,管腔狭窄,模型大鼠出现了明显的肺血管构型重建。同时,各分流组肺小动脉管壁厚度占血管外径百分比(WT%)、肺小动脉管壁面积占血管总面积百分比(WA%)也逐步升高,各组之间WT%、WA%值的差异有统计学意义(P<0.05)。3.免疫组化结果显示随着分流时间的延长,各组大鼠肺组织中5-HTT的表达呈逐步升高的趋势,各组之间的差异有统计学意义(P<0.05)。4.原位末端标记法检测结果提示各组大鼠肺组织血管内均存在一定比例的凋亡细胞。随着分流时间的延长,大鼠肺动脉平滑肌凋亡细胞逐步减少,凋亡指数(AI)也逐步下降。统计学分析显示各组间均有显著性差异(P<0.05)。5. Western Blot法检测结果提示随着分流时间的延长,各组大鼠肺组织中5-HTT蛋白、bcl-2蛋白的水平呈逐步升高的趋势,分流11周组与其他各组间指标的差异有统计学意义(P<0.05)。6. RT-PCR检测结果提示随着分流时间的延长,各组大鼠肺组织中5-HTTmRNA表达水平呈逐步升高的趋势,其中分流11周组最为明显,与其他各组之间的差异有统计学意义(P<0.05)。7.随着分流时间的延长,各组大鼠肺组织中5-HTT水平逐步升高,而凋亡指数(AI)逐步下降,相关性分析提示5-HTT水平与AI值之间呈明显负相关关系(P<0.05)。8.瑞舒伐他汀干预组大鼠肺组织中5-HTT、bcl-2蛋白水平较分流组明显降低,而干预组大鼠肺动脉平滑肌TUNEL阳性细胞百分比较分流组明显升高,统计分析显示两组间差异有统计学意义(P<0.05)。1.腹腔分流法能使模型大鼠出现肺动脉压明显升高、右心室肥厚、肺血管构型重建等肺动脉高压的病理改变。2.左向右分流大鼠肺高压模型中存在着分流时间依赖的5-HTT、bcl-2蛋白高表达,而肺动脉平滑肌凋亡细胞百分比却低于正常水平,5-HTT介导的5-HT信号通路可能通过上调bcl-2蛋白表达,抑制肺动脉平滑肌细胞凋亡参与肺血管重构。3.瑞舒伐他汀能明显缓解肺高血流大鼠肺动脉压力升高及肺血管重构,瑞舒伐他汀抑制模型大鼠肺血管构型重建可能与抑制肺组织中5-HTT、bcl-2蛋白的表达水平,提高肺动脉平滑肌细胞的凋亡数量有关。更多还原

【Abstract】 Pulmonary hypertension(PAH) is a group of diseases characterized by elevation of pulmonary arterial pressure and pulmonary vascular resistence,leading to right ventrieular failure and death.PAH has a complex pathobiology involving many abnormal aspects in cellular structure, molecular signaling pathways , gene expression and so on.The causes are not entirely clear.Study found that vascular active substances and cytokines are generated unusually by inflammation, abnormal blood shearing force, hypoxia and focus on endothelial cells,PASMCs, causing the extracellular matrix accumulation, vasoconstriction, abnormal growth and remodeling and so on .They all contribute to pulmonary hypertension finally.Pulmonary vascular remodeling is the main pathological feature of pulmonary hypertension.Abnormal proliferation of cells and reducing of apoptotic cells was the main cause of the tissue remodeling. Apoptosis is a kind of orderly inflammatory death of nuclear cells. It is launched by intracellular specific program and implemented through activated DNA enzymes in the case of some physiology and pathology. It is also called programmed cell death. Apoptosis and proliferation are two opposites.The balance of them maintain the normal state of the tissue structure and physiological function. It has also been demonstrated that either increased proliferation or decreased apoptosis of PASMCs contribute to pulmonary vascular medial hypertrophy and vascular remodeling,which is an important mechanism of PAH. Under normal circumstances, hemal wall structure is stable via balance of apoptosis and proliferation. Various internal and external stimulating effects promot proliferation genes and/or apoptosis inhibition genes of pulmonary smooth muscle cells to express throught a series of signal transductic process, causing PASMCs proliferation and/or apoptotic reducing, migration.It contributs to pulmonary ascular wall thickening and pulmonary arterial reconstruction. Therefore, many scholars focus on promoting PASMCs apoptosis as therapeutic approach for patient with PAH in the future.Serotonin transporter (5-HTT) is a kind of membrane protein.It is expressed in neurons, platelets, PASMCs.The main function of serotonin transporter is transfering serotonin (5-HT) into cells to exert biological effects. 5 - HT is mediated into pulmonary smooth muscle cells by serotonin transporter and cause pulmonary arterial smooth muscle cell proliferation and hypertrophy through multiple signal transduction pathways.Then it cause pulmonary vascular reconstruction. Several study results indicate that serotonin transporter plays an important role in many types of pulmonary hypertension.Is there also a high expression of 5-HTT in pulmonary hypertension with left to right shunt? the relation between 5-HTT high expression and apoptosis of PASMCs has not been reported.Statins is inhibitors of HMG-CoA reductase, They have so many functions such as lipid lowering, improving endothelial function, stable atheromatous plaque, inhibiting platelet aggregation, anti-inflammatory, anti-oxidation, immune that they have been widely applied to Cardiology and Neurology. Recently, more and more scholars begin to pay close attention to "non- lipid lowering effect" of statins.Domestic and international studies have found that statins affect every aspect of pulmonary hypertension through many mechanisms. Statins reduce pulmonary artery pressure, improve pulmonary arterial diastolic function and restrain pulmonary vascular reconstruction through inducing pulmonary smooth muscle cells or vascular endothelial apoptosis and inhibiting their proliferation.It is a bright future to apply statins to treat PAH. Rosuvastatin is a new type of HMG-CoA reductase inhibitor.It is a new member of the statins family.Its role in pulmonary hypertension and mechanism has less been reported.There is even no report about its function in pulmonary hypertension with left to right shunt.In this study, we used abdominal aorta- inferior vena cava shunt method to establish the high blood flow rat model. Through observating 5-HTT expression and apoptosis of PASMCs,we explored whether these factors were involved in PAH with high blood flow and the possible mechanism of PAH with left to right shunt in rats.Meanwhile, we used rosuvastatin to intervene the model rats .We investigated whether rosuvastatin affected the expression of 5-HTT and apoptosis of PASMCs in rats to explore preliminarily the mechanism of rosuvastatin about pulmonary vascular remodeling of high blood flow rat.1. Forty SD rats used in the experiment were randomly divided into four groups: control group,4 weeks group, 8 weeks group and 11 weeks group. We established rat model with high lung blood flow through abdominal aorta- inferior vena cava shunt in 4 weeks group, 8 weeks group and 11 weeks group.The rats of controle group received laparotomy without shunt.2. Before executing rats, we measured pulmonary average pressure (mPAP) of rats with a mini polyethylene catheter.The ratio of RV weight to S﹢LV weight was calculated as index of the right ventricular hypertrophy.3.After paraffin embedding , the lung sections were stained with Hematoxyline-eosine(HE) and observed the chang of pulmonary vascular.We calculated vascular wall thickness/vascular diameter(WT % ) and vascular area/total vascular Area(WA % ) as the index about pulmonary vascular remodeling.4.Protein expression of 5-HTT in each rat was measured with immunohistochemical stain(SP). 5.Apoptosis of PASMCs in each rat was measured by TUNEL assay and apoptosis index(AI) was calculated.6.Protein expression and of 5-HTT and bcl-2 in each rat were measured by Western Blot.7.The expression of 5-HTTmRNA in each rat was measured by RT-PCR.8.According to above experiment thirty SD rats were randomly divided into three groups: controle group,shunt group, shunt+ rosuvastatin group. After operation the rats of shunt + rosuvastatin group were fed by rosuvastatin with dose of 10mg/kg/day for 11 weeks.Western Blot and TUNEL assay were applied to evaluate the affect of rosuvastatin on 5-HTT and bcl-2 protein expression and PASMCs apoptosis of rat with high lung blood flow.9.All data were expressed as mean士SD.Statistical analysis were performed by SPSS 13.0. Grouping t-test was used to compare the difference of two groups.ANOVA was used to compare the differences of many groups.A value of P<0.05 was statistically significant.1.With the increasing of shunt time, both mPAP and RVI of each shunt group were gradually elevated.Obvious elevation of pulmonary vascular pressure and right ventricular index appeared in model rats at the last 11 th week. Statistical analysis showed that there were significant differences between 11 weeks group and other groups(P<0.05).2.With the increasing of shunt time, pulmonary vascular remodelling appeared in rats of shunt group.The smooth muscle cells of pulmonary vascular proliferated and became hypertrophy.The intima became thicken and the lumen became stenosis. Meanwhile,both WT% and WA% were gradually elevated in shunt groups. Statistical analysis showed that there was a significant difference between each two groups(P<0.05).3.Immunohistochemistry indicated that with the increasing of shunt time, the expression of 5-HTT in shunt groups were gradually elevated. Statistical analysis showed that there was a significant difference between each two groups(P<0.05)4.TUNEL assay indicated that there are a certain proportion of the apoptotic cells in each group. With the increasing of shunt time, apoptosis of PASMCs in shunt groups gradually decreased with apoptosis index decreasing. Statistical analysis showed that there was a significant difference between each two groups(P<0.05)5.Western Blot showed that with the increasing of shunt time, the expression of 5-HTT and bcl-2 protein in shunt groups were gradually elevated.Statistical analysis showed that there were significant differences between 11 weeks group and other groups(P<0.05).6.RT-PCR indicated that with the increasing of shunt time, the expression of 5-HTTmRNA in shunt groups were gradually elevated.Statistical analysis showed that there were significant differences between 11 weeks group and other groups(P<0.05).7. With the increasing of shunt time, the level of 5-HTT gradually increased and apoptosis index of PASMCs gradually decreased.Correlation analysis showed that there was a significant negative correlation between level of 5-HTT and apoptosis index.8. Compared with shunt group, the expression of 5-HTT and bcl-2 protein in shunt + rosuvastatin group significantly decreased.The percentage of apoptotic PASMCs was obviously elevated. Statistical analysis showed that there was a significant difference between each two groups(P<0.05).1.Abdominal aorta - inferior vena cava shunt leads to obvious high pressure of pulmonary artery, right ventricular hypertrophy and pulmonary arterial reconstruction of rat2.The expressions of 5-HTT and bcl-2 protein dependence on shunt time are obviously enhanced in left to right shunt-induced PAH rats as well as the PASMCs apoptosis percentage is lower than normal percentage. The 5-HT signal transductic process via 5-HTT participates in pulmonary vascular remodeling through up-regulating expression of bcl-2 protein and suppressing apoptosis of PASMCs.3.Rosuvastatin protects rats with high lung blood flow against elevation of PAP and pulmonary vascular remodeling . It is considered that this function might relate to suppressing expression of 5-HTT and bcl-2 protein and promoting apoptosis of PASMCs.更多还原