【作者】 胡静；

【导师】 徐永健；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 研究背景吸烟不仅可引起慢性支气管炎、慢性阻塞性肺气肿,而且还可进一步导致肺动脉高压和慢性肺源性心脏病。肺动脉高压与慢性阻塞性肺病(COPD)患者的临床预后恶化、生存率降低密切相关,目前尚缺乏特异而有效的治疗。既往对COPD合并肺动脉高压发病机制的研究多集中于缺氧性肺血管收缩和缺氧性肺血管重建。近年发现吸烟可直接导致肺血管重建、参与肺动脉高压的形成,其主要机制可能是吸烟促进肺动脉平滑肌细胞(PASMC)的增殖,但具体机制尚不清楚。细胞内信号转导通路的激活、生长因子的产生和释放在吸烟致PASMC增殖中可能起着重要作用。蛋白激酶C(PKC)是细胞内生物信号转导途径中的一类关键酶,参与调控血管平滑肌细胞的增殖、凋亡、分化、迁移和基因表达等。我们实验室以往的研究发现PKC的活化,PKCα亚型的表达上调在低氧致PASMC增殖中起重要作用。前期研究工作还发现无论是慢性吸烟大鼠,还是吸烟非COPD患者和吸烟COPD患者其肺小动脉PKC-αmRNA及蛋白质的表达都增加,提示PKC-α可能参与了慢性吸烟所致的肺血管重建。但PKC特别是PKC-α在吸烟对PASMC增殖的影响中所起的作用有待在细胞培养水平进一步予以研究。碱性成纤维细胞生长因子(bFGF)作为一种生长因子和丝裂原,可促进肺动脉内皮细胞增殖,外膜成纤维细胞生长,强烈地刺激中膜平滑肌细胞增殖、迁移、合成分泌胶原和纤维连接蛋白等细胞外基质,在低氧和野百合碱所致的肺血管重建和肺动脉高压中发挥着重要作用。有研究发现,在吸烟合并COPD的患者中,bFGF在肺小动脉平滑肌细胞的表达增加,可能参与了吸烟所致的肺血管重建,但目前尚无相关的动物在体实验和离体实验的研究。本研究从PKC信号转导通路和bFGF两个方面探讨吸烟致PASMC增生和肺血管重建的可能机制,为COPD合并肺动脉高压的发病机制研究和防治提供基础资料。第一部分香烟烟雾提取物对肺动脉平滑肌细胞增殖的影响及蛋白激酶C相关作用的研究目的:探讨香烟烟雾提取物(CSE)对体外培养的大鼠和人PASMC增殖的影响及PKC信号转导途径在其中所起的作用。方法:组织块法培养正常Wistar大鼠PASMC,复苏培养人PASMC细胞系。(1)不同浓度的CSE作用于PASMC 24h,四甲基偶氮唑盐(MTT)比色法检测PASMC增殖,台盼蓝排斥法检测活细胞率。(2)PASMC与PKC激活剂12-肉豆蔻酰-13-乙酸佛波酯(PMA)预孵育24h下调PKC或与PKC抑制剂马来酰亚胺甲磺酸盐(Ro31-8220)预孵育30min然后暴露于5%CSE,MTT法、流式细胞术和增殖细胞核抗原(PCNA)染色等方法观察细胞增殖的变化。(3)5%CSE作用PASMC 24h,逆转录-聚合酶链反应(RT-PCR)和Western Blot分别检测PKC-αmRNA和蛋白质表达量的变化。免疫荧光细胞化学及激光共聚焦扫描检测大鼠PASMC内PKC-α的分布。(4)大鼠PASMC暴露于5%CSE前6h转染PKC-α的反义寡核苷酸,观察PKC-α蛋白表达量和细胞增殖的变化。结果:( 1)20%和30%CSE对PASMC有细胞毒性作用,5%和10%CSE不影响PASMC的活细胞率。5%CSE组PASMC MTT的吸光度(A)值较对照组明显增高。(2)5%CSE组PASMC的MTT A值、增殖指数(PI)、S期细胞分数(SPF)和PCNA染色平均光密度值增高,与对照组比较差异有统计学意义(P<0.05)。PMA+5%CSE组和Ro+5%CSE组以上指标均降低,与5%CSE组比较,差异有统计学意义(P<0.05),与对照组比较,差异无显著性。(3)5%CSE组PKC-αmRNA和蛋白表达量增加,与对照组比较,差异有统计学意义(P<0.01)。5%CSE处理后,大鼠PASMC中PKC-α由细胞浆转位至细胞核周或核内(。4)PKC-α反义寡核苷酸转染组大鼠PASMC中PKC-α蛋白质表达量降低(转染前后分别为1.180±0.056、0.713±0.047),MTT A值降低(0.252±0.041),与5%CSE组(0.302±0.021)比较,差异有统计学意义(P<0.01),与对照组(0.244±0.016)比较,差异无显著性。结论:高浓度的CSE对大鼠和人PASMC有细胞毒性作用,低浓度(5%)的CSE则能促进大鼠和人PASMC的增殖。PKC特别是PKC-α在CSE促PASMC增殖的信号转导机制中起重要作用。第二部分香烟烟雾对大鼠肺动脉平滑肌细胞碱性成纤维细胞生长因子表达的影响分题1烟雾暴露大鼠肺动脉bFGF表达的变化及其与肺血管重建的关系目的:探讨烟雾暴露对大鼠肺动脉bFGF表达的影响及这种影响与肺血管重建的关系。方法:健康雄性Wistar大鼠30只,随机分为正常对照组(C组)、烟雾暴露组(S2w组、S4w组、S8w组、S12w组)。取动脉血检测血氧分压(PaO2),右心导管法测定平均肺动脉压(mPAP)。α平滑肌肌动蛋白(α-SM-actin)染色显示肺血管的重建,PCNA染色检测细胞增殖。RT-PCR检测肺动脉bFGF mRNA表达,免疫组织化学染色检测bFGF蛋白表达。结果:与C组大鼠比较,烟雾暴露各组大鼠PaO2和mPAP无明显改变。S4w组、S8w组和S12w组大鼠肺泡管旁肺小动脉肌化比例和烟雾暴露各组大鼠肺小血管壁细胞的增殖指数明显高于C组(P<0.05)。烟雾暴露各组大鼠肺动脉bFGF mRNA[(0.327±0.076)、(0.483±0.131)、(0.828±0.207)、(0.743±0.159)]和蛋白的表达[(0.184±0.012)、(0.230±0.017)、(0.292±0.013)、(0.325±0.020)]均较C组明显增强(P<0.05)。大鼠肺小动脉肌化比例、肺小血管壁细胞的增殖指数与bFGF mRNA和蛋白表达水平成显著正相关。结论:烟雾暴露能诱导肺小血管壁细胞增殖,导致肺血管重建,其程度随暴露时间延长而加重,bFGF在此过程中可能起重要作用。分题2香烟烟雾提取物通过PKC途径促进大鼠肺动脉平滑肌细胞bFGF表达目的:研究CSE对大鼠PASMC中bFGF表达的影响及PKC途径在其中所起的作用。方法:组织块法培养正常Wistar大鼠PASMC。5%CSE作用于PASMC不同时间或PASMC先与PKC特异性抑制剂Ro31-8220预孵育,然后再暴露于5%CSE,RT-PCR检测PASMC bFGF mRNA表达,免疫细胞化学检测bFGF蛋白表达。结果:对照组PASMC中有少量bFGF mRNA和蛋白表达(分别为0.093±0.034,0.142±0.013)。暴露于5%CSE后4h细胞内bFGF mRNA和蛋白表达即增加,mRNA于8h达高峰(0.436±0.103,P<0.01),蛋白于12h达高峰(0.237±0.031,P<0.01),此后逐渐下降,但24h都仍高于对照组。Ro31-8220预孵育可显著抑制5%CSE诱导的bFGF mRNA和蛋白表达增加。结论:5%CSE可通过激活PKC途径促进大鼠PASMC bFGF的表达。更多还原

【Abstract】 BackgroundCigarette smoking is not only the major cause of chronic bronchitis and emphysema, but also can lead to pulmonary hypertension and cor pulmonale. The presence of pulmonary hypertension is associated with worse clinical outcomes and less survival rates. At the present time, there is no specific and effective treatment for pulmonary hypertension in chronic obstructive pulmonary disease (COPD). In the past, most of the studies on the pathogenesis of COPD-related pulmonary hypertension were focused on hypoxic pulmonary vasoconstriction and hypoxic pulmonary remodeling. However, recent studies suggest that cigarette smoke has a direct effect on the pulmonary vasculature and this effect may lead eventually to pulmonary vascular remodeling and pulmonary hypertension. Smoke-induced proliferation of pulmonary artery smooth muscle cells (PASMC) plays a vital role in the process, but the underlying molecular mechanisms remain largely unclear. Activation of several intracellular pathways and production of numerous growth factors may be involved in the growth-stimulating effect of cigarette smoke on PASMC.PKC is a crucial family of serine-threonine kinases in the intracellular signal transduction pathway. It has been implicated in a variety of cellular functions including proliferation, apoptosis, differentiation of vascular smooth muscle cells. Previous studies in our laboratory showed that activation of PKC and upregulation of PKC-αisozyme play an important role in hypoxia-induced proliferation of PASMC. Recent work within our group also found that PKC-αmRNA and protein expression in small intrapulmonary arteries were markedly increased either in the smoke-exposed animals or in COPD patients and non-COPD smokers, suggesting a possible role for PKC-αin smoke-induced pulmonary vascular remodeling. However, the role of PKC and PKC-αin smoke-induced PASMC proliferation is yet to be investigated in vitro.As an important growth factor and mitogen, bFGF has been reported to stimulate the proliferation of pulmonary artery endothelial cells and adventitial fibroblasts, enable the medial smooth muscle cells to migrate, proliferate and synthesize extracellular matrix such as collagen and fibronectin. It contributes to pulmonary vascular remodeling in rats with hypoxia-induced or monocrotaline-induced pulmonary hypertension. Studies in smokers with COPD showed an unequivocal increase in bFGF expression in vascular smooth muscle of small pulmonary vessels with a luminal diameter under 200μm. The data suggest a role of bFGF in the pathogenesis of COPD-associated vascular remodeling. However, to our knowledge, no studies have examined the involvement of bFGF in smoke-induced PASMC proliferation in animal models and in PASMC cell culture model.In the present study, we investigated the roles of PKC and bFGF in the proliferation of PASMC and pulmonary vascular remodeling caused by cigarette smoke.PartⅠEffect of Cigarette Smoke Extract on Proliferation of Pulmonary Artery Smooth Muscle Cells and the Relevant Roles of Protein Kinase CObjective To investigate the effects of cigarette smoke extract on proliferation of rat and human PASMC and to evaluate the relevant roles of protein kinase C (PKC).Methods Cultured rat and human PASMC were studied in the following conditions: (1) PASMC were exposed to different concentrations of CSE for 24h, then MTT colorimetric assay was used for detection of cell proliferation. Cell viability was assessed by trypan blue exclusion. (2) PASMC were pre-incubated with PMA for 24h or Ro31-8220 for 30min before exposure to 5% CSE for 24h. Cell proliferation was examined by MTT colorimetric assay, cell cycle analysis and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. (3) PASMC were exposed to 5% CSE for 24h, then PKC-αmRNA expression was detected by Reverse transcription-polymerase chain reaction(RT- PCR)and protein expression by Western Blot. In rat PASMC, intracellular distribution of PKC-αwas also observed by immunofluorescence staining and confocal microscopy. (4) Rat PASMC were transfected with specific antisense oligodeoxynucleotides against PKC-α6h before exposure to 5% CSE for 24h. PKC-αprotein expression and cell proliferation were detected by methods described previously.Results (1) At low concentrations (5%) CSE increased proliferation of PASMC, whereas high concentrations (20%, 30%) were inhibitory as a result of cytotoxicity. (2) The absorbance (A) value, proliferation index (PI), S-phase cell fraction (SPF ) and average optical density of PCNA staining in PASMC from 5% CSE exposure group were significantly increased compared to those of control group (P<0.05). PKC down-regulation by PMA pretreatment or PKC inhibition by Ro31-8220 pre-incubation abolished the effect of 5% CSE on PASMC proliferation. (3) After exposure to 5% CSE for 24h, PKC-αmRNA and protein expression in PASMC (1.054±0.078, 1.185±0.041, respectively) were much higher than in untreated cells (0.573±0.054, 0.671±0.055, respectively) (P<0.01). Moreover, in rat PASMC, 5% CSE induced a translocation of PKC-αfrom cytoplasm toward the perinuclear area and into the nucleus. (4) Specific antisense oligodeoxynucleotides against PKC-αreduced 5% CSE-induced expression of PKC-αprotein (0.713±0.047 vs 1.180±0.056), also abolished the effect of 5% CSE on rat PASMC proliferation significantly.Conclusions CSE can be cytotoxic at high concentrations. But at low concentrations, it makes a mitogenic effect on cultured rat and human PASMC. PKC, especially its alpha isozyme, seems to play an important role in CSE-induced proliferation of both rat PASMC and human PASMC. PartⅡSection 1 Basic fibroblast growth factor expression and pulmonary vascular remodeling after cigarette smoke exposure in the ratObjective To investigate basic fibroblast growth factor expression and pulmonary vascular remodeling in rats exposed to cigarette smoke.Methods Thirty male Wistar rats were randomly divided into five groups: control group(C group), smoke exposure groups (S2w, S4w, S8w, S12w group). Mean pulmonary artery pressure (mPAP) was measured by right cardiac catheterization. The percentage of muscularised small pulmonary arteries adjacent to alveolar ducts was determined byα-SM-actin staining and vascular cell proliferation by proliferating cell nuclear antigen staining. RT- PCR and immunohistochemistry staining analysis were used for the detection of bFGF mRNA and protein expression in pulmonary arteries.Results Compared to control group, artery oxygen partial pressure and mPAP were not altered in smoke exposure groups. The percentage of completely muscularised small vessels adjacent to alveolar ducts was significantly increased in S4w, S8w and S12w group. Cigarette smoke caused marked proliferation of pulmonary vascular cells. The expressions of bFGF mRNA (0.327±0.076, 0.483±0.131, 0.828±0.207, 0.743±0.159, respectively) and protein (0.184±0.012, 0.230±0.017, 0.292±0.013, 0.325±0.020, respectively) in pulmonary arteries in smoke exposure groups were higher than those of C group (P<0.05). The percentage of muscularised vessels, proliferation index of vascular wall cells correlated strongly with levels of bFGF mRNA expression and the values of average optical density of bFGF protein.Conclusions These data suggest that smoke causes marked production of bFGF in pulmonary arteries. bFGF appears to play an important role in the pathogenesis of smoke-induced vascular remodeling. Section 2 Cigarette smoke extract induces bFGF expression via protein kinase C in rat pulmonary artery smooth muscle cellsObjective To investigate the effect of CSE on bFGF expression in rat PASMC and the possible role of PKC in this process.Methods Cultured PASMC were prepared by an explant method from intrapulmonary artery in normal male Wistar rats. Sub-confluent PASMC were serum-starved to induce quiescence, then were exposed to 5%CSE for different time periods. Some PASMC were pre-incubated for 30min with PKC inhibitor Ro31-8220 before exposure to 5%CSE for 12h. RT-PCR and immuocytochemistry staining were performed for the detection of bFGF mRNA and protein expression in PASMC.Results The control PASMC expressed bFGF mRNA and protein at low levels (0.093±0.034, 0.142±0.013, respectively). PASMC treated with 5%CSE for various times showed a time-dependent increase in bFGF expression. Increased bFGF mRNA and protein expression were evident at 4h after CSE treatment. Maximal increase in bFGF mRNA occurred at 8h (0.436±0.103, P<0.01), while peak expression of bFGF protein was at 12h (0.237±0.031, P<0.01). Then bFGF mRNA and protein expression were slowly declined and were still elevated at 24h. Pretreatment with Ro31-8220 nearly completely inhibited CSE-induced bFGF mRNA and protein expression.Conclusion These results indicate that 5%CSE stimulates bFGF mRNA and protein expression in cultured rat PASMC. The induction of bFGF seems to occur through activation of the PKC pathway.更多还原