- 學位論文
胎盤生長因子在慢性阻塞性肺病的表現量增加
Increased expression of placenta growth factor in chronic obstructive pulmonary disease
摘要
血管內皮生長因子 (vascular endothelial growth factor, VEGF) 和它的接受體在肺氣腫發病機轉應是扮演重要作用。 但是另一個血管因子：胎盤生長因子 (placenta growth factor, PlGF)對於慢性阻塞性肺病 （COPD）的影響並不是很清楚。 所以本實驗將分成三大部分來了解胎盤生長因子(PlGF)的所有功能：第一部分是臨床實驗，我們在臨床上收集慢性阻塞性肺病(COPD)病人與對照組受試者，來了解是否胎盤生長因子(PlGF)表現量在慢性阻塞性肺病(COPD)病人是較高的，而高的胎盤生長因子(PlGF)表現量是否有一些不好的影響?；第二部分是體外細胞實驗( in vitro cell culture study)，我們使用不同種類的炎性發炎細胞激素(pro-inflammatory cytokines)，來清楚的釐清血管內皮生長因子(VEGF)與胎盤生長因子(PlGF)的相互作用機轉，另外我們將建立一個模擬慢性阻塞性肺病慢性發炎的體外細胞培養，將胎盤生長因子(PlGF)和不同種類的炎性發炎細胞激素(pro-inflammatory cytokines)連續刺激14天來觀察細胞血管內皮生長因子(VEGF)的表現量與細胞存活與細胞凋亡的比例；第三部分是建立一個動物模式(animal model)，建立一種肺氣腫的動物模式。 我們使用的是從氣管內注入彈性蛋白酶(elastase)來破壞肺部實質而產生肺氣腫的變化。我們連續施打四周後觀察肺部肺泡腔室擴大(肺氣腫)的情形，除此以外，我們以基因工程方是篩選出基因剔除鼠(knock out, KO)，用的是胎盤生長因子基因剔除鼠(knock out placenta growth factor mice)來更進一步探究胎盤生長因子(PlGF)於慢性阻塞性肺病(COPD)的致病角色與病理變化。 由臨床的實驗結果可以得知，在慢性阻塞性肺病患者的血中濃度(serum)與肺泡灌洗液(bronchoalveolar lavage, BAL)濃度，胎盤生長因子(PlGF)都高於非慢性阻塞性肺病的吸菸患者；同時胎盤生長因子(PlGF) 的血中濃度與肺泡灌洗液濃度都與肺功能(FEV1)呈現明顯的負相關連性。愈高的胎盤生長因子(PlGF)濃度將會導致愈差的肺功能。證實，在人體如果過度表現的胎盤生長因子(PlGF)是對肺組織是有害處的。 我們模擬在14天慢性刺激發炎的體外細胞模式觀察血管內皮生長因子(VEGF)的表現量在使用治療胎盤生長因子PlGF， 腫瘤壞死因子-alpha (TNF-alpha) 或IL – 8個別或全部一起刺激。單獨使用TNF -alpha或IL - 8刺激使得血管內皮生長因子在最初的7天的表現量逐漸增加，然後在10-14天 治療刺激後卻逐漸下降。當單獨使用胎盤生長因子(PlGF)治療經過14天的刺激並沒有使血管內皮生長因子(VEGF)的水平量有所減少；同樣的發現是當伴隨同時使用TNF -alpha兩者一起治療10天後，血管內皮生長因子(VEGF)的表現量有稍微減少，但並沒有達到統計上的差異 (p=0.43)。隨之而來的治療當同時三者一起治療10天包括胎盤生長因子(PlGF)，腫瘤壞死因子-alpha (TNF-α) 和IL – 8 之後，很明顯的發現血管內皮生長因子(VEGF)的表現量有顯著減少，且有達到統計上的差異 (p<0.05)。 細胞接觸這些刺激物質14天後，細胞生存能力試驗(cell viability test) 發現死細胞的百分比顯著增加。另外，我們同時分析細胞凋亡(apoptosis)的現象。 也同時發現細胞凋亡的狀況與數量是明顯上升的跟前七天比較。 而在老鼠的動物實驗中，在各種不同基因型的老鼠以PPE氣管灌注後檢查不同的時間順序發展成肺氣腫的狀況。在使用PPE灌注四週後，可以明顯發現在胎盤生長因子基因野生型(wild type)老鼠肺泡壁明顯破壞而導致肺泡腔室擴大(airspace enlargement)的現象。然而在胎盤生長因子剔除老鼠(KO mice)和對照組(control)老鼠的肺部並沒有看到肺泡腔室擴大的現象發生，而在胎盤生長因子基因混生型老鼠的肺部(HE) 肺泡腔室擴大的現象是比較減少的。另外，發現MMP-9和TNF-alpha的表現量在胎盤生長因子基因剔除老鼠是比野生型基因老鼠的表現量還低；但是在血管內皮生長因子(VEGF)的表現量在胎盤生長因子基因剔除老鼠是比野生型基因老鼠的表現量還高。另外在胎盤生長因子基因野生型(wild type)老鼠肺泡壁明顯產生細胞凋亡(apoptosis)的數量是比基因剔除老鼠來得更多。同時細胞凋亡的現象在基因剔除鼠的肺部組織是比較少的。額外再給與胎盤生長因子從PPE氣管灌注後在加入發現又將導致成肺氣腫的型成。 而胎盤生長因子的作用都可以被血管內皮生長因子接受體第一型抑制劑所阻斷不管在體外細胞實驗或是動物實驗都會看到此結果。 因此本實驗綜合臨床、體外細胞實驗及動物實驗強烈認為胎盤生長因子與慢性阻塞性肺病的致病機轉有相當的關連性 (PlGF contributes to the pathogenesis of COPD) 。
並列摘要
Vascular endothelial growth factor (VEGF) and its receptor may play an important role in the pathogenesis of emphysema. But the effect of another angiogenic factor, placenta growth factor (PlGF), to chronic obstructive pulmonary disease (COPD) is unknown. The purpose of this study was to elucidate the role of VEGF and PlGF in patients with chronic obstructive pulmonary disease (COPD). At first, we measure the levels of VEGF and PlGF in sera and BAL fluids of COPD patients. We then studied the regulation of PlGF and VEGF in cultured bronchial epithelial cells after exposure to various pro-inflammatory cytokines, as well as the effects of 14 days exposure to heightened PlGF on bronchial epithelial cells. Thirdly, the animal model for emphysema was established and aimed to test this hypothesis by determining whether emphysema could be prevented in mice whose PlGF had been knocked out. It further aimed to elucidate the role of PlGF in the pathogenesis of emphysema. We measured the levels of VEGF and PlGF in serum from patients with COPD (n=184), smokers (n=212), nonsmokers (n=159), and in bronchoalveolar lavage (BAL) fluid from another group (COPD n=20, controls n=18). In vitro cell culture experiments were performed to investigate the effect of PlGF on VEGF. Besides, pulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues. The serum levels of PlGF were significantly higher in COPD than in controls (27.1 (SE, 7.4) pg/ml vs. 12.3 (SE, 5.1) pg/ml in smokers and 10.8 (SE, 6.3) pg/ml in nonsmokers, p=0.005). The levels of PlGF in BAL fluids were also significantly higher in COPD than in controls (45.7 (SE, 12.3) pg/ml vs. 23.9 (SE, 7.6) pg/ml, p=0.005), associated with an increase of all measured cytokines, like tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8). In COPD patients, the levels of PlGF correlated inversely with forced expiratory volume in one second (FEV1) (in serum, r=-0.59, p=0.002; and in BAL fluids, r=-0.51, p=0.001). While the levels of VEGF in serum were the same between COPD and controls, the levels in BAL fluids were significantly lower in COPD than in controls (127.5 (SE, 30.1) pg/ml vs. 237.8 (SE, 36.1) pg/ml, p=0.002). In cultured bronchial epithelial cells, proinflammatory cytokines induced an increase in the protein expression of both PlGF and VEGF. Continuous concomitant treatment with PlGF, TNF-α and IL-8 stimulation reduced VEGF expression and induced cell death. This phenomenon was suppressed by VEGF receptor inhibitor (CBO-P11). After 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs. In this study including clinical data, in vitro cell culture results, and animal model, implying that PlGF contributes to the pathogenesis of emphysema.