本文選題：內(nèi)皮抑素 + 血管生成　； 參考：《山東大學(xué)》2013年博士論文

【摘要】：研究背景與目的： 特發(fā)性肺纖維化(idiopathic pulmonary fibrosis, IPF)是最常見的肺間質(zhì)疾病,也是呼吸系統(tǒng)最嚴重疾病之一,病程一般呈進行性發(fā)展,最終可以引起呼吸衰竭而死亡,其5年生存率不超50%,因此受到許多國內(nèi)外學(xué)者的關(guān)注。IPF發(fā)病機制不明,治療效果差。研究其發(fā)病機制,拓展新的治療措施具有重要意義。 近年來血管生成在肺纖維化發(fā)生中的作用日益受到重視,血管生成可能是肺纖維化形成過程中的一個關(guān)鍵環(huán)節(jié)。很多研究發(fā)現(xiàn)在博來霉素誘導(dǎo)的肺纖維化動物模型中,抑制促血管生成因子或給予抗血管生成因子干預(yù)減輕血管生成能改善肺纖維化的進展。血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)是目前已知的最主要的血管生成因子,能夠促進新血管的生成,增加血管通透性,并參與炎細胞浸潤、維持其存活和促進轉(zhuǎn)化生長因子β1(transforming growth factor β1, TGFβ1)表達等,VEGF受體2(vascular endothelial growth factor receptor2, VEGFR2)是VEGF發(fā)揮功能的主要受體。VEGF在肺纖維化形成中過度表達。抑制VEGF/VEGFR2通路可能通過抑制異常血管生成改善肺纖維化。 VEGF能激活細胞外調(diào)節(jié)蛋白激酶(extracellular signal regulated kinase, ERK)。ERK1/2是目前發(fā)現(xiàn)的一個重要的細胞增殖信號調(diào)節(jié)蛋白,能調(diào)控血管內(nèi)皮形態(tài)及多種炎性介質(zhì)和細胞因子的表達。ERKl/2激活后可以轉(zhuǎn)移到細胞核內(nèi),磷酸化一系列轉(zhuǎn)錄因子包括核因子-κB (nuclear factor-κB, NF-κB),進而調(diào)控下游蛋白轉(zhuǎn)錄表達。NF-κB在炎癥反應(yīng)調(diào)控中起核心作用,參與調(diào)控許多細胞因子表達,包括促炎因子腫瘤壞死因子-α(tumour necrosis factor-α, TNF-α)和促纖維化因子TGFβ1. 內(nèi)皮抑素能抑制VEGF/VEGFR2通路,在其他疾病研究中對于抑制ERK1/2和NF-κB活化、下調(diào)TNT-α和TGF β1表達的作用也有報道�；谝陨贤芳凹毎蜃釉诜卫w維化過程中的重要作用,推測應(yīng)用內(nèi)皮抑素干預(yù)血管形成及炎癥反應(yīng)可能改善肺纖維化進展。本研究將建立博來霉素(bleomycin, BLM)誘導(dǎo)的大鼠肺纖維化模型,給予重組人血管內(nèi)皮抑素(recombinant human endostatin, rhES)皮下注射,觀察血管形成、炎癥及纖維化改變,探討內(nèi)皮抑素對肺纖維化的可能的作用機制。 方法： 1.肺纖維化模型建立：將博來霉素經(jīng)氣管注入SD大鼠,觀察動物一般情況及不同時期肺組織切片HE染色及Masson染色,確定成功制備肺損傷纖維化模型。 2.對血管形成、炎癥及肺纖維化的影響：實驗動物分為正常對照組(生理鹽水,SA group),博來霉素模型組(BLM group),早期rhES組(BLM+前14天rhES, E-ES group)、晚期rhES組(BLM+后14天rhES, L-ES group)及全程rhES組(BLM+28天rhES, W-ES group)。每組15只大鼠,分別于7天、14天、28天各處死5只。應(yīng)用HE染色、Masson染色及羥脯氨酸測定研究肺纖維化大鼠模型肺損傷不同階段肺部炎癥及纖維化改變；測定支氣管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中白細胞計數(shù)及Wright-Giemsa染色觀察白細胞分類改變,并測定BALF蛋白與血清總蛋白濃度,計算血管通透指數(shù)(protein permeability index, PPI);免疫組織化學(xué)方法測量微血管密度(microvascular density, MVD)。 3.機制研究：免疫組織化學(xué)方法測定VEGFR2蛋白表達；酶聯(lián)免疫吸附(enzyme-linked immune sorbent assay, ELISA)分別測量肺組織勻漿VEGF、 TGF-β1及BALF中TNF-α表達。實時熒光定量PCR分析VEGF、VEGFR2mRNA水平表達；Western blot法分析ERK1/2及NF-κB的激活。 4.結(jié)果統(tǒng)計：應(yīng)用軟件SPSS18.0進行數(shù)據(jù)統(tǒng)計,所有計量數(shù)據(jù)都用均數(shù)±標(biāo)準(zhǔn)差來表示,采用雙因素方差分析進行統(tǒng)計學(xué)分析,P0.05表示差異具有統(tǒng)計學(xué)意義。 結(jié)果： 1.動物模型建立：正常大鼠鏡下見肺結(jié)構(gòu)清晰正常,各觀察時期無結(jié)構(gòu)改變,無炎細胞浸潤及膠原纖維沉積。給予博來霉素氣管內(nèi)灌注后,第7天見肺泡間隔增寬,病灶區(qū)域內(nèi)巨噬細胞、中性粒細胞等炎性細胞浸潤明顯,少量膠原纖維沉積；14天肺泡間隔明顯增寬,仍有大量炎性細胞浸潤但較前減輕,成纖維細胞及膠原纖維增多；第28天可見肺泡結(jié)構(gòu)破壞紊亂,肺間質(zhì)纖維成分大量沉積,炎癥細胞較前減少。 2.動物一般情況觀察：SA組大鼠活潑強壯,皮毛光亮,攝食正常,體重逐漸增加；BLM組死亡1只,有咳喘,皮毛無光澤,精神差,攝食減少,體重較SA組明顯下降(P0.001),后期體重略有增加。E-ES和w-ES組大鼠早期有咳喘,攝食減少,體重下降較BLM組減少(P0.05)；L-ES組大鼠表現(xiàn)同BLM組,體重變化較BLM組無統(tǒng)計學(xué)差異。 3.病理學(xué)改變：SA組各觀察時期肺組織無明顯炎癥細胞浸潤和纖維沉積。BLM組早期見肺泡間隔增寬,病灶區(qū)域炎癥細胞浸潤明顯,少量膠原纖維沉積；晚期肺泡結(jié)構(gòu)破壞明顯,肺間質(zhì)內(nèi)大量藍色膠原纖維沉積,炎癥細胞浸潤較早期減少。E-ES和W-ES組HE染色及Masson染色顯示第7、14天肺泡炎較BLM組明顯減輕(day7:P0.001, day14:P0.05),纖維化評分第14、28天較BLM組明顯減低(day14:P0.05, day28:P0.001)。L-ES組大鼠肺泡炎及纖維化評分較BLM組大鼠無明顯統(tǒng)計學(xué)差異。 4.羥脯氨酸測定：SA組大鼠肺羥脯氨酸含量各觀察時間段無明顯變化。BLM造模后各觀察時間段均較SA組明顯升高(P0.001)。E-ES和w-ES組羥脯氨酸含量較BLM組均明顯下降(day7:P0.01, day14和day28：P0.001)。L-ES組羥脯氨酸含量較BLM組低,但尚無統(tǒng)計學(xué)意義(day28：P=0.09) 5. MVD:SA組各觀察時間段肺微血管密度無明顯變化。BLM造模后第7天新生血管明顯增多,第14和28天逐漸下降(P0.001)。E-ES和w-ES組各觀察時間段MVD較BLM組降低,L-ES組MVD較BLM組無明顯變化。 6. BALF炎癥細胞計數(shù)及分類：SA組各觀察時間段BALF炎癥細胞計數(shù)及分類無變化。BLM造模后BALF中白細胞總數(shù)、中性粒細胞、巨噬細胞及淋巴細胞計數(shù)均明顯升高(P值均0.05)。E-ES和w-ES組第7和14天白細胞總數(shù)、中性粒細胞及巨噬細胞計數(shù)較BLM組均明顯下降(P0.05),淋巴細胞計數(shù)在各觀察時間段較BLM組無明顯變化。L-ES組炎癥細胞計數(shù)及分類較BLM組無統(tǒng)計學(xué)差異。 7. PPI:SA組各觀察時間段PPI無變化。BLM組PPI第7天明顯升高,第14和28天逐漸下降(P0.001)。E-ES和W-ES組PPI較BLM組降低(day7和day14:P0.001, day28:P0.05)。L-ES組PPI較BLM組無統(tǒng)計學(xué)差異。 8. VEGF及VEGFR2mRNA與蛋白水平表達變化：BLM組各觀察時段VEGF及VEGFR2mRNA水平較SA組明顯增高,以第7天為著,第14和28天逐漸下降(day7和day14:P0.001, day28:P0.05). E-ES和W-ES組VEGF/VEGFR2mRNA表達較BLM組降低(P值均0.05)。L-ES組VEGF及VEGFR2mRNA表達較BLM組無統(tǒng)計學(xué)差異。肺勻漿VEGF濃度及VEGFR2免疫組化評分變化同mRNA水平變化。 9. TNF-α表達：SA組各觀察時間段TNF-α表達水平無明顯變化。BLM造模后TNF-α第7天明顯升高,第14和28天逐漸下降(P0.001)；E-ES和w-ES組TNF-α水平較BLM組在各觀察時期均明顯下降(P0.001)；L-ES組第28天TNF-α水平較BLM組也下降(P0.05) 10. TGF-β1表達：BLM組TGF-β1表達較SA組明顯升高,第14天最顯著,第28天略有下降(P0.001)；早期應(yīng)用內(nèi)皮抑素能顯著抑制TGF-β1升高(P0.001)；晚期應(yīng)用內(nèi)皮抑素后TGF-β1表達較BLM組降低,但尚無統(tǒng)計學(xué)差異(day28：P=0.142) 11. pERK1/2和總ERK1/2:BLM造模后各組pERK1/2表達均較SA組明顯升高(P值均0.05)；F-ES和W-ES組pERK1/2表達第7、14天明顯低于BLM組和L-ES組(day7:P0.001, day14:P0.05),第28天各組間無統(tǒng)計學(xué)差異：L-ES組和BLM組各觀察時間段pERKl/2表達無顯著差異�？侲RKl/2在各組各觀察時間段內(nèi)表達穩(wěn)定無顯著變化。 12. NF-κB激活：SA組NF-κB活化單位p65少量表達且各時期無明顯變化。BLM造模后p65表達明顯增多且第14天達到高峰,第28天有所下降。E-ES和w-ES組各觀察時間段p65表達較BLM組明顯下降(P0.05)；L-ES組和BLM組各觀察時間段p65表達尚無統(tǒng)計學(xué)差異(day28：P=0.139) 結(jié)論： 本實驗通過大鼠氣管內(nèi)注入博來霉素后成功建立肺纖維化動物模型,肺組織病理特點說明可用于肺間質(zhì)纖維化的研究。早期開始應(yīng)用內(nèi)皮抑素能明顯抑制肺纖維化過程中異常的血管生成、炎癥反應(yīng)及膠原纖維沉積。內(nèi)皮抑素能降低VEGF/VEGFR2基因及蛋白水平表達,能抑制ERK1/2磷酸化和NF-κB激活,減少促炎因子TNF-α及促纖維化因子TGF-β1生成,從而抑制異常血管生成及血管通透性增加,減輕肺泡炎癥反應(yīng)及BALF炎細胞滲出�；谝陨峡赡軝C制,內(nèi)皮抑素最終減少膠原沉積并改善肺纖維化形成,從而為深入研究臨床工PF等肺纖維化疾病治療措施提供更為廣闊的前景。
[Abstract]:Research background and purpose:
Idiopathic pulmonary fibrosis (IPF) is the most common pulmonary interstitial disease and one of the most serious diseases of the respiratory system. The course of the disease is generally progressive, and eventually can cause respiratory failure and death. The 5 year survival rate is not more than 50%. Therefore, many domestic and foreign scholars have paid attention to the pathogenesis of.IPF and the therapeutic effect. It is of great significance to study its pathogenesis and expand new therapeutic measures.
Angiogenesis plays an increasingly important role in the development of pulmonary fibrosis in recent years. Angiogenesis may be a key link in the process of pulmonary fibrosis. Many studies have found that in the animal model of pulmonary fibrosis induced by bleomycin, inhibiting angiogenic factors or giving anti angiogenic factors to reduce angiogenesis can be found. The progress of pulmonary fibrosis is improved. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor known at present. It can promote the formation of new blood vessels, increase vascular permeability, and participate in inflammatory cell infiltration, maintain its survival and promote transforming growth factor beta 1 (transforming growth factor beta 1,) TGF beta 1) expression and so on. VEGF receptor 2 (vascular endothelial growth factor receptor2, VEGFR2) is the major receptor for VEGF playing the function of.VEGF in pulmonary fibrosis formation over expression. Inhibition of VEGF/VEGFR2 pathway may improve pulmonary fibrosis by inhibiting abnormal angiogenesis.
VEGF activates extracellular signal regulated kinase (ERK).ERK1/2 is an important regulation protein of cell proliferation signal, which can regulate the morphology of vascular endothelial cells and the expression of various inflammatory mediators and cytokines, and can be transferred to the nucleus after activation of.ERKl/2, and a series of transcriptional causes are phosphorylated. It includes nuclear factor kappa B (nuclear factor- kappa B, NF- kappa B), and then regulates the core role of the downstream protein transcriptional expression of.NF- kappa B in the regulation of inflammatory response, and participates in regulating the expression of many cytokines, including proinflammatory factor TNF - alpha (tumour necrosis factor- alpha, TNF- alpha) and fibrotic factor beta 1.
Endostatin inhibits VEGF/VEGFR2 pathway and is also reported to inhibit the activation of ERK1/2 and NF- kappa B and down regulation of TNT- - and TGF beta 1 expression in other disease studies. Based on the important role of the above pathway and cytokines in the process of pulmonary fibrosis, it is speculated that endostatin intervention in angiogenesis and inflammatory response may improve lung fiber This study will establish a rat model of pulmonary fibrosis induced by bleomycin (BLM) and give recombinant human angioendostatin (recombinant human endostatin, rhES) subcutaneously, to observe angiogenesis, inflammation and fibrosis, and to explore the possible mechanism of endostatin on pulmonary fibrosis.
Method:
1. the model of pulmonary fibrosis was established. Bleomycin was injected into the SD rats. The general condition of the animals and the HE staining and Masson staining of the lung tissue sections at different stages were observed. The pulmonary fibrosis model was successfully prepared.
The effect of 2. on vascular formation, inflammation and pulmonary fibrosis: the experimental animals were divided into normal control group (normal saline, SA group), BLM group, early rhES group (rhES, E-ES group) in the first 14 days of BLM+, and the late rhES group (rhES, 14 days after BLM+), and 15 rats in each group, respectively 5 rats were killed at 7 days, 14 days and 28 days. HE staining, Masson staining and hydroxyproline assay were used to study pulmonary inflammation and fibrosis in different stages of lung fibrosis rat model. Leukocyte count in bronchoalveolar lavage fluid (BALF) and Wright-Giemsa staining were used to observe the changes of white blood cells. The concentration of BALF protein and serum total protein was measured and the vascular permeability index (protein permeability index, PPI) was calculated. The microvascular density (microvascular density, MVD) was measured by immunohistochemical method.
The 3. mechanism study: the immunohistochemical method was used to determine the expression of VEGFR2 protein; enzyme-linked immune sorbent assay (ELISA) was used to measure the expression of TNF- alpha in lung homogenate VEGF, TGF- beta 1 and BALF respectively.
4. results statistics: the application software SPSS18.0 for data statistics, all the measured data are expressed with mean standard deviation, and the two factor analysis of variance is used for statistical analysis. P0.05 shows that the difference has statistical significance.
Result:
1. animal models were established: normal rat lung structure was clear and normal, no structural changes, no inflammatory cell infiltration and collagen fibrous deposition during the observation period. After intratracheal perfusion of bleomycin, the alveolar septum was broadened on the seventh day, and macrophages, neutrophils and other inflammatory cells were infiltrated in the lesion area, and a small amount of collagen fiber was deposited. In the 14 day, the alveolar septum was obviously broadened, a large number of inflammatory cells were still infiltrated, but the fibroblasts and collagen fibers increased. The alveolar structure was destroyed on the twenty-eighth day, the fibrous composition of the pulmonary interstitial fibers was deposited, and the inflammatory cells were decreased.
2. animal general observation: the rats in group SA were lively and strong, the fur was bright, the feeding was normal, the body weight was gradually increased, and the BLM group died 1, with cough and asthma, poor fur, poor ingestion, and decreased body weight compared with the SA group (P0.001). The later body weight slightly increased in the.E-ES and w-ES group, and the intake decreased and the weight decrease was lower than the BLM group. There was no significant difference in body weight between L-ES group and BLM group (P0.05); in group BLM, there was no significant difference in body weight.
3. pathological changes: in group SA, there was no obvious inflammatory cell infiltration and fibrous deposition in the lung tissue of group.BLM, the alveolar septum increased in the early stage of the lung tissue, the infiltration of inflammatory cells in the lesion area was obvious, a small amount of collagen fibrils were deposited, the late alveolar structure was destroyed obviously, a large number of blue gel fibrils were deposited in the interstitial lung, and the infiltration of inflammatory cells decreased by.E in the early stage. HE staining and Masson staining in group -ES and W-ES showed that lung alveolitis in day 7,14 was significantly lower than that in BLM group (day7:P0.001, day14:P0.05). The fibrosis score was significantly lower than that in the BLM group (day14:P0.05, day28:P0.001), and there was no significant difference in pulmonary alveolitis and fibrosis scores in rats.
4. hydroxyproline determination: there was no obvious change in the content of hydroxyproline in SA group. The observation time of.BLM was significantly higher than that of group SA (P0.001). The content of hydroxyproline in group.E-ES and w-ES was significantly lower than that in group BLM (day7:P0.01, day14 and day28:P0.001), and the content of hydroxyproline in.L-ES group was lower than that in BLM group, but there was no statistics yet. Learning significance (day28:P=0.09)
5. MVD:SA group had no obvious changes in the density of pulmonary microvessel in each observation period. Seventh days after.BLM model, the neovascularization increased significantly, and the fourteenth and twenty-eighth day decreased gradually (P0.001) in group.E-ES and w-ES group, MVD was lower than that in BLM group, and L-ES group MVD was not significantly changed in BLM group.
The count and classification of 6. BALF inflammatory cells: the count of BALF inflammatory cells in each observation time of group SA and the number of leukocytes in BALF, neutrophils, macrophages and lymphocyte counts were significantly increased (P value was 0.05).E-ES and w-ES group seventh and fourteenth days white fine cell count, neutrophils and macrophage count was more BLM than BLM The group was significantly decreased (P0.05). There was no significant difference in lymphocyte count between the group of BLM and the group of.L-ES. There was no statistical difference in the count and classification of the inflammatory cells in the group of the group of BLM.
7. PPI:SA group PPI without change.BLM group PPI seventh days increased significantly, fourteenth and twenty-eighth days gradually decreased (P0.001).E-ES and W-ES group PPI lower than the BLM group (Day7 and day14:P0.001, day28:P0.05) no statistical difference compared with those in the group.
8. VEGF and VEGFR2mRNA and protein level expression changes: the level of VEGF and VEGFR2mRNA in each observation period in group BLM was significantly higher than that in the SA group, with a decrease of fourteenth and twenty-eighth days (Day7 and day14:P0.001, day28:P0.05). The expression of VEGF/VEGFR2mRNA expression in E-ES and W-ES group was lower than that in the group of E-ES and W-ES (0.05). Statistical difference. VEGF concentration and VEGFR2 immunohistochemical score in lung homogenate changed with mRNA level.
9. TNF- alpha expression: there was no obvious change in the expression level of TNF- a in each observation time of group SA, TNF- alpha seventh days after.BLM modeling, and gradually decreased on fourteenth and twenty-eighth days (P0.001), and the level of TNF- alpha in E-ES and w-ES groups was significantly lower than that in the BLM group (P0.001), and the level of alpha in L-ES group was also lower than that of the L-ES group at twenty-eighth days.
10. TGF- beta 1 expression: the expression of TGF- beta 1 in group BLM was significantly higher than that in group SA, the most significant in Fourteenth days, and a slight decrease in twenty-eighth days (P0.001); early application of endostatin could significantly inhibit the increase of TGF- beta 1 (P0.001). The expression of TGF- beta 1 in late application of endostatin was lower than that in the BLM group, but the Shang Wutong difference (day28:P=0.142)
The expression of pERK1/2 in each group was significantly higher than that in group SA after 11. pERK1/2 and total ERK1/2:BLM (P value was 0.05). PERK1/2 expression in F-ES and W-ES groups was significantly lower than BLM and L-ES (day7:P0.001,). There was no significant difference between the twenty-eighth days. There was no significant change in the expression level in each group.
12. NF- kappa B activation: the activation unit p65 of NF- kappa B in group SA was a small amount of expression and there was no obvious change in each period, the expression of p65 was significantly increased and the peak was reached on the fourteenth day. The p65 expression in the twenty-eighth days of the twenty-eighth days and the p65 expression of each observation time section of the w-ES group was significantly lower than that of the BLM group. (day28:P=0.139)
Conclusion:
In this experiment, the animal model of pulmonary fibrosis was successfully established after intratracheal injection of bleomycin in rats. The pathological characteristics of lung tissue showed that it could be used in the study of pulmonary fibrosis. Early application of endostatin could obviously inhibit the abnormal angiogenesis in the process of pulmonary fibrosis, inflammatory reaction and collagen fibrous deposition. Endostatin can reduce VEGF. The expression of /VEGFR2 gene and protein level can inhibit the activation of ERK1/2 phosphorylation and NF- kappa B, reduce the formation of pro-inflammatory factor TNF- alpha and fibrotic factor TGF- beta 1, thus inhibiting abnormal angiogenesis and increased vascular permeability, and alleviates alveolar inflammatory reaction and BALF inflammatory cell exudation. Based on the possible mechanism, endostatin eventually reduces collagen deposition And improve the formation of pulmonary fibrosis, so as to provide a broader perspective for in-depth study of treatment measures such as clinical work PF and other pulmonary fibrosis.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R563;R-332

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Anti-adjuvant arthritis of recombinant human endostatin in rats via inhibition of angiogenesis and proinflarnmatory factors[J];Acta Pharmacologica Sinica;2004年09期

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本文編號：2090428