重组人血管内皮抑素在博来霉素诱导肺纤维化大鼠模型中的作用及机制研究

发布时间：2018-07-02 14:33

本文选题：内皮抑素 + 血管生成　；参考：《山东大学》2013年博士论文

【摘要】：研究背景与目的：特发性肺纤维化(idiopathic pulmonary fibrosis, IPF)是最常见的肺间质疾病,也是呼吸系统最严重疾病之一,病程一般呈进行性发展,最终可以引起呼吸衰竭而死亡,其5年生存率不超50%,因此受到许多国内外学者的关注。IPF发病机制不明,治疗效果差。研究其发病机制,拓展新的治疗措施具有重要意义。近年来血管生成在肺纤维化发生中的作用日益受到重视,血管生成可能是肺纤维化形成过程中的一个关键环节。很多研究发现在博来霉素诱导的肺纤维化动物模型中,抑制促血管生成因子或给予抗血管生成因子干预减轻血管生成能改善肺纤维化的进展。血管内皮生长因子(vascular endothelial growth factor, VEGF)是目前已知的最主要的血管生成因子,能够促进新血管的生成,增加血管通透性,并参与炎细胞浸润、维持其存活和促进转化生长因子β1(transforming growth factor β1, TGFβ1)表达等,VEGF受体2(vascular endothelial growth factor receptor2, VEGFR2)是VEGF发挥功能的主要受体。VEGF在肺纤维化形成中过度表达。抑制VEGF/VEGFR2通路可能通过抑制异常血管生成改善肺纤维化。 VEGF能激活细胞外调节蛋白激酶(extracellular signal regulated kinase, ERK)。ERK1/2是目前发现的一个重要的细胞增殖信号调节蛋白,能调控血管内皮形态及多种炎性介质和细胞因子的表达。ERKl/2激活后可以转移到细胞核内,磷酸化一系列转录因子包括核因子-κB (nuclear factor-κB, NF-κB),进而调控下游蛋白转录表达。NF-κB在炎症反应调控中起核心作用,参与调控许多细胞因子表达,包括促炎因子肿瘤坏死因子-α(tumour necrosis factor-α, TNF-α)和促纤维化因子TGFβ1. 内皮抑素能抑制VEGF/VEGFR2通路,在其他疾病研究中对于抑制ERK1/2和NF-κB活化、下调TNT-α和TGF β1表达的作用也有报道。基于以上通路及细胞因子在肺纤维化过程中的重要作用,推测应用内皮抑素干预血管形成及炎症反应可能改善肺纤维化进展。本研究将建立博来霉素(bleomycin, BLM)诱导的大鼠肺纤维化模型,给予重组人血管内皮抑素(recombinant human endostatin, rhES)皮下注射,观察血管形成、炎症及纤维化改变,探讨内皮抑素对肺纤维化的可能的作用机制。方法： 1.肺纤维化模型建立：将博来霉素经气管注入SD大鼠,观察动物一般情况及不同时期肺组织切片HE染色及Masson染色,确定成功制备肺损伤纤维化模型。 2.对血管形成、炎症及肺纤维化的影响：实验动物分为正常对照组(生理盐水,SA group),博来霉素模型组(BLM group),早期rhES组(BLM+前14天rhES, E-ES group)、晚期rhES组(BLM+后14天rhES, L-ES group)及全程rhES组(BLM+28天rhES, W-ES group)。每组15只大鼠,分别于7天、14天、28天各处死5只。应用HE染色、Masson染色及羟脯氨酸测定研究肺纤维化大鼠模型肺损伤不同阶段肺部炎症及纤维化改变；测定支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中白细胞计数及Wright-Giemsa染色观察白细胞分类改变,并测定BALF蛋白与血清总蛋白浓度,计算血管通透指数(protein permeability index, PPI);免疫组织化学方法测量微血管密度(microvascular density, MVD)。 3.机制研究：免疫组织化学方法测定VEGFR2蛋白表达；酶联免疫吸附(enzyme-linked immune sorbent assay, ELISA)分别测量肺组织匀浆VEGF、 TGF-β1及BALF中TNF-α表达。实时荧光定量PCR分析VEGF、VEGFR2mRNA水平表达；Western blot法分析ERK1/2及NF-κB的激活。 4.结果统计：应用软件SPSS18.0进行数据统计,所有计量数据都用均数±标准差来表示,采用双因素方差分析进行统计学分析,P0.05表示差异具有统计学意义。结果： 1.动物模型建立：正常大鼠镜下见肺结构清晰正常,各观察时期无结构改变,无炎细胞浸润及胶原纤维沉积。给予博来霉素气管内灌注后,第7天见肺泡间隔增宽,病灶区域内巨噬细胞、中性粒细胞等炎性细胞浸润明显,少量胶原纤维沉积；14天肺泡间隔明显增宽,仍有大量炎性细胞浸润但较前减轻,成纤维细胞及胶原纤维增多；第28天可见肺泡结构破坏紊乱,肺间质纤维成分大量沉积,炎症细胞较前减少。 2.动物一般情况观察：SA组大鼠活泼强壮,皮毛光亮,摄食正常,体重逐渐增加；BLM组死亡1只,有咳喘,皮毛无光泽,精神差,摄食减少,体重较SA组明显下降(P0.001),后期体重略有增加。E-ES和w-ES组大鼠早期有咳喘,摄食减少,体重下降较BLM组减少(P0.05)；L-ES组大鼠表现同BLM组,体重变化较BLM组无统计学差异。 3.病理学改变：SA组各观察时期肺组织无明显炎症细胞浸润和纤维沉积。BLM组早期见肺泡间隔增宽,病灶区域炎症细胞浸润明显,少量胶原纤维沉积；晚期肺泡结构破坏明显,肺间质内大量蓝色胶原纤维沉积,炎症细胞浸润较早期减少。E-ES和W-ES组HE染色及Masson染色显示第7、14天肺泡炎较BLM组明显减轻(day7:P0.001, day14:P0.05),纤维化评分第14、28天较BLM组明显减低(day14:P0.05, day28:P0.001)。L-ES组大鼠肺泡炎及纤维化评分较BLM组大鼠无明显统计学差异。 4.羟脯氨酸测定：SA组大鼠肺羟脯氨酸含量各观察时间段无明显变化。BLM造模后各观察时间段均较SA组明显升高(P0.001)。E-ES和w-ES组羟脯氨酸含量较BLM组均明显下降(day7:P0.01, day14和day28：P0.001)。L-ES组羟脯氨酸含量较BLM组低,但尚无统计学意义(day28：P=0.09) 5. MVD:SA组各观察时间段肺微血管密度无明显变化。BLM造模后第7天新生血管明显增多,第14和28天逐渐下降(P0.001)。E-ES和w-ES组各观察时间段MVD较BLM组降低,L-ES组MVD较BLM组无明显变化。 6. BALF炎症细胞计数及分类：SA组各观察时间段BALF炎症细胞计数及分类无变化。BLM造模后BALF中白细胞总数、中性粒细胞、巨噬细胞及淋巴细胞计数均明显升高(P值均0.05)。E-ES和w-ES组第7和14天白细胞总数、中性粒细胞及巨噬细胞计数较BLM组均明显下降(P0.05),淋巴细胞计数在各观察时间段较BLM组无明显变化。L-ES组炎症细胞计数及分类较BLM组无统计学差异。 7. PPI:SA组各观察时间段PPI无变化。BLM组PPI第7天明显升高,第14和28天逐渐下降(P0.001)。E-ES和W-ES组PPI较BLM组降低(day7和day14:P0.001, day28:P0.05)。L-ES组PPI较BLM组无统计学差异。 8. VEGF及VEGFR2mRNA与蛋白水平表达变化：BLM组各观察时段VEGF及VEGFR2mRNA水平较SA组明显增高,以第7天为著,第14和28天逐渐下降(day7和day14:P0.001, day28:P0.05). E-ES和W-ES组VEGF/VEGFR2mRNA表达较BLM组降低(P值均0.05)。L-ES组VEGF及VEGFR2mRNA表达较BLM组无统计学差异。肺匀浆VEGF浓度及VEGFR2免疫组化评分变化同mRNA水平变化。 9. TNF-α表达：SA组各观察时间段TNF-α表达水平无明显变化。BLM造模后TNF-α第7天明显升高,第14和28天逐渐下降(P0.001)；E-ES和w-ES组TNF-α水平较BLM组在各观察时期均明显下降(P0.001)；L-ES组第28天TNF-α水平较BLM组也下降(P0.05) 10. TGF-β1表达：BLM组TGF-β1表达较SA组明显升高,第14天最显著,第28天略有下降(P0.001)；早期应用内皮抑素能显著抑制TGF-β1升高(P0.001)；晚期应用内皮抑素后TGF-β1表达较BLM组降低,但尚无统计学差异(day28：P=0.142) 11. pERK1/2和总ERK1/2:BLM造模后各组pERK1/2表达均较SA组明显升高(P值均0.05)；F-ES和W-ES组pERK1/2表达第7、14天明显低于BLM组和L-ES组(day7:P0.001, day14:P0.05),第28天各组间无统计学差异：L-ES组和BLM组各观察时间段pERKl/2表达无显著差异。总ERKl/2在各组各观察时间段内表达稳定无显著变化。 12. NF-κB激活：SA组NF-κB活化单位p65少量表达且各时期无明显变化。BLM造模后p65表达明显增多且第14天达到高峰,第28天有所下降。E-ES和w-ES组各观察时间段p65表达较BLM组明显下降(P0.05)；L-ES组和BLM组各观察时间段p65表达尚无统计学差异(day28：P=0.139) 结论：本实验通过大鼠气管内注入博来霉素后成功建立肺纤维化动物模型,肺组织病理特点说明可用于肺间质纤维化的研究。早期开始应用内皮抑素能明显抑制肺纤维化过程中异常的血管生成、炎症反应及胶原纤维沉积。内皮抑素能降低VEGF/VEGFR2基因及蛋白水平表达,能抑制ERK1/2磷酸化和NF-κB激活,减少促炎因子TNF-α及促纤维化因子TGF-β1生成,从而抑制异常血管生成及血管通透性增加,减轻肺泡炎症反应及BALF炎细胞渗出。基于以上可能机制,内皮抑素最终减少胶原沉积并改善肺纤维化形成,从而为深入研究临床工PF等肺纤维化疾病治疗措施提供更为广阔的前景。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563;R-332

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