沙利度胺对博莱霉素致大鼠肺纤维化的干预作用及机制

发布时间：2018-05-18 14:50

本文选题：肺间质纤维化 + 博莱霉素　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:肺间质纤维化(pumlonary interstitial fibrosis)是一种慢性渐进性疾病,以炎症反应、成纤维细胞增殖和细胞外基质蛋白沉积为特点,诱因众多。而特发性肺间质纤维化(idiopathic pulmonary fibrosis,IPF)被定义为一种特殊形式的慢性纤维变性的间质性肺炎,组织病理学表现为寻常性间质肺炎。现已证实它是一种限制性通气功能障碍和弥散功能障碍疾病,患者最终死于呼吸衰竭。尽管IPF预后不良,但发病机制仍为未知,并且尚无有效的治疗措施。目前的治疗方法通常包括皮质类固醇激素或联合使用一种细胞毒药物。但是免疫抑制疗法的作用有限,并且副作用不容忽视。因此,迫切需要一种替代治疗方案。人肺成纤维细胞的体外实验研究表明,沙利度胺能减少IL-6、TGF-β_1、VEGF、Ang-1的产生及胶原的合成,并能抑制依赖IL-6的成纤维细胞增殖和依赖TGF-β_1的成纤维细胞转分化,这些可能是沙利度胺预防肺纤维化的潜在作用机制。本实验旨在研究沙利度胺对博来霉素致大鼠纤维化的干预作用。通过测量肺组织内羟脯氨酸(HYP)含量,以及肺组织和血清中TNF-α、TGF-β_1的表达水平探讨可能的作用机制。方法:选取清洁级健康、雄性SD大鼠(河北医科大学实验动物中心提供)90只,体重220±10g,适应性饲养一周后,随机分为:(1)对照组(A组):30只,气管内注入0.1ml/100g生理盐水,于假造模后第一日用生理盐水1ml/100g灌胃,1次/d,分别于7、14、28天各处死10只。(2)模型组(B组):30只,气管内一次性注入博来霉素A_5(5mg/kg),于造模后第一日用生理盐水1ml/100g灌胃,1次/d,分别于7、14、28天各处死9只、10只、9只。(3)沙利度胺组(C组):30只,气管内一次性注入博来霉素A_5(5mg/kg),于造模后第一日用沙利度胺溶液(沙利度胺与生理盐水配成1%溶液)100mg/kg灌胃,1次/d,分别于7、14、28天各处死10只。各组剪断双侧股动脉取血并放血处死,用酶联免疫吸附试验(ELISA)法测定血清中TNF-α、TGF-β_1的含量。取右肺下叶固定于4%多聚甲醛溶液中,用免疫组化法检测TNF-α、TGF-β_1,并用图像分析系统半定量测定TNF-α和TGF-β_1的阳性面积百分比。通过HE染色、Masson胶原染色来评价肺泡炎和肺纤维化的程度,并按照Szapiel法分为4级。右肺上叶制成匀浆测定肺组织中羟脯氨酸浓度。采用SPSS13.0统计软件处理数据。结果: 1肺组织病理分析结果:在同一时间点,B组肺泡炎程度显著高于A组(P0.01),B组肺纤维化程度亦显著高于A组,7天时(P0.05),14天、28天时(P0.01),表明动物模型复制成功。C组肺泡炎及纤维化程度较模型组减轻,7天:C组肺泡炎程度较B组显著减轻(P0.01);14天:C组肺泡炎及纤维化程度较B组显著减轻(P0.01);28天:C组纤维化程度较B组显著减轻(P0.01)。 2肺组织羟脯氨酸(HYP)含量变化:B组大鼠肺组织HYP含量随造模时间延长逐渐升高,至28天达高峰,7天时与A组相比差异有统计学意义(P0.01),与A组相比各时间点HYP含量的差异均具有统计学意义(P0.01)。C组大鼠HYP含量呈低水平升高趋势,造模后7、14、28天HYP含量均显著低于B组(P0.01)。 3肺组织TNF-α,TGF-β_1含量变化:B组大鼠肺组织中TNF-α、TGF-β_1表达量于7、14、28天均高于A组、C组,各时间点比较均有统计学意义(P0.01)。 4血清中TNF-α,TGF-β_1含量变化:B组大鼠血清中TNF-α的表达量除28天外,其余各时间点均明显高于A和C组,差异具有统计学意义(P0.01);B组大鼠血清中TGF-β_1表达量于7、14、28天均高于A组和C组,各时间点比较均有统计学意义(P0.01)。结论: 1沙利度胺在博莱霉素诱导的大鼠肺纤维化模型中,能够减少肺间质的胶原沉积,对肺间质纤维化具有一定的治疗作用。 2沙利度胺对TNF-α和TGF-β_1的产生具有抑制作用,进而减轻早期的炎症反应,抑制晚期的纤维化改变,这可能是沙利度胺对肺纤维化防治作用的潜在机制。
[Abstract]:Objective: pulmonary interstitial fibrosis (pumlonary interstitial fibrosis) is a chronic progressive disease characterized by inflammatory response, fibroblast proliferation and extracellular matrix protein deposition. And idiopathic pulmonary fibrosis (idiopathic pulmonary fibrosis, IPF) is defined as a special form of chronic fibrous degeneration. Interstitial pneumonia, histopathological manifestation of common interstitial pneumonia. It has been proved to be a restrictive ventilation dysfunction and diffuse dysfunction disease, and the patient eventually died of respiratory failure. Although the prognosis of IPF is poor, the pathogenesis is still unknown, and there is no effective treatment. The current treatment methods usually include cortex. Steroid hormones or combined use of a cytotoxic drug. But immunosuppressive therapy is limited and side effects can not be ignored. Therefore, an alternative therapy is urgently needed. In vitro experimental study on human lung fibroblasts showed that thalidomide could reduce the production of IL-6, TGF- beta _1, VEGF, Ang-1, and collagen synthesis and inhibit the dependence of human lung fibroblasts. The proliferation and the fibroblast transdifferentiation of TGF- beta _1 may be the potential mechanism of thalidomide in preventing pulmonary fibrosis in Lai IL-6.
The aim of this study was to investigate the effect of thalidomide on bleomycin induced fibrosis in rats. The possible mechanism of the expression of hydroxyproline (HYP) in lung tissue and the expression level of TNF- alpha and TGF- beta _1 in lung tissue and serum was investigated.
Methods: 90 male SD rats (Hebei Medical University experimental animal center), weighing 220 + 10g, were randomly divided into two groups: (1) the control group (group A): 30 rats were injected with 0.1ml/100g saline in the trachea. The first day after the artificial model was given to the stomach with physiological saline 1ml/100g and 1 times /d, respectively, to death 1 days, respectively, were killed 1 days respectively, and respectively to the 7,14,28 days to death 1 respectively. 0 (2) the model group (group B): 30 rats were injected with bleomycin A_5 (5mg/kg) in the trachea at one time. After the first day, the normal saline 1ml/100g was administered to the stomach, and 1 times /d, respectively, on 7,14,28 days, respectively, and 9. (3) the thalidomide group (group C): 30, and the bleomycin A_5 (5mg/kg) was injected into the trachea for the first time, and the first day was dissolved in thalidomide after the model. The solution (thalidomide and physiological saline mixed with 1% solution) was gavage with 100mg/kg, 1 times /d, and 10 rats were killed on 7,14,28 days respectively. Each group was cut off the bilateral femoral arteries to take blood and put to death. The content of TNF- alpha and TGF- beta _1 in serum was determined by enzyme linked immunosorbent assay (ELISA). The right lower lobe was fixed in 4% polycondensation Formaldehyde Solution and examined by immunohistochemical method. TNF- alpha, TGF- beta _1 were measured and the percentage of positive area of TNF- A and TGF- beta _1 was semi quantified by image analysis system. The degree of pulmonary alveolitis and pulmonary fibrosis was evaluated by HE staining and Masson collagen staining, and divided into 4 grades according to Szapiel method. The concentration of hydroxyproline in lung tissue was measured by the upper lobe of right lung, and SPSS13.0 statistics software was used. Data.
Result:
1 pathological analysis of lung tissue: at the same time point, the degree of alveolitis in group B was significantly higher than that in group A (P0.01), and the degree of pulmonary fibrosis in group B was also significantly higher than that in group A, 7 days (P0.05), 14 days, and 28 days (P0.01), indicating that animal model replicating of pulmonary alveolitis and fibrosis process was less than that in model group, 7 days: the degree of pulmonary alveolitis in C group was significantly less than that in B group (P0.0) 1); 14 days: alveolar inflammation and fibrosis in group C were significantly lower than those in group B (P0.01); on the 28 day: fibrosis in group C was significantly lower than that in group B (P0.01).
2 changes in the content of hydroxyproline (HYP) in lung tissue: the HYP content in lung tissue of rats in group B increased gradually with the duration of mould making, to the peak of 28 in Tianda. The difference between the 7 days and the A group was statistically significant (P0.01). The difference of HYP content at each time point compared with the A group had statistical significance (P0.01) the HYP content of the.C group was low level, and the model was created. The content of HYP in the post 7,14,28 days was significantly lower than that in the B group (P0.01).
3 the changes in the content of TNF- alpha and TGF- beta _1 in lung tissue: the expression of TNF- alpha and TGF- beta _1 in the lung tissue of the rats in group B was higher than that in the A group, and the C group was statistically significant (P0.01) at all time points.
4 the changes of the content of TNF- A and TGF- beta _1 in serum: the expression of TNF- alpha in the serum of B rats was significantly higher than that of the A and C groups, and the difference was statistically significant (P0.01). The _1 expression of TGF- beta in the serum of the group B rats was higher than that in the 7,14,28 day group and the group, and the time points were statistically significant.
Conclusion:
1 thalidomide in bleomycin induced rat pulmonary fibrosis model can reduce the collagen deposition in the interstitial lung, and have a certain therapeutic effect on pulmonary fibrosis.
2 thalidomide inhibits the production of TNF- A and TGF- beta _1, thus alleviates the early inflammatory response and inhibits late fibrosis, which may be a potential mechanism for the prevention and treatment of pulmonary fibrosis by thalidomide.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R965

【参考文献】