肺纤方提取物干预肺间质纤维化大鼠血管生成及VEGF/VEGFR信号通路研究

By clinical summarize,we know that Feixianfang is the effective prescriptions in treating idiopathic pulmonary fibrosis. Experimental evidence proved that Feixianfang can play a role in resisting pulmonary fibrosis and ground substance remodeling.In our study ,we found that micro angiogenesis is the most important factor in pulmonary fibrosis,so we want to study how the extractive from Feixianfang interfering pulmonary micro angiogenesis and signal pathway in rats pulmonary fibrosis.. In our study ,we will use the whole animal to study how the extractive from Feixianfang interfering the lung microvascular density and ultrastructure of the rats of pulmonary fibrosis had been managed 14 days and 28 days, test the expression of the inherent membrane protein of CD31, CD34 in the microvascular endothelial cell surface ,detect the expression of mRNA of factors such as vascular endothelial growth factor (VEGF) and its receptor of VEGFR-1, 2, vascular endothelial adhesion molecules, coagulation protease receptor and fibrinolysis plasminogen activator inhibitor, and examine the expression of VEGF / VEGFR ,p38MAPK , PI3K and protein ALK. In addition,we will use cells in vitro test to study how the extractive from Feixianfang affecting pulmonary microvascular endothelial cell adhesion, migratory and angiogenesis of the rats of pulmonary fibrosis by primary cultured. Further up,we will explore how Feixianfang resisting pulmonary interstitial fibrosis which will provide the basis and lay the foundation in treating idiopathic pulmonary fibrosis safely and effectively by traditional Chinese medicine.

肺纤方是治疗特发性肺间质纤维化临床总结出的有效方剂，既往动物试验研究显示其具有抗肺纤维化、抗基质重塑等作用。肺微血管生成是肺间质纤维化的重要环节，本课题将开展肺纤方提取物干预大鼠肺间质纤维化血管生成及其信号通路机制研究。 .拟通过整体动物实验研究肺纤方提取物干预博莱霉素肺间质纤维化大鼠模型14天、28天的肺病理微血管密度、超微结构，检测微血管内皮细胞表面固有型膜蛋白CD31、CD34表达，检测血管内皮生长因子VEGF及其受体VEGFR-1、2、血管内皮粘附因子、凝血蛋白酶受体、纤维蛋白溶解酶原激活物抑制因子等mRNA表达，检测VEGF/VEGFR通路中信号分子p38MAPK 、PI3K和ALK蛋白表达。通过体外细胞试验研究肺纤方提取物对肺间质纤维化大鼠原代培养的肺微血管内皮细胞的粘附、增殖、游走及血管形成影响。深入探讨肺纤方抗肺间质纤维化的作用机制,为中药安全有效治疗肺间质纤维化提供依据

特发性肺间质纤维化（IPF）慢性炎症损伤及肺间质过度纤维沉积的病变，为呼吸难治疾病。肺纤方是临床治疗肺间质纤维化的有效方剂，针对痰瘀痹阻肺络、肺肾亏虚为肺间质纤维化的病机关键。本项目通过整体动物实验和体外细胞实验，研究肺纤方提取物抗肺间质纤维化血管生成和VEGF/VEGFR信号通路研究。. 本课题整体动物实验研究结果显示：肺纤方大中剂量组有抑制大鼠肺泡炎和肺纤维增生作用，电镜观察肺纤方大中剂量组均能抑制肺泡炎和纤维化程度及微血管密度。 肺纤方大中剂量组下调VEGF 及VEGF/VEGFR信号通路表达水平、从而抑制血管新生密度，显示肺纤方通过减少肺泡炎时的异常血管新生而具有抗肺间质纤维化作用。肺纤方大中剂量组可以抑制CD31、CD34免疫组化蛋白表达提示肺纤方大、中剂量组均能抑制增生的微血管密度。肺纤方大剂量组减轻VEGFR2/ALK/PI3K及 VEGFR1介导的P38MAPK/ PI3k信号通路蛋白表达，从而抑制胶原的合成和沉积、抑制细胞外基质纤维沉积而具有抗肺间质纤维化作用。肺纤方大剂量组可以显著抑制VCAM-1，PAI-1的基因表达具有抗粘附、抗凝血、抗纤维蛋白沉积作用，也是肺纤方具有抗肺间质纤维化通肺络及活血化瘀作用基础。. 体外实验研究结果显示：肺纤方大剂量组可以显著抑制肺纤维化大鼠体外培养的肺微血管内皮细胞的黏附和游走迁移及新生血管形成，肺纤方大、中、小剂量均能抑制肺微血管形成，其中肺纤方大剂量几乎完全抑制血管形成。还探讨研究了肺纤方提取物对肺间质纤维化大鼠微血管内皮细胞表达VEGF、VEGFR、PAI-1、 VCAM-1基因的影响，实验结果提示肺纤方能够抑制大鼠肺纤维化模型体外培养的肺微血管内皮细胞VEGF、VCAM-1、和PAI-1mRNA表达，从而改善血凝及纤溶抑制和抑制血管形成而发挥抗肺纤维化作用。. 本项目通过实验研究，初步揭示了肺纤方提取物干预肺间质纤维化肺微血管生成机制，为临床肺间质纤维化的中医药治疗提供了新的思路。项目资助发表核心论文7篇，待发表2篇，参加国际会议学术交流2篇，已培养硕士生2名，均已毕业并取得学位。