【作者】 樊炼；

【导师】 潘立群；

【作者基本信息】 南京中医药大学 ， 中医外科学， 2010， 博士

【摘要】 研究背景：传统中医学有“气血相关”、“行气通脉”、“补气活血”、“脉者血之府也”的理论,因此,具有行气活血作用的中药可能具有促血管生成作用。这为研究中药促血管生成作用奠定了理论基础。在祖国医学中,黄芪被誉为“疮家圣药”,具有补气生血利水之功效,大量的临床应用及方剂证明,黄芪促进组织愈合；在实验中发现黄芪具有良好的促血管生成作用。我们前期研究亦发现黄芪注射液载入胶原后具有促血管新生作用,其机制可能与促进血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)等生长因子的表达,提高组织细胞血红蛋白和羟脯氨酸含量有关。本课题将使用黄芪的主要成分黄芪多糖修饰Ⅰ型胶原,在体外、鸡胚尿囊膜模型和大鼠损伤模型中验证其促血管新生作用,并初步探讨其发生机制。本实验分为两部分。第一部分黄芪多糖与Ⅰ型胶原协同促血管新生的疗效筛选与蛋白组学机制探讨目的：通过中药黄芪多糖修饰Ⅰ型胶原,观察黄芪多糖与Ⅰ型胶原促进血管生成疗效,并探讨黄芪多糖与Ⅰ型胶原是否有协同作用。方法：首先通过人脐静脉血管内皮细胞(HUVEC)增殖实验测定不同浓度黄芪多糖和胶原对细胞的增殖作用；并进一步通过细胞迁移实验测量细胞迁移率；Western Blot方法验证血管生成素Ⅰ(angiopoitin I)、整合素蛋白·αvβ3 (Integrinαvβ3)、VEGF的表达。所有数据以均数±标准差(x±s)表示,采用SPSS 11.0统计软件进行统计学one-way ANOVA分析,以P<0.05具有统计学意义。结果：黄芪多糖浓度在20ug／ml时促进细胞生长作用最明显,此后随着浓度的升高,促生长的作用逐渐降低,但均显著高于控制组(P<0.01);而黄芪多糖和胶原合用,对细胞的促生长有协同作用,显著高于20ug／ml胶原组(P=0.0139)。在20ug／ml黄芪多糖修饰胶原组HUVEC的吸光度值、细胞迁移率均明显高于其他各组(P<0.05)。Wester Blot结果提示血管生成素I(angiopoitin I)和VEGF、Integrinαvβ3的表达随着黄芪多糖和胶原浓度的增加而升高；20ug／ml胶原+20ug／ml黄芪多糖比例为1：1时的黄芪多糖胶原处理组达到最高。结论：黄芪多糖能促进HUVEC的增殖和迁移,并上调血管生成素Ⅰ、VEGF和Integrinαvβ33的表达,推测黄芪多糖具有促进血管生成的作用,且黄芪多糖和胶原具有协同作用。最佳量效配比为1：1第二部分黄芪多糖胶原促血管新生的实验研究目的：研究黄芪多糖胶原的促血管新生作用,并对其协同作用及发生机制进行初步的探讨。方法：将黄芪多糖胶原植入模型中,分别在鸡胚绒毛尿囊膜(chick chorioallantoic membrance, CAM)模型及SD大鼠损伤模型中观察研究新生微血管密度、胶原中血红蛋白、肉芽组织中羟脯氨酸含量及CD34抗原免疫组化来验证其促血管新生作用,并对其最佳剂量进行观察。通过VEGF及整合素蛋白αvβ3的表达探讨促血管新生作用的机制。所有数据采用以均数士标准差(x±s)表示,采用SPSS 11.0统计软件进行统计学one-way ANOVA分析,以P<0.05具有统计学意义。结果：鸡胚CAM模型中,20ug/ml黄芪多糖+胶原组微血管明显高于其他各组(p<0.05),具有统计学意义；大鼠损伤模型中,大鼠创面胶原中血红蛋白含量及肉芽组织中羟脯氨酸含量明显高于对照组(p<0.01)；创面组织CD34免疫组化提示黄芪多糖胶原组血管发生活跃,与对照组相比有显著差异；在创面组织中,VEGF及整合素蛋白αvβ3表达明显,提示了其在促血管新生的作用机制中占重要地位。结论：中等剂量(20ug/ml)黄芪多糖胶原能明显促进血管新生,且二者具有协同作用,其发生机制与VEGF及整合素蛋白αvβ3的表达相关。中药在促血管新生的应用中具有良好的前景。更多还原

【Abstract】 Background:There are many theories such as blood related with QI, therefore, the Chinese medicine which has the role of qi and blood may have a role in promoting angiogenesis. This angiogenesis effect of Chinese herbal medicine has laid a theoretical foundation. In Traditional Chinese medicine, the astragalus was thinked as the "magic medicine in treated sore", with the effect of tonifying Qi, hemogenesis and alleviate water retention.A large number of clinical applications and prescriptions show that astragalus promote tissue healing; and the promote angiogenesis effect of astragalus was discovred in several experiments. Our previous study also found that astragalus would promote angiogenesis, and the mechanism of which may be related to the promotion of vascular endothelial growth factor and other growth factors that increase the content of cell hemoglobin and hydroxyproline acid content. This project will study the angiogenesis function using the main constituents of astragalus APS load type 1 collagen in vitro, in Chick chorioallantoic membrane model and rat damage model, and discuss its mechanisms. This experiment is divided into two parts.Part I:The Synergy Effects of Astragalus Polysaccharide and Collagen I to Promote Angiogenesis on cellular levelObject:To observe the promote angiogenesis effect of the astragalus polysaccharide and collagen type I, and to discuss the synergistic effect between the astragalus polysaccharide and collagen. Methods:Firstly, to determinate the proliferation effect on human umbilical vein endothelial cells (HUVEC) of different concentrations of astragalus polysaccharides and collagen I; secondly, to survery the cell migration rate by experiment of cell migration. Then, to verificate the expression of angiopoitin I, integrinαvβ3 and VEGF. All the data were showed as mean±standard deviation and were statistical analysis of one-way ANOVA by SPSS 11.0 statistical software. P<0.05 was considered to has statistically significant. Results:The most significant effect of promote cell growth of astragalus polysaccharide was determined on the concentration of 20ug/ml.the role of promoting growth was gradually decreased as the concentration of astragalus polysaccharide increased.while the synergistic effect was discovered when combination of astragalus and collagen I, the promoting growth effect was significantly higher than 20ug/ml collagen group.(P=0.0139).The value of OD and migration rate of HUVEC was significantly higher than other groups(P<0.05).The expression of angiopoietin I.VEGF and integrinαvβ3 was increased as the increasing of the concentration of astragalus polysaccharide and collagen I, The peak value was discovered when the 20ug/ml astragalus and 20ug/ml collagen I was combinated. Conclusions:Astragalus polysaccharide can promote HUVEC proliferation and migration, and increases the expression of angiopoietin-Ⅰ, VEGF and the Integrinαvβ3.The results suggested the role of angiogenesis by astragalus polysaccharide’s treatment. And the synergistic effect was discovered when the astragalus polysaccharide and collagen I was combinated.Part II:Experimental Study of Astragalus Polysaccharide Modified-collagen on AngiogenesisObject:To study the promote angiogenesis effect of Astragalus polysaccharide modified-collagen on CAM and SD rat model respectively, and approach its mechanism of synergistic effect preliminarily.Methods:Firstly, the type-I collagen was modified by astragalus polysaccharide,then the effect of promote angiogenesis was observed in the chick embryo chorioallantoic membrane (chick chorioallantoic membrance, CAM) model and damage model of SD rats.Then the new microvessel density, content of hemoglobin, prolyl hydroxyl acid content in granulation tissue and CD34 antigen staining were used to verify its role in promoting angiogenesis, and its optimal dose were observed. The expression of VEGF and of integrinαvβ3 protein in CAM was verified by Western blot to explore the role of promoting angiogenesis mechanism.The expression of VEGF and intrgrin in the granulation tissue was verified by RT-PCR. All data were showed as mean±standard deviation (±s),and was analysised by SPSS 11.0 statistical software for statistical analysis of one-way ANOVA.P<0.05 was thinked to be has statistically significant.Results:In the chick embryo CAM model, the promote angiogenesis effect of the 20ug/ml collagen group of astragalus polysaccharide+collagen was significantly higher than other groups (p<0.05), with statistical significance,and the 20ug/ml was the best dose. The content of hemoglobin and hydroxyproline in the SD rat injury model in rats wound collagen content and granulation tissue was significantly higher (p<0.01)than the group of collagen only; CD34 positive cells was observed more intensive in the granulation tissue of group of astragalus polysaccharide+collagen as compared with the control group were significantly different; in wound tissue, the expression of VEGF and integrinαvβ3 was verified by RT-PCR, that suggesting its role in promoting angiogenesis accounts for an important position. Conclusion:The moderate dose (20ug/ml) astragalus polysaccharides modified-collagen can significantly promote angiogenesis, and there was a synergistic effect between them, its mechanism was associated with the expression of VEGF and integrinαvβ3.All the results would suggest that Chinese medicine in promoting the application of angiogenesis have good prospects.更多还原