【作者】 高连印；

【导师】 韦艾凌；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的：从微环境角度,通过体内试验和体外实验,研究癌痛消通过调节肝细胞癌缺氧微环境中细胞因子HIF-1α、VEGF及PHD2的表达水平治疗肝癌的机理。方法：1体内试验：采用适宜临床研究的Walker-256移植性肝癌大鼠模型。采用ELISA方法观察癌痛消高、中、低剂量组对移植性肝癌模型大鼠血清AFP、HIF-1α、VEGF表达水平的影响；采用免疫组织化学法、蛋白印迹法、实时荧光定量PCR法,分别观察癌痛消对移植性肝癌模型大鼠肝癌旁组织HIF-1α、VEGF及PHD2表达水平的影响。2体外实验：制备癌痛消高、中、低剂量及5-FU含药血清,采用蛋白印迹法观察癌痛消对缺氧微环境微血管内皮细胞(HMEC-1)中HIF-1α、VEGF及PHD2蛋白表达水平的影响。结果：1体内试验：癌痛消各组均可以明显抑制血清中AFP、 HIF-1α、VEGF的表达水平；癌痛消各组对肝癌旁组织中HIF-1α、 VEGF及PHD2蛋白和基因的表达均有调节作用,在缺氧微环境中过表达的HIF-1α、VEGF的蛋白和基因均有不同程度的下调,而缺氧微环境中低表达水平的PHD2蛋白和基因均有不同程度的提高,其中癌痛消高剂量组疗效较为显著。2体外试验：癌痛消高、中、低剂量含药血清作用于缺氧微环境HMEC-1后,结果显示癌痛消各组HMEC-1细胞中的HIF-1α、 VEGF蛋白的表达显著低于模型组；癌痛消各组HMEC-1细胞中PHD2蛋白的表达显著高于模型组,其中癌痛消高剂量组效果最显著。结论：1癌痛消可降低移植性肝癌模型大鼠血清中AFP、HIF-1α、 VEGF的表达量,以癌痛消高剂量组效更佳。2癌痛消可降低移植性肝癌模型大鼠肝癌旁织中HIF-1α、VEGF蛋白和基因的过表达,提升PHD2蛋白和基因的低表达,其中癌痛消高剂量组疗效较为显著。3癌痛消含药血清可降低缺氧微环境HMEC-1中HIF-1α、 VEGF蛋白过表达、提升PHD2蛋白的低表达,其中癌痛消高剂量组疗效较为显著。癌痛消通过调节肝细胞癌缺氧微环境中细胞因子HIF-1α、VEGF及PHD2的表达水平治疗肝癌。更多还原

【Abstract】 Objective:To study on the mechanism of liver cancer treatment, from the perspective of the microenvironment, that Aitongxiao regulate the expression levels of hepatocellular carcinoma cytokines HIF-la, VEGF and PHD2in the hypoxic microenvironment, via vivo and vitro experiments.Methods:1vivo experiment:We used Walker-256transplanted liver cancer model rats which was appropriate for clinical studies. We observed the effects what Aitongxiao high, medium and low dose made to the expressions of serum AFP, HIF-1α and VEGF of transplanted liver cancer model rats by ELISA. We observed the expressions of HIF-1α, VEGF and PHD2by immunohistochemistry, Western blot and real-time quantitative PCR (real-time PCR) method.2vitro experiment:We prepared the serums containing Aitongxiao high, medium and low dose and5-FU, and used Western blot to observe the impacts Aitongxiao had on HIF-1α, VEGF, PHD2expressions of micro-vascular endothelial cells in hypoxic microenvironment.Results:1vivo experiment:we could see the expressions of serum AFP, HIF-la and VEGF in Aitongxiao groups could be inhibited. The expressions of HIF-la, VEGF and PHD2in HCC adjacent tissues could be adjusted in Aitongxiao groups. It could also reduce the overexpressed HIF-1α, VEGF protein and gene and improve the low expression of PHD2protein with different degrees in hypoxic microenvironment, especially the Aitongxiao high-dose group.2vitro experiment:After serum-containing Aitongxiao high, medium and low dose acted on HMEC-1in hypoxic microenvironment, the results showed that the expression of HIF-1α and VEGF protein in the HMEC-1cells in Aitongxiao groups was significantly lower than that in the model group, while the expression of PHD2protein was higher. The Aitongxiao high-dose group had the most significant effect.Conclusion:1Aitongxiao could reduce the expression of serum AFP, HIF-1α and VEGF in the transplanted liver cancer model rats, especially the Aitongxiao high-dose group.2Aitongxiao could depress over-depression of HIF-1α, VEGF protein and gene in the hypoxic microenvironment, and increase low-expression of PHD2protein and genic. The Aitongxiao high-dose group performed the best among all model groups.3Serum containing Aitongxiao granules could depress over-depression of HIF-1α, VEGF protein and gene in HMEC-1, and increase low-expression of PHD2protein. Among all the Aitongxiao groups, the high-dose performed the best. Aitongxiao could regulate the expression of cytokines HIF-1α, VEGF and PHD2in the hypoxic microenvironment so as to exert its therapeutic effect.更多还原