黄芪、莪术配伍调控肝癌血管生成的体视学及分子机制研究

The preliminary study results proved that compatibility of Astragalus mongholicus and Rhizoma curcumae can effectively inhibit tumor growth and metastasis by inhibiting angiogenesis to some extent.It can contribute to the regulation of angiogenesis, inhibit tumor metastasis and may enhance the sensitivity to chemotherapeutics by improving the structure and morphology of tumor angiogenesis and creating local aerobic conditions according to the latest data. To understand the anti-tumor mechanism of compatibility of Astragalus mongholicus and Rhizoma curcumae, a Hepatocellular Carcinoma model of nude rat will be established, tumor-derived vascular endothelial cells and vascular structure will be observed by stereological method, signaling pathway of tumor angiogenesis and thrombosis, expression and functional changes of protein molecules (eg. HIF-1,VEGF,TF, and FVII etc.) will be detected. We hope to find out the use of drugs for nourishing qi and activating blood in the treatment of Hepatocellular Carcinoma. The main purpose is not to kill tumor cells , but to improve the sensitivity of chemotherapeutics and the micro-environment, to inhibit the micro-thrombosis and hematogenous metastasis of cancer cell through inhibition of tumor angiogenesis and improvement of microvascular structure.At the same time, we hope to provide guidance and evidence for the rational use of drugs of clinical cancer treatment and supplement compatibility theory of traditional Chinese medicine. The study found that the tumor cells and tissue hypoxia can upregulate the expression of HIF-1 and VEGF(vascular endothelial growth factor). HIF-1 is different (source) dimer composed of HIF-1α and HIF-1β. It is rapidly degraded in normal oxygen environment, but it is stable in the anoxic environment. VEGF is the most important tumor angiogenic factors.It is secreted by the tumor cells, It has become the most popular target molecules in the anti-angiogenic treatment. Recently, the drug application using hypoxia regulatory factors as targets has aroused great interest for cancer therapy. Natural and synthetic drugs have been used in cancer therapy. HIF-1α is a new target of the anti-angiogenic. The experiments has proved that curcumin ,green tea extract, resveratrol, vitexin, and rapamycin can inhibit hypoxia-induced angiogenesis. Inhibition of tumor blood vessel formation is becoming the new hot spot in the cancer treatment. The continued growth of the tumor vasculature promotes tumor growth and metastasis. Tumor cell proliferation relys on contiguous vessels to obtain nutrients and oxygen, until the tumor growth is more than a certain size.Tumor growth is slow down and some cells even undergo apoptosis or death when blood vessel growth is insufficient and nutrition or oxygen are scarce. Thereby Inhibition of tumor angiogenesis can inhibit tumor growth to a certain extent.

前期实验研究显示：经典药对黄芪、莪术相使配伍，补气破血，荣脉固本，能有效抑制肿瘤生长转移，对新生血管生成亦有一定抑制作用。然申请者据前沿动态信息和相关性研究积累，提出假说：二者配伍后抗肿瘤或不以抑杀肿瘤细胞及抑制新生血管生成为主要目的，而是调控肿瘤新生血管形态结构，纠正乏氧状态，抑制微血栓形成，阻碍肿瘤细胞逸落沿血道转移，更可使化疗药直达病所，达到增敏效果。本课题拟以肝原位癌裸鼠模型为研究对象，用体视学法观察用药前后瘤体内免疫荧光双染标记的新生血管形态结构变化；为揭示其与氧、酸碱内环境、调控因子的正负相关性，及控制肝癌血行转移和使化疗药物增敏的药效基础，检测信号分子指标（HIF-1、HGF、VEGF、TF和CD105等）及miRNA（miR-122等）表观遗传学改变，以期深入探究补气活血药配伍抗肿瘤的分子机制与作用靶点。为临床肿瘤治疗合理用药提供指导和依据，同时丰富中药药性配伍理论。

目的：观察黄芪、莪术配伍，及联合顺铂对肝癌原位移植瘤血管结构及血管生成的影响，探究其提高化疗药物抗肿瘤敏感性机制。方法：一、构建人肝癌裸鼠原位移植瘤模型。用TUNEL法观察其对肝癌细胞的凋亡作用；ELISA法检测其对bcl-2、FGF-2、MMP-2的影响；采用免疫组化染色CD34观察微血管数目；采用Western Blotting和Real-time RT-PCR检测其对TF、HGF、FVII蛋白及mRNA表达的影响；采用IHC和Real time-PCR检测其对HIF-1α、VEGF 、CD147、iNOS蛋白和mRNA以及miR-122、miR-221、miR-151的影响。二、在活体状态下利用光声层析成像对原位移植瘤血管成像。三：将黄芪、莪术配伍水煎剂与斑马鱼胚胎孵育24 h，采用立体显微镜统计体节间血管根数和长度，采用激光共聚焦成像统计体节间血管面积，并观察体节间血管结构。结果：一、黄芪、莪术配伍对原位移植瘤具有生长抑制作用，平均瘤重及瘤体积均低于模型对照组；高、中剂量组能降低bcl-2、FGF-2、MMP-2含量，降低TF、FVII 、HGF蛋白及mRNA的表达，下调HIF-1α、VEGF、CD147、iNOS蛋白和mRNA的表达，显著上调miR-122的表达，对miR-221、miR-151表达有抑制作用(P＜0.05或P＜0.01)；联合顺铂，瘤重及瘤体积均明显降低，能显著下调bcl-2含量，诱导肿瘤细胞凋亡作用明显增加，降低肿瘤组织内微血管数目，降低TF、FVII 、HGF mRNA表达，抑制HIF-1α、VEGF、CD147、iNOS蛋白及mRNA表达。二、黄芪、莪术高剂量组肿瘤新生血管较稀，但均匀生长，走行自然，侧枝血管分叉较少，形态规则。与模型组比较，肿瘤血管分叉数、面积显著减少（P＜0.01）。三、黄芪、莪术配伍后体节间血管变短，甚至缺失，但管壁完整，血管形态规则，均匀等距。黄芪、莪术配伍能够抑制顶端细胞丝状伪足的伸展。与空白对照组比较，黄芪、莪术配伍组体节间血管根数、长度和面积均减少（P＜0.05或P＜0.01），且呈浓度相关性。结论：黄芪、莪术配伍，对血管生成具有抑制作用，能明显改善肿瘤和斑马鱼血管结构，与顺铂联合后抑制肿瘤生长，诱导肿瘤细胞凋亡效应增强。结果为补气活血药促进肿瘤血管正常化，提高化疗药敏感性提供依据。