肿瘤治疗新思路："沙漠化疗法"对肺癌生长及血管生成的影响和相关机制研究

In tumor growth and development, angiogenesis totally nourishes the tumor cells, while aquaporin is the main channel of water permeation, which provides positive effects on tumor growth and metastasis. Because of the pivotal roles of angiogenesis and aquaporin (AQP) in tumor microenvironment, we hypothesize that: with the double challenges of insufficient nutrients and water deprivation by angiogenesis inhibitor and aquaporin-siRNA (Desertification therapy), the desertification of tumor microenvironment won't suitable for tumor growth. To validate this hypothesis, we try to prepare the siRNA of AQP-1 to transfect the H157 cells and human umbilical vein endothelial cells (HUVEC). With the combinational administration of endostar and siRNA-AQP 1，the cell proliferation, cell cycle, cell migration/invasion, angiogenesis in vitro and cytoskeleton remodel will be detected with MTT staining, PI(propidium iodide) staining flowcytometry, tubeformation assay,chick embryo chorioallantoic membrane (CAM) assay, transwell assay and wound healing assay. Western blot will be performed to explain the possible synergistic mechanism in vitro. Meanwhile, the in vivo inhibitory effects on lung cancer will be investigated by measuring the tumor volumes and tumor weights of H157 subcutaneous xenograft with the Kaplan-Meier of survival curve. The expressions of Ki-67, CD31 and VEGF of the tumor sections also will be detected with immunohistochemistry to evaluate the anti-proliferate and anti-angiogenesis effects of 'desertification therapy'. The in vitro and in vivo results will confirm our hypothesis from the three levels (molecule～cell～integrity). Our conception will enlighten the experimental and clinic therapies of lung cancer with new insights.

在肿瘤微环境中，血管生成是肿瘤获得营养的主要方式，水通道蛋白对肿瘤生长和转移具有重要作用，因此我们可设想：在血管生成抑制剂导致的营养缺乏和水通道蛋白siRNA抑制引起的水剥夺双重打击下（沙漠化疗法），处于"缺水断粮"沙漠化环境中"又渴又饿"的肿瘤将无法生存！为此，我们拟制备水通道蛋白1的siRNA，转染H157和人脐静脉内皮细胞，加恩度处理，以MTT染色、PI染色、小管形成、鸡胚尿囊膜、Tranwell和划痕法探索"沙漠化疗法"对细胞增殖、周期、侵袭、血管生成和细胞骨架的影响，再以Western印迹分析二者联合的可能机制，最后以H157皮下移植瘤进行Kaplan-Meier分析、肿瘤进展和免疫组化指标检测(Ki-67、CD31和VEGF)，以期从分子～细胞～整体水平判断"沙漠化疗法"对肺癌生长和血管生成的影响。该研究将对肺癌实验研究和临床治疗提供新思路和新方法，具有重要的理论与应用价值。

在肿瘤微环境中，血管生成和水分运输在肿瘤生长与转移中具有重要作用，协同阻断这两个过程以人为模拟肿瘤细胞“缺水断粮”沙漠化环境（沙漠化疗法）将使肿瘤细胞无法生存！为此，我们构建了人水通道蛋白1的siRNA，转染H157 和人脐静脉内皮细胞，加恩度处理，以CCK-8染色、小管形成、鸡胚尿囊膜、划痕法和Transwell探索“沙漠化疗法”对细胞增殖、迁移、和血管生成的影响，再以Western blot分析二者联合对血管生成信号通路的影响；体内实验中，我们利用构建的慢病毒载体进行AQP1的干扰抑制，H157 和HCT116的裸鼠皮下移植瘤模型建立成功后，给予不同处理，进行肿瘤生长分析和微血管密度分析(CD31)，从分子～细胞～整体水平判断“沙漠化疗法” 对肺癌和结肠癌生长和血管生成的影响。CCK-8检测表明AQP1的siRNA抑制可明显增强Endostar所导致的HUVEC细胞增殖，但对肿瘤细胞的体外增殖无明显影响。Wound healing 与transwell分析表明AQP1抑制可增强Endostar对血管内皮细胞迁移的抑制作用。血管生成抑制实验（Tubeformation和CAM）表明AQP1抑制可显著增强Endostar对血管的生长抑制作用，这一作用与二者联合调节细胞增殖和血管生成信号通路的相关蛋白eNOS、MMP-2、MMP-9、Akt、VEGFR2表达与活性有关。H157和HCT116的裸鼠移植瘤实验表明：水通道蛋白1抑制联合血管生成抑制Endostar可有效抑制肿瘤的生长，这一作用可能与肿瘤血管生成受到明显抑制有关。同时，我们也应用人黑色素细胞瘤Ａ375细胞裸鼠移植瘤模型和B16/F10的小鼠皮下移植瘤模型初步探索了沙漠化疗法在黑色素瘤治疗中的应用，实验结果初步证明水通道蛋白1抑制联合temozolomide可有效抑制黑色素瘤的生长。该研究将为肿瘤的实验研究和临床治疗提供新思路和新方法， 具有重要的理论与应用价值。