张雅兰　陈文发　吴春玲

[摘要]研究壁虎对肝癌干细胞特性的影响，并探讨其分子机制。采用MTT法检测肝癌细胞增殖；肿瘤球培养和流式细胞术检测肝癌干性；Western blot检测Wnt通路及干性相关蛋白的表达；免疫共沉淀检测相互作用蛋白；皮下移植瘤模型检测肝癌细胞的干性。结果显示，壁虎对Huh7细胞的IC50为（0750±0112） g·mL-1，其对Hep3B细胞的IC50为（0454±0039） g·mL-1。壁虎处理的肝癌细胞培养形成的肿瘤球的数目和大小均缩小（P<005），CD44，CD133阳性的肝癌细胞比例减少（P<005）。守宫处理肝癌细胞后，βcatenin，CD44，cMyc，CCND1，Sox2，Oct4，Nanog，ABCG2等表达均下调。免疫共沉淀结果显示，壁虎能抑制LRP6与Frizzled6的相互作用。表明，壁虎能够抑制肝癌细胞的增殖、肿瘤球的形成及肿瘤干细胞的比例，其通过靶向LRP6，阻止LRP6与Frizzled6复合物的形成而抑制Wnt信号通路。

[关键词]壁虎； 肝细胞癌； 干细胞特性； Wnt

[Abstract]To explore the effects and mechanism of aqueous extracts of gecko on cancer stem cells properties of hepatocellular carcinoma In vitro， MTT assay was used to detect the cells growth in Huh7 and Hep3B Spheroidforming assay and flow cytometry were performed to observe the the stemness of Huh7 and Hep3B cells The protein expressions of βcatenin， CD44， cMyc， CCND1， Sox2， Oct4， Nanog and ABCG2 were detected by Western blot Interacting proteins were detected by coimmunoprecipitation； and a subcutaneous xenograft model was used to detect the stemness of hepatoma carcinoma cells The results indicated that aqueous extracts of gecko induced cell growth inhibition in a dose and timedependent manner， with the IC50 of （0750±0112） g·mL-1 for Huh7 and （0454±0039） g·mL-1for Hep3B， respectively The number and size of tumor spheres formed by hepatoma carcinoma cells were decreased after treatment by aqueous extracts of gecko（P<005）； the proportions of cells staining with putative markers for cancer stem cells， such as CD133 and CD44， were decreased（P<005） After treatment with aqueous extracts of gecko， the expression levels of βcatenin， CD44， cMyc， CCND1， Sox2， Oct4， Nanog and ABCG2 were decreased Coimmunoprecipitation results showed that the aqueous extracts of gecko could inhibit the interaction between LRP6 and Frizzled6， indicating that the aqueous extracts of gecko could inhibit the proliferation of hepatoma cells， the formation of tumor spheres and the proportion of tumor stem cells， and inhibit the Wnt signaling pathway by targeting LRP6 to prevent the formation of LRP6 and Frizzled6 complexes.

[Key words]gecko； hepatocellular carcinoma； stemness； Wnt

肝細胞癌（hepatocellular carcinoma，HCC）是全球发病率居第5位的恶性肿瘤，也是我国最常见的恶性肿瘤之一。近年来，尽管多种HCC的治疗模式不断进展，但HCC的远期效果仍不理想，其中癌症的复发转移是导致远期效果不理想的重要原因。肿瘤干细胞（CSC）对放化疗的敏感性差，被认为是造成癌症转移、肿瘤化疗和放疗抵抗的主要原因之一。最初在《本草纲目》中记载，壁虎，亦称守宫或天龙，是一种传统的中药材。在现代医学中，壁虎被证实在肝癌中具有抗肿瘤作用[15]，但其对肿瘤干性的影响未见相关报道。本研究探讨壁虎对肝癌干性的影响，并阐述其作用机制，以望为其临床治疗肝癌提供新的依据。

1材料

11药材鲜壁虎Gekko swinhonis Gunther购自河北祁奥中药饮片有限公司，经由作者鉴定后使用。经冷冻、研磨、溶解、过滤等步骤后制成鲜壁虎水提取物，经BCA法定量后，制成质量浓度100 g·L-1的溶液。endprint

12动物BALB/c裸小鼠45只，雄性，4周龄，购自上海斯莱克实验动物有限责任公司，许可证号SCXK（沪）20120002。裸鼠饲养环境稳定，实验动物相关操作符合动物伦理学要求。……