槐果碱靶向肿瘤干细胞诱导分化治疗肝癌及其机制研究

Differentiation therapy of tumor cell, in particular tumor stem cells, can treat malignant tumor effectively. Sophocarpine was the effective component of chinese native medicine, we first observe the inhibitory effect of sophocarpine in vitro through the cell proliferation and cloning experiments. On the basis of this study, we use flow cytometry to select EpCAM, CD133 positive tumor stem cells from human liver cancer samples, to futher clear the proliferation and spheres formation of cancer stem cells can be inhibited by sophocarpine. RT-PCR will be used to detect the expression of liver function gene P450, ALB, G-6-P, CYP1A2 and the expression of stem cells proliferation and self-renewal related genes NANOG, OCT3/4, Sox2, c-myc. We will found that sophocarpine could be induced hepatoma cells and liver cancer stem cell differentiation into mature hepatic cells in vivro and in vivo; At the same time, the mouse orthotopic liver tumor model of planting will be preparated to clarify the preventive and therapeutic effect of sophocarpine on experimental liver cancer. With continuous affinity chromatography and competition chromatography separation of binding protein with sophocarpine, and to futhure discuss the relationship between sophocarpine and SHP-2, to explore the effects of SHP2 anti-cancer , and futher to clarify the mechanism of sophocarpine and specific binding protein SHP-2 in the differentiation therapy cancer stem cells and provide a new method for differentiation treatment of liver cancer.

诱导肿瘤细胞尤其是肿瘤干细胞分化，可达到有效治疗恶性肿瘤的目的。本课题采用中药有效成分槐果碱，研究其对肝癌细胞的作用，发现其体外具有抗肝癌的作用；在此基础上利用流式细胞仪从人肝癌标本中分选出EpCAM、CD133阳性的肿瘤干细胞，明确槐果碱抑制肿瘤干细胞的增殖、成球能力；并利用RT-PCR检测肝脏功能基因P450、ALB、G-6-P、及干细胞增殖和自我更新相关基因NANOG、OCT3/4、Sox2、c-myc的表达，了解槐果碱可诱导肝癌细胞及肝癌干细胞向成熟肝细胞分化；根据以上研究结果，我们将制备小鼠皮下成瘤及原位肝脏种植瘤模型，进一步验证槐果碱抗肿瘤的效果；通过连续亲和色谱法和竞争色谱法分离槐果碱的特异结合蛋白为SHP2；着重研究槐果碱与SHP-2的关系,并了解SHP-2抗肝癌的作用，阐明槐果碱通过特异结合蛋白SHP2诱导分化治疗肝癌的作用机制，为诱导分化治疗肝癌提供新的思路。

肝细胞癌是世界上最常见的恶性肿瘤之一，临床上迫切需要寻找更加安全有效的治疗方法。肿瘤分化治疗为肝癌提供了新的方向。肿瘤干细胞在维持肿瘤细胞的增殖、复发和转移中起着重要的作用，是原发性肿瘤起始、复发与转移的根源。槐果碱是从豆科槐属植物苦参及豆科槐属植物苦豆子中的干燥根及其地面部分提取的有效单体，是一种药源丰富，廉价、低毒的药物，具有抑制内毒素活性、减少炎症因子释放、减轻细胞损伤作用。本课题通过肝癌细胞提取蛋白质组，采用连续亲和色谱法和竞争色谱法分离槐果碱的特异结合蛋白；采用MALDI-TOF-TOF质谱鉴定特异结合蛋白的多肽序列，蛋白质芯片技术分析特异结合蛋白为SHP-2。研究槐果碱对肝癌及肝癌干细胞的作用，并探讨槐果碱诱导分化治疗肝癌的效果，阐明槐果碱通过特异结合蛋白Shp2诱导分化治疗肝癌的作用机制，为诱导分化治疗肝癌提供新的方法，同时为临床上肝癌的治疗提供新的思路。