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目的:从Wnt/β-catenin信号通路调控探讨白花蛇舌草(HDW)抑制大肠癌细胞及其干细胞生长的作用机制。方法:MTT法检测HDW对HT-29细胞活力的影响;建立HT-29皮下移植瘤模型,通过测量瘤体体积观察HDW对瘤体生长的影响,采用免疫组化检测HDW干预后对移植瘤组织中ki-67表达的影响;采用SP法分析HDW对HT-29细胞干细胞比例的影响;同时,采用Western blotting及免疫组化法检测HDW干预后对HT-29细胞中干细胞标记物LGR-5、OCT-4表达和Wnt/β-catenin通路中APC、survivin、β-catenin及p-βcatenin表达的影响。结果:MTT检测结果显示不同浓度的HDW干预可显著降低HT-29细胞活力,呈现一定的剂量依赖;HDW显著抑制HT-29移植瘤瘤体的生长且能够下调ki-67的表达;HDW可降低HT-29细胞中干细胞的比例,体内外实验结果均显示HDW可明显抑制干细胞标记物LGR-5和OCT-4的表达,并且上调APC及p-β-catenin的表达,下调survivin的表达。结论:HDW对大肠癌细胞和大肠癌干细胞的生长具有显著抑制作用,抑制Wnt/β-catenin信号通路的活化及调控关键因子的表达是其重要作用机制之一。
OBJECTIVE: To investigate the mechanism of inhibition of proliferation of colorectal cancer cells and stem cells by Hedyotic diffusa (HDW) from Wnt / β-catenin signaling pathway. Methods: The effect of HDW on the viability of HT-29 cells was detected by MTT assay. The model of HT-29 subcutaneously transplanted tumor was established. The effect of HDW on tumor growth was observed by measuring the tumor volume. The effect of HDW on the proportion of stem cells in HT-29 cells was analyzed by SP method. Western blotting and immunohistochemistry were used to detect the effect of HDW on the expression of stem cell markers LGR-5, OCT-4 expression and the expression of APC, survivin, β-catenin and p-βcatenin in Wnt / β-catenin pathway. Results: The results of MTT assay showed that different concentrations of HDW could significantly reduce the viability of HT-29 cells in a dose-dependent manner. HDW significantly inhibited the growth of HT-29 tumor and down-regulated the expression of ki- The proportion of stem cells in HT-29 cells in vitro and in vivo showed that HDW could significantly inhibit the expression of stem cell markers LGR-5 and OCT-4, up-regulate the expressions of APC and p-β-catenin, and down-regulate the expression of survivin. CONCLUSION: HDW has a significant inhibitory effect on the growth of colorectal cancer cells and colorectal cancer stem cells. Inhibiting the activation of Wnt / β-catenin signaling pathway and regulating the expression of key factors are one of the important mechanisms.