西黄丸经Wnt信号转导通路关键蛋白β-catenin调控人肺癌干细胞增殖

The mechanism of Xihuang Pill regulating the growth of the lung cancer stem cells by controlling β-catenin of Wnt pathway

目的:研究西黄丸经Wnt信号转导通路关键蛋白β-catenin调控人肺癌干细胞增殖的机制。方法:培养人肺腺癌细胞系LAC,应用流式细胞荧光激活(FACS)技术,筛选鉴定具干细胞特性的SP细胞;50只C57BL/6小鼠分为西黄丸高剂量组、中剂量组、低剂量组,环磷酰胺组、模型组5组,SP细胞调整细胞密度1×107/ml,每只小鼠右腋皮下接种0.2ml瘤细胞悬液,成瘤后,分组按32g/kg、16g/kg、8g/kg给以西黄丸药液灌胃,CTX组7.5mg/kg,模型组予等量生理盐水,连续给药14d,处死小鼠,剥取瘤块称瘤重,并计算抑瘤率。日本大耳白兔随机分为5组,按上述剂量和方法给药,各组连续给药3 d后腹主动脉采血,离心,获得含药血清。SP细胞密度调至2×107/ml,种入培养瓶,分为西黄丸高、中、低剂量组,CTX组、空白组5组,分别加入如前所述制备的各组含药血清(血清、培养液体积比30%),培养24h后用逆转录-聚合酶链反应(RT-PCR)法和蛋白印迹(Western Blot)法分别检测β-catenin mRNA及蛋白表达。结果:西黄丸32g/kg、16g/kg可明显抑制C57BL/6小鼠体内肺癌移植瘤,同时可明显降低β-catenin mRNA及蛋白表达。结论:西黄丸可经由抑制β-catenin的表达,阻止肺癌干细胞Wnt信号转导通路激活,从而抑制肺癌干细胞增殖为肺癌细胞。

Objective： To observe the mechanism of Xihuang Pill（ 西黄丸） regulating the growth of the lung cancer stem cells by controlling β-catenin of Wnt pathway. Methods： SP cells of the LAC were sorted by Fluorescence-activated cell sorting assay（ FACS）. SP cells were regulated to 1 × 107/ml and sc,Xihuang Pill liquid（ 32,16,8g /kg） and CTX（ 7. 5mg /kg） were given lasted 2weeks. Then the tumor growth inhibition rate were calculated. Drug serum of rabbits which intervened with Xihuang Pill liquid（ 32,16,8g /kg） and CTX（ 7. 5mg /kg） were prepared. SP cells were regulated to 2 × 107/ml,divided into control group,Xihuang Pill low,middle and high-dose groups,and the CTX group. Drug serum of groups were put into corresponding SP cells（ drug serum occupies 70 % of cell medium）. The expression of β-catenin mRNA and protein was detected by Reverse Transcription-Polymerase Chain Reaction（ RT-PCR） and Western Blot. Results： The Xihuang Pill high and middle-dose groups（ 32g /kg,16g /kg） functioned significantly to inhibit the transplanted tumor on mice of lung cancer stem cells（ P ＆lt;0.05 or P ＆lt;0.01）; The Xihuang Pill high and middle-dose groups（ 32g/kg、16g/kg） could reduce down the expression of β-catenin mRNA and protein of the lung cancer stem cell（ P ＆lt; 0. 05,P ＆lt; 0. 01）. Conclusion： Xihuang Pill could block the activation of Wnt pathway by regulating down the expression of β-catenin. So Xihuang Pill could inhibit the growth of the lung cancer stem cells.