攻癌肠清方对宫颈癌干细胞增殖、放疗敏感性的影响

Effect of Gong-ai-chang-qing-fang on Proliferation and Radiosensitivity of Cervical Cancer Stem Cells

目的探讨攻癌肠清方对宫颈癌干细胞增殖、放疗敏感性的影响及其机制。方法采用无血清培养基分离培养宫颈癌细胞CASKI的干细胞;流式细胞仪检测干细胞表面标记物CD133的表达;微球体形成实验检测干细胞的成球情况;噻唑蓝(MTT)检测攻癌肠清方联合放疗处理后,宫颈癌干细胞增殖能力;克隆形成实验检测宫颈癌干细胞联合放疗干细胞克隆形成能力的差异;Western blot法检测宫颈癌干细胞中p-GSK-3β、β-catenin、cyclin D1蛋白的表达水平;荧光定量聚合酶链式反应(PCR)检测宫颈癌干细胞中Sox2、C-myc及p-GSK-3β、β-catenin、cyclin D1 mRNA表达的水平。结果流式细胞术分选后CD133阳性率为(98.42±5.71)%,显著高于分选前的(3.82±0.81)%(P<0.05)。与分选前相比,分选后干细胞标志物OCT4、Sox2和Nanog的蛋白表达水平显著增加(P<0.05)。在24、48、72 h的时间点上,3组细胞增殖抑制率比较差异均有统计学意义(P<0.05)。尤以作用72 h的攻癌肠清方联合放疗组的细胞增值抑制率最高。与对照组比较,攻癌肠清方联合放疗组及放疗组的宫颈癌干细胞克隆形成率显著降低(P<0.05);且攻癌肠清方联合放疗组的克隆形成率显著低于攻癌肠清方组及放疗组(P<0.05)。与对照组比较,攻癌肠清方组、放疗组及攻癌肠清方联合放疗组宫颈癌干细胞的凋亡率显著增加(P<0.05)。且攻癌肠清方联合放疗组的细胞凋亡率显著高于攻癌肠清方及放疗组(P<0.05)。攻癌肠清方联合放疗组宫颈癌干细胞中C-myc及Sox2 mRNA及蛋白的表达均显著低于其他各组(P<0.05)。攻癌肠清方组、放疗组及攻癌肠清方联合放疗组宫颈癌干细胞中p-GSK-3β、β-catenin、cyclin D1蛋白表达水平均显著低于对照组(P<0.05)。且以攻癌肠清方联合放疗组各指标蛋白水平最低。结论攻癌肠清方能有效抑制宫颈癌干细胞的增殖并促进其放疗敏感性,其机制可能与攻癌肠清方抑制肿瘤干细胞p-GSK-3、β-catenin、cyclin D1蛋白的表达,降低C-myc、Sox2的表达,从而抑制了干细胞增殖,促进细胞凋亡有关。

OBJECTIVE To investigate the effect of Gong-ai-chang-qing-fang on the proliferation and radiosensitivity of cervical cancer stem cells and its mechanism. METHODS Stem cells of cervical cancer cell line CASKI were isolated and cultured in serum-free medium. Flow cytometry was used to detect the expression of stem cell surface marker CD133. Microsphere formation assay was used to detect stem cell formation. MTT assay was used to detect the proliferation ability of cervical cancer stem cells. Clone formation assay was used to detect the difference in stem cell formation ability. Western blot was used to detect p-GSK-3β, β-catenin, cyclin D1 protein expression in cervical cancer stem cells. Fluorescence quantitative PCR was used to detect the levels of Sox2, C-myc and p-GSK-3β, β-catenin and cyclin D1 mRNA expression in cervical cancer stem cells. RESULTS The stable passage of cervical cancer stem cells could be isolated and cultured in serum-free medium. The positive rate of CD133 after flow cytometry was(98.42±5.71)%, which was significantly higher than that before sorting(3.82±0.81)%(P<0.05). At 24, 48, and 72 h, the inhibition rates of cell proliferation in the three groups were statistically significantly different(P<0.05). In particular, at 72 h the Gong-ai-chang-qing-fang combined with radiotherapy group had the highest cell proliferation inhibition rate. Compared with the control group, the colony formation rate of cervical cancer stem cells was significantly lower in the Gong-ai-chang-qing-fang combined with radiotherapy group and radiotherapy group(P<0.05);the colony formation rate of the cancer-reinforcing intestinal clearing group combined with radiotherapy group was significantly lower than those of Gong-ai-chang-qing-fang group and radiotherapy group(P<0.05). Compared with the control group, the apoptotic rate of cervical cancer stem cells was significantly increased in the Gong-ai-chang-qing-fang group, the radiotherapy group and the attacking cancer clearing group combined with the radiotherapy group(P<0.05). The apoptotic rate of the Gong-ai-chang-qing-fang and radiotherapy was significantly higher than those of Gong-ai-chang-qing-fang and radiotherapy groups(P<0.05). The expressions of C-myc and Sox2 mRNA and protein in cervical cancer stem cells were significantly lower than those in the other groups(P<0.05). The expression levels of p-GSK-3β, β-catenin and cyclin D1 in cervical cancer stem cells were significantly lower than those in the control group(P<0.05). And the protein level of each index in the combination of attacking cancer and clearing combined with radiotherapy was the lowest. CONCLUSION Gong-ai-chang-qing-fang can effectively inhibit the proliferation of cervical cancer stem cells and promote the sensitivity of radiotherapy. The mechanism may be inhibiting the expressions of p-GSK-3, β-catenin, cyclin D1 and decreasing C-myc, Sox2 in cancer stem cells to inhibit the stem cells proliferation and promote apoptosis.