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中华结直肠疾病电子杂志 ›› 2019, Vol. 08 ›› Issue (06) : 574 -579. doi: 10.3877/cma.j.issn.2095-3224.2019.06.006

所属专题: 文献

论著

从Wnt通路抑制因子表观遗传修饰调控研究重楼皂苷Ⅶ抗结直肠癌机制
陈鸣旺1, 高舒影1, 刘海光1, 唐清珠1, 罗宏标1,()   
  1. 1. 423000 郴州市第一人民医院肛肠外科
  • 收稿日期:2018-12-29 出版日期:2019-12-25
  • 通信作者: 罗宏标

Epigenetic modification regulation of Wnt pathway inhibitors to study the mechanism of polyphyllinⅦ against colorectal cancer

Mingwang Chen1, Shuying Gao1, Haiguang Liu1, Qingzhu Tang1, Hongbiao Luo1,()   

  1. 1. Department of Anorectal Surgery, First People′s Hospital of Chenzhou City, Chenzhou 423000, China
  • Received:2018-12-29 Published:2019-12-25
  • Corresponding author: Hongbiao Luo
  • About author:
    Corresponding author: Luo Hongbiao, Email:
引用本文:

陈鸣旺, 高舒影, 刘海光, 唐清珠, 罗宏标. 从Wnt通路抑制因子表观遗传修饰调控研究重楼皂苷Ⅶ抗结直肠癌机制[J/OL]. 中华结直肠疾病电子杂志, 2019, 08(06): 574-579.

Mingwang Chen, Shuying Gao, Haiguang Liu, Qingzhu Tang, Hongbiao Luo. Epigenetic modification regulation of Wnt pathway inhibitors to study the mechanism of polyphyllinⅦ against colorectal cancer[J/OL]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2019, 08(06): 574-579.

目的

从Wnt通路抑制因子表观遗传修饰调控研究重楼皂苷Ⅶ(PPLVⅡ)抗结直肠癌机制。

方法

实验分三组,空白对照组、结直肠癌模型组和PPLVII治疗组。采用TUNEL法检测小鼠的细胞凋亡情况,TOPglow/FOPglow TCF试剂盒用于检测Wnt信号传导途径活性,应用双抗体夹心法ELISA(sandwich ELISA)检测cleaved caspase-3、cleaved caspase-8、cleaved caspase-9、Bax、Bcl-2以及Cdk-4、Cdk-6、Cyclin D1和p21的表达,用免疫蛋白印迹检测细胞中的β-连环蛋白,实时荧光定量PCR检测干细胞相关基因OCT4、SOX2、c-Myc和TCF4的表达,检测lgG、β-catenin和H3k9Ac与MMP-9启动子的结合。

结果

TUNEL免疫组织化学分析与对照组相比,PPLVII诱导肿瘤细胞凋亡;PPLVII治疗后cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9和Bax表达增加,而Bcl-2则降低,差异有统计学意义(P<0.05);Cyclin D1、CDK-4、CDK-6表达下降,而p21表达上调,差异有统计学意义(P<0.01);PPLVII干预治疗后检测治疗组的TOP/FOP比率降低,差异有统计学意义(P<0.05);在PPLVII进行干预治疗后β-连环蛋白灰度值下降,差异有统计学意义(P<0.05);PPLVII进行干预治疗后OCT4、SOX2、c-Myc和TCF4相关基因的表达下调,差异有统计学意义(P<0.05)。

结论

重楼皂苷Ⅶ干预治疗通过降低Wnt通路抑制因子的表达对结直肠癌有一定的改善和治疗作用。

Objective

Inquiry PolyphyllinⅦ resistance mechanism of colorectal cancer from Wnt pathways inhibitory factor the epigeNETsic modifications regulation.

Methods

The experiment is divided into three groups: blank control group, model group and colorectal cancer polyphyllinⅦ treatment group. TUNEL assay was used to detect apoptosis in mice. TOPglow/FOPglow TCF kit was used to detect activity of Wnt signal transduction pathway. Sandwich ELISA was used to detect expression of caspase-3, caspase-8, caspase-9, Bax, bcl-2, cdk-4, cdk-6, Cyclin D1 and p21. The expression of stem cell related genes OCT4, SOX2, c-myc and TCF4 was detected by real-time fluorescence quantitative PCR, and the binding of lgG, beta-catenin and H3k9Ac to mmp-9 promoter was detected.

Results

Compared with the control group, TUNEL immunohistochemical analysis showed that PPLVII significantly induced apoptosis of tumor cells. After PPLVII treatment, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax expression were increased, while Bcl-2 expression was decreased, the difference was significant (P<0.05). The expression of Cyclin D1, CDK-4 and CDK-6 decreased, while the expression of p21 was up-regulated, the difference was significant (P<0.01). The TOP/FOP ratio in the treatment group was decreased after PPLVII intervention. Betacatenin was detected by western blotting, The results showed that the gray level of PPLVII intervention decreased, the difference was significant (P<0.05). The expressions of OCT4, SOX2, c-Myc and TCF4 related genes were down-regulated after PPLVII intervention, the difference was significant (P<0.05).

Conclusion

Treatment by reducing PPLVII Wnt pathways of inhibiting factor expression in colorectal cancer have certain improvement and treatment effect.

图1 TUNEL染色确定细胞凋亡水平(×100)。1A:对照组,1B:重楼皂苷VII治疗组
表1 PPLVII影响细胞凋亡相关蛋白的相对表达量(双抗体夹心法ELISA)(±s,ng/mL)
表2 PPLVII影响细胞周期相关蛋白的相对表达量(双抗体夹心法ELISA)(±s,ng/mL)
图2 蛋白印迹检测β-连环蛋白水平(WB)
表3 PCR相关基因表达检测结果(±s
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