多纳非尼抑制胆管癌TFK-1细胞增殖、迁移和侵袭及促进凋亡的机制研究

doi:10.11958/20221485

天津医药 ›› 2022, Vol. 50 ›› Issue (12): 1259-1263.doi: 10.11958/20221485

多纳非尼抑制胆管癌TFK-1细胞增殖、迁移和侵袭及促进凋亡的机制研究

刘猛¹^,²^,³(), 姜玖良², 付立跃², 李俊俊², 朱海涛¹^,²^,³^,^△()

1 贵州医科大学附属医院肝胆外科（邮编550004）
2 贵州医科大学临床医学院
3 贵州医科大学附属医院临床医学研究中心

收稿日期:2022-09-15 修回日期:2022-10-08 出版日期:2022-12-15 发布日期:2022-12-30
通讯作者: 朱海涛 E-mail:1115839288@qq.com;zhuhaitao@gmc.edu.cn
作者简介:刘猛（1993），男，硕士在读，主要从事肝胆胰肿瘤的基础方面研究。E-mail：1115839288@qq.com
基金资助:
国家自然科学基金资助项目(81960429)

Mechanism of donafenib inhibiting proliferation, migration and invasion and promoting apoptosis of cholangiocarcinoma TFK-1 cells

LIU Meng¹^,²^,³(), JIANG Jiuliang², FU Liyue², LI Junjun², ZHU Haitao¹^,²^,³^,^△()

1 Department of Hepatobiliary Surgery, the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China
2 School of Clinical Medicine, Guizhou Medical University
3 Clinical Medical Research Center, the Affiliated Hospital of Guizhou Medical University

Received:2022-09-15 Revised:2022-10-08 Published:2022-12-15 Online:2022-12-30
Contact: ZHU Haitao E-mail:1115839288@qq.com;zhuhaitao@gmc.edu.cn

刘猛, 姜玖良, 付立跃, 李俊俊, 朱海涛. 多纳非尼抑制胆管癌TFK-1细胞增殖、迁移和侵袭及促进凋亡的机制研究[J]. 天津医药, 2022, 50(12): 1259-1263.

LIU Meng, JIANG Jiuliang, FU Liyue, LI Junjun, ZHU Haitao. Mechanism of donafenib inhibiting proliferation, migration and invasion and promoting apoptosis of cholangiocarcinoma TFK-1 cells[J]. Tianjin Medical Journal, 2022, 50(12): 1259-1263.

摘要/Abstract

摘要：

目的研究多纳非尼抑制人胆管癌TFK-1细胞增殖、迁移和侵袭及促进凋亡的机制。方法将人胆管癌TFK-1细胞分为对照组（加入等量的二甲基亚砜处理），2、5、10 μmol/L多纳非尼组。CCK-8法检测细胞增殖抑制率；平板克隆实验检测细胞增殖情况；流式细胞术检测细胞凋亡；Transwell实验检测细胞迁移和侵袭能力；蛋白质免疫印迹法检测通路蛋白Wnt、β-catenin、细胞周期蛋白-D1（Cyclin D1）、抗凋亡蛋白Bcl-2、促凋亡蛋白Bax的表达；免疫荧光实验检测β-catenin进入细胞核的变化。结果随着多纳非尼浓度增加，TFK-1细胞增殖抑制率、凋亡率升高，平板克隆形成数、细胞迁移和侵袭数量逐渐减少，Wnt、β-catenin、Cyclin D1、Bcl-2表达均逐渐下降，Bax表达逐渐增高（P<0.05）；免疫荧光显示，与对照组比较，2 μmol/L多纳非尼组能够抑制β-catenin进入细胞核（P<0.05）。结论多纳非尼可以抑制人胆管癌TFK-1细胞增殖、迁移和侵袭，其机制可能与抑制Wnt/β-catenin通路活化及促进细胞凋亡相关。

关键词: 胆管肿瘤, Wnt信号通路, 细胞增殖, 细胞运动, 细胞凋亡, 多纳非尼

Abstract:

Objective To study the mechanism of donafenib inhibiting the proliferation, migration and invasion of human cholangiocarcinoma TFK-1 cells and promoting apoptosis. Methods Human cholangiocarcinoma TFK-1 cells were divided into the control group (treated with the same amount of dimethyl sulfoxide) and the 2, 5 and 10 μmol/L donafenib groups. CCK-8 assay was used to detect the inhibition rate of cell proliferation. Cell proliferation was detected by plate cloning assay. Cell apoptosis was detected by flow cytometry. Transwell assay was used to detect cell migration and invasion. Western blot assay was used to detect the expression of pathway proteins Wnt, β-catenin, Cyclin D1, anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax. Immunofluorescence assay was used to detect changes of β-catenin into the nucleus. Results With the increase of donafenib concentration, the proliferation inhibition rate and apoptosis rate of TFK-1 cells increased, the number of plate clone formation, cell migration and invasion decreased gradually, the expression levels of Wnt, β-catenin, Cyclin D1 and Bcl-2 decreased gradually, and the expression of Bax increased gradually (P<0.05). Immunofluorescence results showed that 2 μmol/L donafenib could inhibit β-catenin from entering the nucleus compared with the control group (P<0.05). Conclusion Donafenib can inhibit the proliferation, migration and invasion of human cholangiocarcinoma TFK-1 cells, and the mechanism may be related to the inhibition of Wnt/β-catenin pathway activation and the promotion of apoptosis.

Key words: bile duct neoplasms, Wnt signaling pathway, cell proliferation, cell movement, apoptosis, donafenib

中图分类号:

R735.8

图/表 7

图1 各组克隆形成实验结果

Fig.1 Colony formation assay results

图2 4组TFK-1细胞凋亡流式图

Fig.2 The cell apoptosis in four groups of TFK-1 cells detected by flow cytometry

图3 4组TFK-1细胞迁移和侵袭能力变化（结晶紫染色，×200）

Fig.3 Effects of donafenib on migration and invasion of TFK-1 cells (crystal violet staining, ×200)

表1 各组TFK-1细胞迁移和侵袭数量比较（个/视野，$\bar{x}\pm s$）

Tab.1 Comparison of the migration and invasion of TFK-1 cells between the four groups

组别	n	细胞迁移	细胞侵袭
对照组	3	167.67±5.31	216.67±4.64
2 μmol/L多纳非尼组	3	77.33±4.50^a	116.33±3.40^a
5 μmol/L多纳非尼组	3	40.67±2.87^ab	33.67±2.49^ab
10 μmol/L多纳非尼组	3	27.67±2.49^abc	19.33±2.62^abc
F		507.400^＊＊	1 427.000^＊＊

图4 Western blot检测多纳非尼作用后β-catenin通路蛋白和凋亡相关蛋白表达变化 A：对照组；B：2 μmol/L多纳非尼组；C：5 μmol/L多纳非尼组；D：10 μmol/L多纳非尼组。

Fig.4 Expression changes of β-catenin pathway protein and apoptosis related protein after treatment with donafenib detected by Western blot assay

表2 各组Wnt、β-catenin、Cyclin D1、Bax、Bcl-2蛋白表达水平比较（n=3，$\bar{x}\pm s$）

Tab.2 Comparison of expression levels of Wnt, β-catenin, Cyclin D1, Bax and Bcl-2 between the four groups

组别	Wnt	β-catenin	Cyclin D1	Bax	Bcl-2
对照组	0.72±0.05	0.69±0.03	0.80±0.04	0.15±0.03	0.53±0.02
2 μmol/L多纳非尼组	0.53±0.02^a	0.46±0.03^a	0.63±0.03^a	0.27±0.02^a	0.32±0.02^a
5 μmol/L多纳非尼组	0.33±0.04^ab	0.34±0.02^ab	0.34±0.02^ab	0.42±0.01^ab	0.23±0.02^ab
10 μmol/L多纳非尼组	0.17±0.04^abc	0.18±0.01^abc	0.31±0.02^abc	0.90±0.03^abc	0.13±0.03^abc
F	514.400^＊＊	438.000^＊＊	594.300^＊＊	990.800^＊＊	475.000^＊＊

图5 免疫荧光检测多纳非尼处理后β-catenin进入细胞核的变化（免疫荧光染色，×200）

Fig.5 Changes in β-catenin entry into the nucleus after treatment with donafenib detected by immunofluorescence assay (immunofluorescence staining, ×200)

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多纳非尼抑制胆管癌TFK-1细胞增殖、迁移和侵袭及促进凋亡的机制研究

Mechanism of donafenib inhibiting proliferation, migration and invasion and promoting apoptosis of cholangiocarcinoma TFK-1 cells

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