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  • 张超峰,罗天航,钮宏文,邓琳,方国恩.GW4064激活法尼酯X受体抑制结肠癌细胞生长浸润及基质细胞衍生因子1的表达[J].第二军医大学学报,2019,40(2):185-190    [点击复制]
  • ZHANG Chao-feng,LUO Tian-hang,NIU Hong-wen,DENG Lin,FANG Guo-en.Activation of farnesoid X receptor by GW4064 inhibits invasive growth of colon cancer cells and expression of stromal cell-derived factor 1[J].Acad J Sec Mil Med Univ,2019,40(2):185-190   [点击复制]
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GW4064激活法尼酯X受体抑制结肠癌细胞生长浸润及基质细胞衍生因子1的表达
张超峰1,罗天航1,钮宏文2,邓琳2,方国恩1*
0
(1. 海军军医大学(第二军医大学)长海医院普通外科, 上海 200433;
2. 上海中医药大学附属曙光医院急诊创伤外科, 上海 201203
*通信作者)
摘要:
目的 探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞生长浸润的机制。方法 体外培养人结肠癌细胞系HT-29,用浓度为0、0.1、1、3、5、7、10 μmol/L的GW4064分别处理HT-29细胞72 h,采用MTT法检测细胞活力;用浓度为0、1、5 μmol/L的GW4064分别处理HT-29细胞24 h,用相差显微镜观察细胞形态;用浓度为0、1 μmol/L的GW4064分别处理HT-29细胞24 h,采用细胞划痕实验检测细胞浸润的变化;用浓度为0、1、5 μmol/L的GW4064分别处理HT-29细胞24 h,采用PCR检测FXR mRNA和基质细胞衍生因子1(SDF-1)mRNA表达的变化;用浓度为0、1、5、7 μmol/L的GW4064分别处理24 h,采用ELISA法检测细胞培养上清液中SDF-1表达的变化。于裸鼠皮下接种HT-29细胞建立裸鼠成瘤模型,灌胃给予GW4064或溶剂DMSO,16 d后检测肿瘤生长情况及瘤体中FXRSDF-1 mRNA的表达。结果 GW4064处理HT-29细胞后,细胞生长受到抑制,且呈剂量依赖性,1、3、5、7、10 μmol/L GW4064组HT-29细胞的生长活力与对照组(0 μmol/L)相比差异均有统计学意义(P均<0.05)。用相差显微镜观察可见GW4064处理HT-29细胞后,细胞收缩变圆,从瘦长的细胞向表皮样细胞改变。细胞划痕实验结果显示GW4064处理HT-29细胞后,细胞迁移距离变短,与对照组(0 μmol/L)相比差异有统计学意义(P<0.05)。PCR检测结果显示GW4064处理后FXR mRNA表达呈剂量依赖性增加,而SDF-1 mRNA表达则相反。ELISA检测结果显示细胞培养上清液中SDF-1的表达量随着GW4064浓度的增加而逐渐下降,1、5、7 μmol/L GW4064组与对照组(0 μmol/L)相比差异均有统计学意义(P均<0.05)。给予GW4064后,荷瘤小鼠肿瘤体积变小,与对照组(给予DMSO)相比差异有统计学意义(P<0.05),并且瘤体内FXR mRNA表达增加、SDF-1 mRNA表达减少。结论 FXR激活后抑制了结肠癌细胞生长浸润,同时抑制了结肠癌细胞SDF-1的表达分泌。
关键词:  结肠肿瘤  法尼酯X受体  GW4064  基质细胞衍生因子1
DOI:10.16781/j.0258-879x.2019.02.0185
投稿时间:2018-10-31修订日期:2019-02-12
基金项目:国家自然科学基金(81472277).
Activation of farnesoid X receptor by GW4064 inhibits invasive growth of colon cancer cells and expression of stromal cell-derived factor 1
ZHANG Chao-feng1,LUO Tian-hang1,NIU Hong-wen2,DENG Lin2,FANG Guo-en1*
(1. Department of General Surgery, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Emergency and Traumatic Surgery, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
*Corresponding author)
Abstract:
Objective To explore the mechanism by which specific agonist of farnesoid X receptor (FXR) GW4064 inhibits the growth and invasion of colon cancer cells. Methods Human colon cancer cell lines HT-29 were in vitro cultured. After treatment with GW4064 of 0, 0.1, 1, 3, 5, 7 and 10 μmol/L for 72 h, cell viability was measured by MTT assay. After treatment with GW4064 of 0, 1 and 5 μmol/L for 24 h, the cell morphology was observed under phase contrast microscope, and the mRNA expression levels of FXR and stromal cell-derived factor 1 (SDF-1) were detected by PCR. After treatment with GW4064 of 0 and 1 μmol/L for 24 h, the cell invasive ability was detected by cell scratch test. After treatment with GW4064 of 0, 1, 5 and 7 μmol/L for 24 h, the SDF-1 expression in the culture medium was detected by ELISA. Nude mouse tumorigenesis model was established by subcutaneous inoculation of HT-29 cells. After intragastric administration with GW4064 or DMSO for 16 d, the tumor growth and the mRNA expression of FXR and SDF-1 in the tumors were determined. Results GW4064 inhibited the growth of HT-29 cells in a dose-dependent manner, and there was significant difference in the cell viability of HT-29 cells between the GW4064 groups (1, 3, 5, 7 and 10 μmol/L) and the control group (0 μmol/L, all P<0.05). After treatment with GW4064, phase contrast microscopy showed contracted and rounded colon cancer cells and slender cells transforming into epidermoid cells. The cell scratch test showed that the invasion ability of the colon cancer cells was significantly reduced after treatment with GW4064 compared with the control group (0 μmol/L, P<0.05). PCR results showed that the mRNA expression level of FXR was increased in a dose-dependent manner after GW4064 treatment, while the expression of SDF-1 mRNA changed in the opposite way. ELISA results showed that the SDF-1 expression in the cell culture supernant was decreased with the increase of GW4064 concentrations, and there were significant differences between the GW4064 (1, 5 and 7 μmol/L) groups and the control group (0 μmol/L, P<0.05). GW4064 significantly reduced tumor size compared with the control group (DMSO, P<0.05). In addition, the mRNA expression of FXR in the tumors was increased, and the mRNA expression of SDF-1 was decreased. Conclusion The activation of FXR can inhibit the invasive growth of colon cancer cells and the expression of SDF-1.
Key words:  colonic neoplasms  farnesoid X receptor  GW4064  stromal cell-derived factor 1