二甲双胍对转化生长因子β1诱导黑素瘤1205Lu细胞上皮-间质转化及侵袭的影响

中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (6): 426-430.

二甲双胍对转化生长因子β1诱导黑素瘤1205Lu细胞上皮-间质转化及侵袭的影响

梁美琪¹,梁官钊²,段昕所³,孙立新⁴,陆洁¹

1. 承德医学院附属医院皮肤科
2. 中国医学科学院北京协和医学院皮肤病研究所
3. 河北承德医学院附属医院皮肤科
4. 承德医学院附属医院中心实验室

收稿日期:2016-09-18 修回日期:2016-12-04 发布日期:2017-05-31
通讯作者: 陆洁 E-mail:chengdejiel@163.com

Effects of metformin on transforming growth factor?beta1?induced epithelial?mesenchymal transition and invasion in the human melanoma cell line 1205Lu

Received:2016-09-18 Revised:2016-12-04 Published:2017-05-31

摘要/Abstract

摘要： 目的探讨二甲双胍在转化生长因子β1（TGF?β1）诱导下，对黑素瘤1205Lu细胞上皮-间质转化（EMT）过程及侵袭的影响。方法体外培养1205Lu细胞，分3组处理：空白对照组、TGF?β1（5 ng/ml）组、TGF?β1（5 ng/ml） + 二甲双胍（1 mmol/L）组。处理48 h后，显微镜下观察各组细胞形态变化并采集照片。Transwell侵袭实验检测细胞侵袭能力，采用蛋白质印迹法和实时荧光定量RT?PCR技术分别测定细胞内N?钙黏蛋白及基质金属蛋白酶9（MMP?9）在蛋白质和mRNA水平上的表达，通过单因素方差分析及LSD?t法对结果进行统计学分析。结果与空白对照组相比，TGF?β1诱导1205Lu细胞向成纤维细胞样形态转变（同上皮-间质转化样改变），联合二甲双胍可逆转上述形态改变。3组穿膜细胞数为空白对照组194.1 ± 8.295、TGF?β1组412.2 ± 13.427、TGF?β1 + 二甲双胍组175.3 ± 8.693。N钙黏蛋白水平3组分别为0.603 ± 0.029、1.963 ± 0.016、0.207 ± 0.009，mRNA水平3组分别为1.000 ± 0.000、6.678 ± 0.043、1.035 ± 0.015。MMP?9蛋白水平3组分别为0.575 ± 0.009、1.243 ± 0.027、0.629 ± 0.008，mRNA水平3组分别为1.000 ± 0.000、5.351 ± 0.604、0.930 ± 0.130。结果经方差分析显示，3组差异有统计学意义（P < 0.05）。结论二甲双胍可显著抑制TGF?β1诱导的黑素瘤1205Lu细胞EMT样形态改变、细胞侵袭及N钙黏蛋白、MMP?9在蛋白和mRNA水平上的表达。

Abstract: Liang Meiqi, Liang Guanzhao, Guo Jian, Duan Xinsuo, Lu Jie Department of Dermatology, Affiliated Hospital of Chengde Medical University, Chengde 067020, Hebei, China （Liang MQ, Liang GZ ［current affiliation: Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China］, Guo J, Duan XS, Lu J） Corresponding author: Lu Jie, Email: chengdejiel@163.com 【Abstract】 Objective To evaluate the effects of metformin on transforming growth factor?beta1 （TGF?β1）?induced epithelial?mesenchymal transition （EMT） in and invasion of the human melanoma cell line 1205Lu. Methods In vitro cultured 1205Lu cells were divided into 3 groups to be treated with serum?free culture medium （blank control group）, serum?free culture medium containing 5 ng/ml TGF?β1 （TGF?β1 group） and serum?free culture medium containing 5 ng/ml TGF?β1 and 1 mmol/L metformin （TGF?β1 + metformin group）, respectively. After 48?hour treatment, morphological changes of 1205Lu cells in the above groups were observed by using a microscope, and photos were taken at the same time. Transwell invasion assay was performed to estimate cellular invasive activity, Western blot analysis and real?time fluorescence?based quantitative RT?PCR were conducted to determine the mRNA and protein of N?cadherin and matrix metalloproteinase?9 （MMP?9）, respectively. Statistical analysis was carried out by one?way analysis of variance （ANOVA） and least significant difference （LSD） test. Results Compared with the blank control group, the stimulation of TGF?β1 could induce the epithelial?mesenchymal transition （EMT）?like morphological changes in 1205Lu cells, while TGF?β1 combined with metformin could reverse the EMT?like morpho?logical changes. The number of 1205Lu cells crossing the transwell Matrigel per high?power field （× 200） was significantly higher in the TGF?β1 group （412.2 ± 13.427） than in the blank control group （194.1 ± 8.295） and TGF?β1 + metformin group （175.3 ± 8.693）. Compared with the blank control group and TGF?β1 + metformin group, the TGF?β1 group also showed significantly increased mRNA and protein of N?cadherin （mRNA: 6.678 ± 0.043 vs. 1.000 ± 0.000, 1.035 ± 0.015; protein: 1.963 ± 0.016 vs. 0.603 ± 0.029, 0.207 ± 0.009） and MMP?9 （mRNA: 5.351 ± 0.604 vs. 1.000 ± 0.000, 0.930 ± 0.130; protein: 1.243 ± 0.027 vs. 0.575 ± 0.009, 0.629 ± 0.008）. Conclusion Metformin can obviously inhibit TGF?β1?induced EMT?like morphological changes in, the capacity to penetrate Matrigel?coated transwell chambers of and the mRNA and protein of N?cadherin and MMP?9 in 1205Lu cells.

中图分类号:

R739.5

梁美琪梁官钊段昕所孙立新陆洁. 二甲双胍对转化生长因子β1诱导黑素瘤1205Lu细胞上皮-间质转化及侵袭的影响[J]. 中华皮肤科杂志, 2017,50(6):426-430. doi:

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