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浙江大学学报（医学版）
浙江大学学报（医学版）  2018, Vol. 47 Issue (2): 181-186    DOI: 10.3785/j.issn.1008-9292.2018.04.12
原著     
异甘草素通过下调基质金属蛋白酶抑制人脑胶质瘤干细胞迁移和侵袭
党莹1,2(),林瑜亮1,孙红军3,孙建军1,李长栋1,荔志云1,*()
1. 兰州军区总医院神经外科, 甘肃 兰州 730050
2. 西安市红会医院功能神经外科, 陕西 西安 710054
3. 甘肃省第二人民医院神经外科, 甘肃 兰州 730030
Isoliquiritigenin can inhibit migration and invasion of human glioma stem cells by down-regulating matrix metalloproteinases
DANG Ying1,2(),LIN Yuliang1,SUN Hongjun3,SUN Jianjun1,LI Changdong1,LI Zhiyun1,*()
1. Department of Neurosurgery, PLA Lanzhou General Hospital, Lanzhou 730050, China
2. Department of Functional Neurosurgery, Xi'an Honghui Hospital, Xi'an 710054, China
3. Department of Neurosurgery, Second Gansu Provincial People's Hospital, Lanzhou 730030, China
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摘要：

目的: 探讨异甘草素对人脑胶质瘤干细胞迁移和侵袭能力的影响及相关机制。方法: 免疫荧光法检测SHG44人脑胶质瘤干细胞的干性特征；transwell实验观察SHG44人脑胶质瘤干细胞的迁移和侵袭能力；实时定量RT-PCR法和蛋白质印迹法分别检测SHG44人脑胶质瘤干细胞中基质金属蛋白酶（MMP）2和MMP-9的mRNA和蛋白表达量。结果: SHG44细胞中干细胞标志物CD133和Nestin呈阳性表达。20 μmol/L和80 μmol/L的异甘草素作用于胶质瘤干细胞48 h后，迁移细胞数分别为76±5和42±4，与阴性对照组（85±6）差异有统计学意义（均P < 0.01），且80 μmol/L异甘草素组迁移细胞数少于20 μmol/L异甘草素组（P < 0.01）；穿透滤膜至小室背面的细胞数分别为190±13和130±9，与阴性对照组（230±14）差异有统计学意义（均P < 0.01），且80 μmol/L异甘草素组穿透滤膜至小室背面的细胞数少于20 μmol/L异甘草素组（P < 0.01）；MMP-2、MMP-9 mRNA和蛋白表达量均较阴性对照组下调（P < 0.05或P < 0.01），且80 μmol/L异甘草素组MMP-2、MMP-9 mRNA和蛋白表达量低于20 μmol/L异甘草素组（P < 0.05或P < 0.01）。结论: 异甘草素可通过下调MMP-2和MMP-9的表达抑制胶质瘤干细胞迁移和侵袭。
关键词： 神经胶质瘤/药物疗法;  干细胞;  异甘草素;  基质金属蛋白酶9;  基质金属蛋白酶2;  肿瘤侵润;  细胞运动    
Abstract:

Objective: To investigate the effects of isoliquiritigenin on the migration and invasion of human glioma stem cells and the underlying mechanism. Methods: The stem cell markers CD133 and Nestin in SHG44 human glioma stem cells were examined with immunofluorescence microscopy. The migration and invasion ability of glioma stem cells was determined by transwell method. The mRNA and protein expression of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by real-time RT-PCR and Western blot, respectively. Results: CD133 and Nestin were positive in SHG44 cells. The number of migrated cells in SHG44 cells treated with 20 and 80 μmol/L isoliquiritigenin for 48 h were significantly lower than that in control group (76±5 and 42±4 vs. 85±6, all P < 0.01), and the number of migrated cells in 80 μmol/L isoliquiritigenin group was lower than that in 20 μmol/L isoliquiritigenin group (P < 0.01). The numbers of cells crossing through membrane in 20 and 80 μmol/L isoliquiritigenin groups were 190±13 and 130±9, respectively, which were significantly lower than that in control group (230±14, all P < 0.01), and the number of crossed cells in the 80 μmol/L isoliquiritigenin group was lower than that in 20 μmol/L isoliquiritigenin group (P < 0.01). The mRNA and protein expression levels of MMP-2 and MMP-9 were decreased compared with control group (P < 0.05 or P < 0.01), and the expression levels in 80 μmol/L isoliquiritigenin group were lower than those in 20 μmol/L isoliquiritigenin group (P < 0.05 or P < 0.01). Conclusion: Isoliquiritigenin exhibits antitumor effects on glioma stem cells by inhibiting cell migration and invasion, which may be related to the down-regulation of MMP-2 and MMP-9.
Key words: Glioma/drug therapy    Stem cells    Isoliquiritigenin    Matrix metalloproteinase 9    Matrix metalloproteinase 2    Neoplasm invasiveness    Cell movement
收稿日期: 2017-09-01 出版日期: 2018-07-24
:  R739.41  
基金资助: 甘肃省重点研发计划(17YF1FA136)
通讯作者: 荔志云     E-mail: dangy052@qq.com;lizhiyun456@163.com
作者简介: 党莹(1989-), 女, 硕士研究生, 主要从事颅内肿瘤的临床和基础研究; E-mail:dangy052@qq.com; https://orcid.org/0000-0001-8423-4039
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党莹
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引用本文:

党莹,林瑜亮,孙红军,孙建军,李长栋,荔志云. 异甘草素通过下调基质金属蛋白酶抑制人脑胶质瘤干细胞迁移和侵袭[J]. 浙江大学学报（医学版）, 2018, 47(2): 181-186.

DANG Ying,LIN Yuliang,SUN Hongjun,SUN Jianjun,LI Changdong,LI Zhiyun. Isoliquiritigenin can inhibit migration and invasion of human glioma stem cells by down-regulating matrix metalloproteinases. J Zhejiang Univ (Med Sci), 2018, 47(2): 181-186.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.04.12    或    http://www.zjujournals.com/med/CN/Y2018/V47/I2/181

基因 引物序列(5′→3′)
MMP-2 正向：GAGTTGGCAGTGCAATACCT
反向：CCAAAGAACTTCTGCATCTTCT
MMP-9 正向：TCCAAGGCCAATCCTACT
反向：CGTCGAGTCAGCTCGGGT
GAPDH 正向：TGGGTGTGAACCACGAGAA
反向：GGCATGGACTGTGGTCATGA
表 1  实时定量RT-PCR引物序列
图 1  荧光显微镜下观察SHG44胶质瘤干细胞球中特异性标志物CD133和Nestin的表达
图 2  异甘草素对SHG44人脑胶质瘤干细胞迁移能力的影响
图 3  异甘草素对SHG44人脑胶质瘤干细胞侵袭能力的影响
图 4  各组MMP-2和MMP-9 mRNA表达量比较
图 5  各组MMP-2和MMP-9蛋白表达及比较
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