超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

doi:10.13481/j.1671-587x.20150105

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (01): 21-26.doi: 10.13481/j.1671-587x.20150105

超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

王子航^1,2, 张宇虹², 夏稻子², 礼广森², 武俊², 由悦², 黄冬梅², 薄华颖², 胡滨², 毛鑫²

1. 大连医科大学研究生院, 辽宁大连 116044;
2. 大连医科大学附属第二医院超声科, 辽宁大连 116023

收稿日期:2014-07-30 发布日期:2015-01-30
通讯作者: 张宇虹,教授,硕士研究生导师(Tel:0411-84671291,E-mail:zhangyh_66@163.com) E-mail:zhangyh_66@163.com
作者简介:王子航(1987-),男,黑龙江省牡丹江市人,在读医学硕士,主要从事超声分子影像学方面的研究。
基金资助:
国家自然科学基金资助课题(81250018,81371582);辽宁省科技厅科技计划项目资助课题(2012225021)

Inhibition of expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion mediated by ultrasound-targeted microbubble destruction

WANG Zihang^1,2, ZHANG Yuhong², XIA Daozi², LI Guangsen², WU Jun², YOU Yue², HUANG Dongmei², BO Huaying², HU Bin², MAO Xin²

1. Graduate School, Dalian Medical University, Dalian 116044, China;
2. Department of Dignostic Ultrasound, Second Affiliated Hospital, Dalian Medical University, Dalian 116023, China

Received:2014-07-30 Published:2015-01-30

摘要/Abstract

摘要：

目的: 探讨应用超声靶向破坏微泡 (UTMD) 技术介导小鼠肝癌细胞株JNK1基因的表达、细胞迁移和侵袭抑制的作用,阐明其作用机制。方法: 构建并筛选RNA干扰效果最好的短发夹RNA(shRNA)。将小鼠肝癌细胞株Hca-F分为正常Hca-F细胞组、shRNA质粒组、脂质体组、超声微泡结合超声辐照组及脂质体结合超声微泡加超声辐照组。采用倒置荧光显微镜观察各组细胞转染率,荧光定量PCR和Western blotting 法检测JNK1基因mRNA和蛋白表达水平,CCK-8法检测各组细胞的细胞活性,应用Transwell 实验检测各组细胞的体外迁移能力。结果: 脂质体结合超声微泡加超声辐照组细胞转染率高于shRNA质粒组、脂质体组和超声微泡结合超声辐照组(均P<0.05),脂质体组和超声微泡结合超声辐照组比较差异无统计学意义(P>0.05)。脂质体结合超声微泡加超声辐照组JNK1 mRNA和蛋白表达水平低于其他各组(P<0.05);脂质体结合超声微泡加超声辐照组细胞活性和平均穿膜细胞数均低于其他各组(P<0.05)。结论: UTMD技术结合脂质体转染法可以提高小鼠肝癌细胞株JNK1 shRNA的转染效率,增强其对基因表达、细胞活力、迁移和侵袭能力的抑制。

关键词: 超声, 微泡, 肝肿瘤, 实验性, c-Jun N端激酶, 短发夹RNA, 转染, 淋巴转移

Abstract:

Objective To explore the inhibition of the expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion mediated by ultrasound-targeted microbubble destruction (UTMD), and to clarify its mechanism. Methods The best short hairpin RNA(shRNA) vector was established and screened.The hepatocellular cell line Hca-F were cultured in vitro and divided into normal Hca-F cells group, shRNA plasmid group, and lipofection group, ultrasound microbubbles combined with ultrasound irradiation group, lipofection combined with ultrasound microbubbles and ultrasound irradiation group.The transfection rates were observed by inverted flurescence microscope.The expression levels of JNK1 mRNA and protein were detected by fluorescence quantitative PCR and Western blotting method.The activities of the cells in varions groups were detected by CCK-8 method.The abilities of migration in vitro of the cells in various groups were detected by Transwell assay. Results The transfection rate of the cells in lipofection combined with ultrasound microbubbles and ultrasound irradiation group was higher than those in shRNA plasmid group, lipofection group and ultrasound microbubbles combined with ultrasound irradiation group (all P<0.05);there was no signifant difference between lipofection group and ultrasound microbubbles combined with ultrasound irradiation group (P>0.05).The expression levels of JNK1 mRNA and protein in lipofection combined with ultrasound microbubbles and ultrasound irradiation group were lower than those in other groups (all P<0.05). The cell activity and the average amount of the cells passed the membrane in lipofection combined with ultrasound microbubbles and ultrasound irradiation group were lower than those in other groups (all P<0.05). Conclusion Combination of lipofection and UTMD in mouse hepatocellular cell lines can improve the transfection rate of JNK1 shRNA, therefore enhance the inhibitory effects on gene expression, the cell activity, migration and invasion.

Key words: ultrasound, microbubble, liver neoplasms,experimental, c-Jun N-terminal kinase, short hairpin RNA, transfection, lympatic metastasis

中图分类号:

R735.7

王子航, 张宇虹, 夏稻子, 礼广森, 武俊, 由悦, 黄冬梅, 薄华颖, 胡滨, 毛鑫. 超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制[J]. 吉林大学学报(医学版), 2015, 41(01): 21-26.

WANG Zihang, ZHANG Yuhong, XIA Daozi, LI Guangsen, WU Jun, YOU Yue, HUANG Dongmei, BO Huaying, HU Bin, MAO Xin. Inhibition of expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion mediated by ultrasound-targeted microbubble destruction[J]. Journal of Jilin University Medicine Edition, 2015, 41(01): 21-26.

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超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

Inhibition of expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion mediated by ultrasound-targeted microbubble destruction

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