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中华乳腺病杂志(电子版) ›› 2011, Vol. 05 ›› Issue (05) : 576 -585. doi: 10.3877/cma.j.issn.1674-0807.2011.05.009

实验研究

壳聚糖纳米粒介导人表皮生长因子受体2小干扰RNA 抑制乳腺癌细胞迁移
张永成1,(), 武晓英2, 陈晓兰1   
  1. 1.510080 广州,广东药学院附属第一医院乳腺科
    2.510006 广州,广东药学院基础学院生物学教研室
  • 收稿日期:2010-04-13 出版日期:2011-10-01
  • 通信作者: 张永成
  • 基金资助:
    广东省科技厅科研基金资助项目(2008B030301033)

Inhibitory effect of chitosan nanoparticles-mediated HER-2 siRNA on migration of breast cancer cell line SK-BR-3

Yong-cheng ZHANG,1(), Xiao-ying WU1, Xiao-lan CHEN1   

  1. 1.Department of Breast Surgery, First Affiliated Hospital of Guangdong Pharmaceutical University,Guangzhou 510080,China
  • Received:2010-04-13 Published:2011-10-01
  • Corresponding author: Yong-cheng ZHANG
引用本文:

张永成, 武晓英, 陈晓兰. 壳聚糖纳米粒介导人表皮生长因子受体2小干扰RNA 抑制乳腺癌细胞迁移[J/OL]. 中华乳腺病杂志(电子版), 2011, 05(05): 576-585.

Yong-cheng ZHANG, Xiao-ying WU, Xiao-lan CHEN. Inhibitory effect of chitosan nanoparticles-mediated HER-2 siRNA on migration of breast cancer cell line SK-BR-3[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2011, 05(05): 576-585.

目的

以壳聚糖纳米粒作为人类表皮生长因子受体2 小干扰RNA(HER-2 siRNA)的载体转染人乳腺癌细胞SK-BR-3,观察HER-2 沉默对其体外迁移能力的影响。

方法

采用离子凝胶法制备壳聚糖-HER-2 siRNA 纳米粒,原子力显微镜观察纳米粒的形态。分4 组进行转染,分别加入转染试剂壳聚糖-HER-2 siRNA 纳米粒(实验组)、LipofectamineTM2000 和HER-2 siRNA(阳性对照组)、壳聚糖-阴性对照siRNA 纳米粒(阴性对照组)和空白壳聚糖纳米粒(空白组)。一步法逆转录-聚合酶链反应(RT-PCR)和Western blot 鉴定HER-2 在SK-BR-3 细胞中的表达变化。Transwell小室法检测HER-2 siRNA 对细胞体外迁移能力的影响。统计分析采用单因素方差分析、K-W H 秩和检验。

结果

成功制备了壳聚糖-siRNA 纳米粒,呈球形,大小约100 nm。实验组能有效抑制HER-2 mRNA 和蛋白的表达以及SK-BR-3 细胞的体外迁移能力,与阴性对照组和空白组相比差异有显著的统计学意义(P 均=0.000)。

结论

壳聚糖纳米粒介导的HER-2 siRNA 能有效抑制人乳腺癌细胞SK-BR-3 中HER-2 的表达及其体外迁移能力。

Objective

To investigate the effect of chitosan nanoparticles loaded with small interference RNA (siRNA) of HER-2(human epidermal growth factor receptor 2)on in vitro migratory activity of SK-BR-3 cells from a human breast carcinoma cell line.

Methods

ChitosansiRNA nanoparticle targeting HER-2 mRNA was prepared by ionic gelation using tripolyphosphate, and the morphology of the nanoparticles was observed with atomic force microscope.The transfected SK-BR-3 cells were divided into 4 groups: the experimental group, which was transfected with chitosan-HER-2 siRNA nanoparticle, the positive control group, which was transfected with HER-2 siRNA by LipofectamineTM 2000, the negative control group, which was transfected with chitosan-negative control siRNA nanoparticle, and the blank group, which was transfected with blank chitosan-nanoparticle.The expression levels of HER-2 mRNA and protein in SK-BR-3 cells were assessed by RT-PCR (reverse transcription polymerase chain) and Western blot respectively.Transwell Chamber assay was performed to detect the effect of chitosan nanoparticles-mediated HER-2 siRNA on the migratory capacity of SK-BR-3 cells.Statistical analysis was performed using one-way analysis of variance or K-W H test.

Results

The chitosan-siRNA nanoparticles were successfully prepared.The nanoparticles were spherical in shape and well distributed, and the mean diameter was about 100 nm.The expressions of HER-2 mRNA and the protein level were significantly decreased and the migratory ability of SK-BR-3 breast cancer cells in vitro was also obviously decreased in the experimental group compared with the negative control group and the blank group (P=0.000).

Conclusion

HER-2 siRNA transfectd by chitosan nanoparticles can effectively inhibit the expression of HER-2 in SK-BR-3 breast cancer cells and the migration of SK-BR-3 cells in vitro.

图1 壳聚糖-siRNA 纳米粒原子力显微镜照片(×40 000)
表1 RT-PCR 检测SK-BR-3 细胞中HER-2/β-actin mRNA 表达
图2 RT-PCR 检测SK-BR-3 细胞HER-2 mRNA 的表达 a:RT-PCR 结果,M: 100 bp DNA 标记条带,1:实验组,2:阳性对照组,3:阴性对照组,4:空白组,βactin:内参对照;b:图像吸光度分析结果
表2 Western blot 检测SK-BR-3 细胞中HER-2/actin 蛋白的表达
图3 Western blot 检测SK-BR-3 细胞中HER-2 蛋白的表达 a: Western blot 结果,1:实验组,2:阳性对照组,3:阴性对照组,4:空白组;b:图像吸光度分析结果
表3 HER-2 siRNA 对SK-BR-3 细胞迁移的影响
图4 HER-2 siRNA 对SK-BR-3 细胞迁移的影响(光镜,HE 染色 ×400) a: 实验组;b: 阳性对照组 ;c: 阴性对照组;d: 空白组
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