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摘要:
目的 观察Notch1蛋白的表达与乳腺癌患者对紫杉醇（paclitaxel，PAC）敏感性的相关性。 方法 分别利用小RNA干扰技术和Notch1胞内片段抑制剂N-[N-（3，5-difluorophenacetyl）-1-alanyl]-S-phenylglycine t-buty lester（DAPT）降低乳腺癌细胞系MDA-MB-231细胞中Notch1的活性，并分别用Western blot和RT-PCR检测两种方法的抑制效果。MTT法检测经过不同浓度紫杉醇（0、1、5、10、15、20、25 μg/mL）处理24 h后，观察对照组和实验组细胞的存活情况。通过肿瘤原代细胞胶原凝胶体包埋化疗药物敏感性检测（collagen gel droplet embedded culture-drug sensitivity test，CD-DST）观察临床病例对紫杉醇的敏感性，并用免疫组织化学方法观察紫杉醇敏感病例和非敏感病例中Notch1的表达情况。 结果 MDA-MB-231细胞对紫杉醇的敏感性增强，且与对照组之间的差异具有统计学意义（P < 0.05），免疫组化的结果显示紫杉醇敏感病例Notch1经过抑制Notch1活性处理的蛋白的表达低于不敏感病例，差异有统计学意义（P < 0.01）。 结论 Notch1显著影响乳腺癌患者对紫杉醇的敏感性，Notch1表达与乳腺癌紫杉醇敏感性呈负相关。 Abstract:Objective To observe the association between Notch1 expression and PAC sensitivity in breast cancer cells. Methods The siRNA interference against Notch1 and endocellular Notch1 inhibitor, N-[N-(3, 5-difluorophenacetyl)-1-alanyl] -S-phenylglycine t-butyl lester (DAPT) were employed to down-regulate Notch1 activity and the inhibitory action was detected through Western blot and RT-PCR, respectively. After 24 hours of exposure to PAC at different concentrations (0, 1, 5, 10, 15, 20, 25 μg/mL), the cell viability of the control group and the experimental group was tested using the MTT colorimetric method, i.e., [3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide]). Immunohistochemistry (IHC) was used to observe the Notch1 expression in PAC-sensitive and non-sensitive cases, and collagen gel droplet embedded culture-drug sensitivity test (CD-DST) was carried out to determine the sensitivity of different cases to the PAC. Results Down-regulation of Notch1 expression by Notch1 siRNA interference or inhibition of Notch1 activity by DAPT significantly increased the sensitivity of MDA-MB-231 cells to PAC (P < 0.05). In the IHC study, the Notch1 expression in PAC-sensitive cases was much lower than in non-sensitive cases (P < 0.01). Conclusion The Notch1 expression significantly influences and negatively correlates with the sensitivity of breast cancer to PAC. -
Key words:
- Notch1 /
- Breast cancer /
- Paclitaxel (PAC) /
- Sensitivity
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图 1 抑制Notch1后乳腺癌细胞系MDA-MB-231不同克隆中Notch1表达情况
A：未处理组MDA-MB-231、空白对照组scr/MDA-MB-231及各低表达克隆37、38、40、41中Notch1表达情况；B：图A各细胞克隆中Notch1表达的相对灰度图
Figure 1. Notch1 expression in MDA-MB-231 cell clonings with downregulation of Notch1
图 2 siRNA抑制Notch1后MDA-MB-231细胞对PAC敏感度的变化（**：P < 0.01）
Figure 2. Changes in sensitivity to PAC of MDA-MB-231 cells after Notch1-targeted siRNA interference of the plasmids(**: P < 0.01)
图 3 DAPT处理后Notch1活性降低，增加MDA-MB-231细胞对紫杉醇的敏感性
A：不同浓度DAPT对MDA-MB-231细胞存活能力的影响；B：MDA-MB-231经DAPT（0、10、15、20 μM）处理24 h后，显著降低Notch1靶基因HES1的表达；C：MDA-MB-231细胞经DAPT（0、20 μ M）处理后，对PAC敏感性显著增强（*：P<0.05，**：P<0.01）。
Figure 3. DAPT reduced Notch1 activity and increased the sensitivity of MDA-MB-231 cells to PAC
图 4 免疫组化检测Notch1在乳腺癌组织中的表达，
A：（-）；B：（+）；C：（++）；D：（+++）；E：对免疫组化结果的统计，乳腺癌紫杉醇敏感组与非敏感组之间Notch1表达具有显著性差异（P < 0.01）
Figure 4. Immunohistochemical staining of Notch1 in breast cancer tissue
表 1 乳腺癌细胞Notch1表达与其对紫杉醇敏感性的相关性分析
Table 1. The correlation between Notch1 expression and PAC sensitivity
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