摘 要 目的: 研究重组腺病毒介导的野生型PTEN基因转染对群司珠单抗(traxtuzumab)耐药性乳腺癌细胞的逆转作用。方法: 构建携带野生型PTEN基因的重组腺病毒AdPTEN，感染群司珠单抗耐药性乳腺癌细胞株BT474， MTT比色法和FCM检测AdPTEN感染对群司珠单抗作用下BT474细胞增殖和凋亡的影响； DNA片段化实验分析BT474凋亡与感染时间的关系； Western blotting法检测AdPTEN感染对BT474细胞中丝氨酸/苏氨酸激酶（Akt）磷酸化水平的影响。建立荷乳腺癌裸鼠模型，观察AdPTEN联合群司珠单抗治疗对乳腺癌移植瘤生长的影响，检测移植瘤细胞中PTEN蛋白的表达、细胞凋亡、超微结构的改变。结果: 成功构建重组腺病毒AdPTEN，其滴度为4.2×10 11T CID50/ml。PCR、RT-PCR和Western blotting法证实PTEN基因可被转导入BT474细胞内并稳定高效表达。AdPTEN联合群司珠单抗处理对BT474细胞增殖的抑制非常显著地强于单用群司珠单抗治疗（P<0.01）；细胞出现明显凋亡，凋亡率为（20.7±5.83）%，并且出现G1期阻滞和S期明显减少,与AdLacZ组和对照组相比差异均有统计学意义(P<0.01）；感染组细胞DNA出现典型的梯形条带, 以感染后24~36 h最为明显； AdPTEN感染明显下调BT474细胞内Akt的磷酸化水平。AdPTEN和群司珠单抗联合治疗可强烈抑制裸鼠移植瘤生长，疗效明显强于群司珠单抗治疗组(P<0.01）；AdPTEN治疗后，移植瘤细胞中PTEN表达明显，TUNEL和电镜检测均证实乳腺癌移植瘤细胞的凋亡。结论: AdPTEN治疗可抑制BT474细胞Akt 的磷酸化，阻断PI3K/Akt信号通路的持续活化，恢复耐药细胞对群司珠单抗的敏感性，引起细胞凋亡。

Abstract Objective: To study the role of adenoviralmediated PTEN gene transfection in reversing drug resistance of trastuzumabresistant human breast cancer. Methods: Recombinant adenoviruses carrying PTEN gene (AdPTEN) were constructed and transfected into trastuzumabresistant human breast cancer cell line BT474. Proliferation and apoptosis of BT474 cells treated with AdPTEN and trastuzumab were measured by MTT assay and FCM. Time course of BT474 cells apoptosis induced by AdPTEN was analyzed by detection of DNA fragmentation. Western blotting was employed to measure phosphorylatedAkt expression levels in AdPTEN treated BT474 cells. The nude mice model of BT474 cells was employed to observe the effects of AdPTEN and trastuzumab on trastuzumabresistant human breast cancer in vivo. The expression of PTEN gene, cancer cells apoptosis and ultrastructural changes were evaluated after the mice were sacrificed. Results: We successfully constructed recombinant AdPTEN; and the titer of the recombinant adenovirus was about 4.2×1011TCID50/ml. PTEN gene expression was identified by PCR, RTPCR and Western blotting assay. Combinatorial AdPTEN and trastuzumab significantly suppressed the proliferation of BT474 cells and induced the apoptosis. The percentage of apopotosis cells treated with AdPTEN was （20.7±5.82）% , companied by cells cycle arrest in G1 phrase (P＜0.01）. DNA of AdPTEN treated BT474 showed a typical DNA ladder at 24-36 h after infection. An obvious downregulation of phosphorylatedAkt expression level in cancer cells was identified by Western blotting. Tumor growth was inhibited in nude mice receiving injection of the recombinant adenoviruses when compared to control mice treated with AdLacZ (P＜0.01）. PTEN protein expression was confirmed by immunohistochemistry. Tumor cell apopotosis was detected by TUNEL and electron microscopy scanning. Conclusion: Our result suggests that PTEN protein can downregulate phosphorylatedAkt and Akt kinase activity, and block the activation of PI3K/Akt pathway, and subsequently reverse trastuzumab resistance and induce cell apoptosis.

上海市科学技术委员会资助项目(No.06DZ19509)