干涉Gankyrin基因表达对小鼠乳腺癌转移的影响  

Effects of knocking down Gankyrin on breast cancer metastasis in mice

作　　者：王少鑫[1] 巩伟丽[1] 韩秋影[1] 满江红[1] 靳宝锋[1] 

机构地区：[1]军事医学科学院国家生物医学分析中心,北京100850

出　　处：《中华医学杂志》2013年第14期1099-1103,共5页National Medical Journal of China

基　　金：国家自然科学基金（31071234）

摘　　要：目的 建立稳定干涉癌基因Gankyrin的4T1-luc细胞株，探讨Gankyrin对小鼠乳腺癌转移的影响。方法采用慢病毒感染和抗性筛选方法获得稳定干涉Gankyrin的乳腺癌细胞株4T1-luc／shGankyrin，通过蛋白质印迹和实时定量PCR方法分别在蛋白质和mRNA水平检测Gankyrin的干涉效果；选用BALB／c小鼠进行4T1细胞原位种植，利用活体成像技术实时观测小鼠体内肿瘤生长和肿瘤转移情况，并对小鼠肺转移瘤进行病理学分析。结果采用免疫印迹和实时定量PCR，检测稳定敲低Gankyrin4T1细胞在蛋白质水平和mRNA水平的表达，均明显低于对照细胞，与对照细胞相比，不同干涉序列的4T1细胞mRNA水平分别为对照细胞的4．9％、25．1％、69．8％。利用活体成像进行细胞数与细胞发光强度的检测，选择细胞数与发光强度基本一致的#2细胞株进行小鼠原位种植，其产生的转移瘤进行实时观测，结果显示敲低Gankyrin的4T1细胞诱导的肺转移瘤荧光强度为3．02×10^6，而对照细胞为10．9×10^6，同时病理学证实了对照细胞产生的肺转移瘤免疫组织化学Gankyrin染色阳性，而敲低Gankyrin的4T1细胞的肺转移瘤免疫组织化学Gankyrin染色阴性。结论采用慢病毒感染的方法可以有效建立稳定敲低Gankyrin表达的细胞株；Gankyrin稳定干涉后对小鼠乳腺癌转移具有明显的抑制作用。Gankyrin有可能成为新的肿瘤治疗靶点。Objective To establish Gankyrin knocking down 4T1-luc cell model and detect the effects of Gankyrin expression on breast cancer metastasis. Methods 4T1-luc cells carryring shGankyrin construct were established by lentivirus infection and antibiotic screening. Western blotting and real-time PCR were used to check the expression levels of Gankyrin. In vivo imaging system was used to monitor the effects of Gankyrin knocked down on cell growth and tumor metastasis after the in situ implantation of Gankyrin knocking down 4T1-luc cells in BALB/c mice. Results The cell expression decreased at the protein and mRNA levels. Gankyrin mRNA expression in different shGankyrin 4T1-luc cells was respectively 4.9% ,25.1% and 69.8% versus the control cells. ShGankyrin#2 4T1-luc cells were chosen for in situ implantation into BAL/c mice because luminescent intensity was consistent with cell numbers. The photon flux of lung metastatic tumor induced by Gankyrin knocking down 4Tl-luc cell was 3.02 × 10^6, while that of lung metastasis induced by control cells was 10. 9 × 10^6. The differences between two groups were significant. In pathology, Gankyrin was detected positive in lung metastasis tumors induced by control group. However, Gankyrin was negative in the Gankyrin knockdown group. Conclusions Lentivirus infection may be effectively used to establish Gankyrin knocking down 4Tl-luc cell model. Because of its involvement in the in vivo pulmonary metastasis of breast cancers, Gankyrin should be a novel target for tumor therapy.

关 键 词：慢病毒感染 肿瘤转移 Gankyrin 

分 类 号：R737.9[医药卫生—肿瘤]