小鼠PRL-3表达载体的构建及对乳腺癌D2F2细胞小鼠移植瘤的治疗作用  

Construction of mouse PRL-3 expression vector and its therapeutic effects on breast cancer D2F2 cell xenografts in mice

作　　者：曹燕飞[1,2] 吕娟[1,3] 黄昊[1] 寿成超[1] 

机构地区：[1]北京大学肿瘤医院,北京市肿瘤防治研究所,生物化学与分子生物学研究室,恶性肿瘤发病机制及转化研究教育部重点实验室 [2]长治医学院生物化学教研室,北京100142 [3]首都医科大学附属北京朝阳医院检验科,山西长治046000

出　　处：《中国肿瘤生物治疗杂志》2013年第3期266-271,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家重点基础研究发展计划(973计划)资助项目(No.2009CB521805)~~

摘　　要：目的:探讨靶向小鼠肝细胞再生磷酸酶-3(mouse phosphatase of regenerating liver-3,mPRL-3)的DNA疫苗对小鼠乳腺癌D2F2细胞体内生长的抑制作用。方法:构建靶向mPRL-3的真核表达载体pVAX1-mPRL-3,并转染至鹌鹑肌成纤维细胞QM7内,Western blotting检测mPRL-3蛋白在QM7细胞中的表达;通过重组慢病毒Lv-mPRL-3或对照载体Lv-Ctrl感染小鼠乳癌D2F2细胞,分别建立高表达mPRL-3的mPRL-3-D2F2细胞或对照NC-D2F2细胞,Western blotting检测小鼠乳腺癌D2F2细胞中mPRL-3蛋白的表达。BALB/c小鼠左侧乳腺脂肪垫下分别接种mPRL-3-D2F2和NC-D2F2细胞后,通过基因枪接种pVAX1-mPRL-3疫苗(mPRL-3-D2F2/pVAX1-mPRL-3),同时设立疫苗对照组(mPRL-3-D2F2/pVAX1-Ctrl)和细胞对照组(NC-D2F2/pVAX1-mPRL-3),检测小鼠的肿瘤体积及生存期。结果:pVAX1-mPRL-3质粒经酶切鉴定及测序验证均正确,并能在QM7细胞中表达。Western blotting检测结果显示,慢病毒感染的mPRL-3-D2F2细胞中mPRL-3蛋白过表达,而NC-D2F2细胞中不表达mPRL-3蛋白。荷瘤小鼠经pVAX1-mPRL-3 DNA疫苗免疫,其肿瘤体积明显低于对照组[(835.3±509.8)vs(1 543.0±578.4)mm3,P<0.01],且pVAX1-mPRL-3疫苗能显著延长荷瘤小鼠的生存期(中位生存期55.5 vs 38 d,P<0.05)。结论:基因枪接种的靶向mPRL-3的DNA疫苗能抑制高表达mPRL-3的小鼠乳腺癌D2F2细胞移植瘤的生长,并延长荷瘤小鼠生存期,提示其对mPRL-3阳性肿瘤有潜在的治疗作用。Objective：To explore the inhibitory effect of mouse phosphatase of regenerating liver-3（mPRL-3）-targeted DNA vaccine on the growth of mouse breast cancer D2F2 cells in vivo.Methods： The eukaryotic expression vector pVAX1-mPRL-3 targeting mPRL-3 was constructed and transfected into quail fibroblasts QM7 cells.The mPRL-3 protein expression in QM7 cells was detected by Western blotting.D2F2 mouse breast cancer cells were infected with recombinant lentivirus（Lv-mPRL-3） or control vector（Lv-Ctrl） to generate cells expressing mPRL-3（mPRL-3-D2F2） or control cells（NC-D2F2）,and the expression of mPRL-3 protein in mouse breast cancer D2F2 cells was analyzed by Western blotting.The mPRL-3-D2F2 and NC-D2F2 cells were respectively inoculated into BALB/c mice＇s left mammary fad pat,then the BALB/c mice were immunized with pVAX1-mPRL-3 DNA vaccine（mPRL-3-D2F2/pVAX1-mPRL-3） by gene gun,and mPRL-3-D2F2/pVAX1-Ctrl and NC-D2F2/pVAX1-mPRL-3 were set as controls.The volume of tumor and the survival time of mice were monitored.Results： The eukaryotic expression vector pVAX1-mPRL-3 was identified by enzymatic digestion and DNA sequencing and the expression of mPRL-3 protein in QM7 cells was also confirmed.Western blotting assay results showed that the over-expression of mPRL-3 protein was detected in mPRL-3-D2F2,but not in NC-D2F2 cells.The tumor volume of the tumor-bearing mice immunized with pVAX1-mPRL-3 vaccine was significantly lower than that of the control group（[835.3±509.8] vs [1543.0±578.4] mm3,P0.01].The pVAX1-mPRL-3 vaccination could significantly prolong the survival time of the tumor bearing mice（median survival time： 55.5 vs 38 d,P0.05）.Conclusion： PRL-3-targeted vaccination mediated by gene gun can inhibit the growth of mouse breast cancer D2F2 cell xenografts expressing mPRL-3,which could be a potential therapy strategy for PRL-3 positive tumor.

关 键 词：小鼠肝细胞再生磷酸酶-3 小鼠 乳腺癌 D2F2细胞 DNA疫苗 基因枪 

分 类 号：R737.9[医药卫生—肿瘤] R730.54[医药卫生—临床医学]