构建单纯疱疹病毒易感的鼠黑色素瘤细胞系B16R^HSV  

Generation of a herpes simplex virus-permissive mouse melanoma cell fine B16R^HSV

作　　者：庄秀芬[1] 周爱萍[2] 施桂兰[1] 韩香萍[3] 李洁[1] 张郁[1] 张友会[1] 张叔人[1] 刘滨磊[1] 

机构地区：[1]中国医学科学院北京协和医学院肿瘤医院肿瘤研究所免疫室,100021 [2]中国医学科学院北京协和医学院肿瘤医院内科,100021 [3]南开大学生命科学学院

出　　处：《中华肿瘤杂志》2012年第3期187-191,共5页Chinese Journal of Oncology

摘　　要：目的构建溶瘤性单纯疱疹病毒（oHSV）易感的C57／BL小鼠黑色素瘤细胞系B16R^HSV，同时仍保留其在小鼠体内诱导肿瘤生长的能力。方法以人黑色素瘤细胞系A375的基因组DNA为模板，采用聚合酶链反应（PCR）扩增出疱疹病毒侵入介体（HVEM），将扩增产物克隆到pGEM—TEasy载体中并测序。将测序正确的HVEM基因克隆入pcDNA3质粒载体，产生pcDNA3-HVEM。以pcDNA3-HVEM质粒载体转染C57／BL小鼠黑色素瘤细胞系B16-F10，使用含有G418的完全培养基筛选可能的阳性克隆，并通过免疫磁珠筛选出表达HVEM的细胞，命名为B16R^HSV。用表达绿色荧光蛋白（GFP）的oHSV（oHSV^GFP）与B16R^HSV细胞体外共培养，观察细胞病变效应和GFP的表达情况。分别以相同剂量的B16-F10和B16R^HSV细胞接种于C57／BL小鼠右侧背部皮下，观察这两种细胞诱导小鼠体内形成肿瘤的能力。结果在荧光显微镜下可观察到B16R^HSV细胞表达绿色荧光并出现细胞病变效应，证明B16R””细胞能够被oHSV感染并在细胞内繁殖，而其亲代细胞B16-F10则不能。B16R^HSV和B16-FI0细胞接种于小鼠5d后，两组小鼠均可观察到肿瘤生长，且肿瘤生长情况基本一致。用oHSV^GFP治疗两种细胞的荷瘤鼠，仅在B16R^HSV细胞荷瘤的小鼠体内显示出明显的溶瘤效应。结论构建的B16R^HSV是对oHSV易感、保持体内成瘤性的小鼠黑色素瘤细胞系。Objective To generate an oncolytic herpes simplex virus （oHSV） permissive mouse melanoma cell line B16R^HSV, preserving the tumorigenic ability in syngeneic mice. Methods The herpes simplex virus entry mediator （HVEM） gene was amplified by PCR from human melanoma cell line A375, and cloned into pGEM-T Easy vector for sequencing. The HVEM gene was then cloned into pcDNA3 vector to generate pcDNA3-HVEM for transfection of mouse melanoma cell line B16-F10 cells. After that, the putative transfected cells were selected in full growth medium containing G418. The HVEM-expressing cells were isolated by immunomagnetic bead separation. The mouse melanoma cell line expressing oHSV receptor- HVEM, designated as B16R^HSV, was generated. The permissibility of B16R^HSV cells to oHSV infection was examined with green fluorescence protein （GFP）-expressing oHSV （oHSV^GFP ）. To investigate the tumorigenic ability of both cells in vivo, 2 ×10^5 cells in 100μl were subcutaneously inoculated into the right flanks of C57/BL mice. Results In vitro, the B16R^HSV mouse melanoma ceils were shown by fluorescence microscopy capable of being infected by oHSV^GFP. In vivo, the B16R^HSV cells, like their wild type counterpart, grew to form melanoma in syngeneic mice. Conclusion A herpes simplex virus-permissive mouse melanoma cell line was established. Its tumorigenicity remained unchanged.

关 键 词：单纯疱疹病毒属 黑色素瘤 疱疹病毒侵入介体 小鼠 

分 类 号：R73[医药卫生—肿瘤]