CAJ | 학술논문

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B16RHSV,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-T Easy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GG418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B16RHSV.用表达绿色荧光蛋白(GFP)的oHSV( oHSVGFP)与B16RHSV细胞体外共培养,观察细胞病变效应和GFP的表达情况.分别以相同剂最的B16-F10和B16RHSV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B16RHSV细胞表达绿色荧光并出现细胞病变效应,证明B16RHSV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.B16RHSV和B16-F10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oHSVGFP治疗两种细胞的荷瘤鼠,仅在B16RHSV细胞荷瘤的小鼠体内显示出明显的溶瘤效应.结论构建的B16RHSV是对oHSV易感、保持体内成瘤性的小鼠黑色素瘤细胞系.
목적 구건용류성단순포진병독( oHSV)역감적C57/BL소서흑색소류세포계B16RHSV,동시잉보류기재소서체내유도종류생장적능력.방법 이인흑색소류세포계A375적기인조DNA위모판,채용취합매련반응(PCR)확증출포진병독침입개체(HVEM),장확증산물극륭도pGEM-T Easy재체중병측서.장측서정학적HVEM기인극륭입pcDNA3질립재체,산생pcDNA3-HVEM.이pcDNA3-HVEM질립재체전염C57/BL소서흑색소류세포계B16-F10,사용함유GG418적완전배양기사선가능적양성극륭,병통과면역자주사선출표체HVEM적세포,명명위B16RHSV.용표체록색형광단백(GFP)적oHSV( oHSVGFP)여B16RHSV세포체외공배양,관찰세포병변효응화GFP적표체정황.분별이상동제최적B16-F10화B16RHSV세포접충우C57/BL소서우측배부피하,관찰저량충세포유도소서체내형성종류적능력.결과 재형광현미경하가관찰도B16RHSV세포표체록색형광병출현세포병변효응,증명B16RHSV세포능구피oHSV감염병재세포내번식,이기친대세포B16-F10칙불능.B16RHSV화B16-F10세포접충우소서5d후,량조소서균가관찰도종류생장,차종류생장정황기본일치.용oHSVGFP치료량충세포적하류서,부재B16RHSV세포하류적소서체내현시출명현적용류효응.결론 구건적B16RHSV시대oHSV역감、보지체내성류성적소서흑색소류세포계.
Objective To generate an oncolytic herpes simplex virus (oHSV) permissive mouse melanoma cell line B16RHSV,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 B16RHSV,was generated.The permissibility of B16RHSV cells to oHSV infection was examined with green fluorescence protein (GFP)-expressing oHSV (oHSVGFP ). To investigate the tumorigenic ability of both cells in vivo,2 × l05 cells in 100 pl were subcutaneously inoculated into the right flanks of CS7/BL mice.Results In vitro,the B16RHSV mouse melanoma cells were shown by fluorescence microscopy capable of being infected by oHSVGFP. In vivo,the B16RHSV 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.