CAJ | 학술논문

目的建立并鉴定表达人乳头瘤病毒16型(HPV 16)E7蛋白的实体瘤模型.方法携带有野生型HPV 16 E7基因的重组质粒pcDNA3.1-E7用限制性内切酶及序列测定进行鉴定.采用脂质体将重组质粒pcDNA3.1-E7转染小鼠淋巴瘤细胞RMA,经G418筛选获得稳定转染细胞单克隆,并用RT-PCR方法检测稳定转染细胞HPV 16 E7 mRNA.将转染细胞接种于C57BL/6小鼠,肿瘤形成后,用Western blot分析检测E7蛋白在小鼠肿瘤组织中的表达.结果酶切及测序证实目的基因DNA序列、插入位点及方向均正确.RT-PCR检测显示HPV 16 E7能在RMA转染细胞中稳定表达.RMA转染细胞可在小鼠体内成瘤,且Western blot分析表明E7蛋白在小鼠肿瘤组织中的表达较体外高.结论 2.5 X 104 个RMA-E7细胞可在小鼠体内成瘤,并且体内E7蛋白表达比体外高,可能导致免疫耐受.
목적 건립병감정표체인유두류병독16형(HPV 16)E7단백적실체류모형.방법 휴대유야생형HPV 16 E7기인적중조질립pcDNA3.1-E7용한제성내절매급서렬측정진행감정.채용지질체장중조질립pcDNA3.1-E7전염소서림파류세포RMA,경G418사선획득은정전염세포단극륭,병용RT-PCR방법검측은정전염세포HPV 16 E7 mRNA.장전염세포접충우C57BL/6소서,종류형성후,용Western blot분석검측E7단백재소서종류조직중적표체.결과 매절급측서증실목적 기인DNA서렬、삽입위점급방향균정학.RT-PCR검측현시HPV 16 E7능재RMA전염세포중은정표체.RMA전염세포가재소서체내성류,차Western blot분석표명E7단백재소서종류조직중적표체교체외고.결론 2.5 X 104 개RMA-E7세포가재소서체내성류,병차체내E7단백표체비체외고,가능도치면역내수.
Objective To construct and identify solid tumor model expressing human papilloma virus type 16 E7.Methods The recombination vector pcDNA3.1-E7 carrying wild type HPV 16 E7 was identified by restriction and sequencing.The recombination vector pcDNA3.1-E7 was transfected into mouse lymphadenoma cell RMA by liposome and the monoclone cells transfected stably were obtained by antibiotics G418 sieving.RT-PCR in vitro was used to detect the HPV 16 E7 mRNA of RMA-E7.RMA-E7 cells were subcutaneously inoculated to syngeneic mice,and the expression of E7 protein in tumor tissue of mice was detected by Western blot after tumor formation.Results Identification of recombination vector by restriction and sequencing showed the length,inserted location and direction of the target gene which was inserted into the recombinant were correct,and the expression of HPV 16 E7 was detected.RMA-E7 could form tumor in syngeneic mice,and E7 protein was expressed higher in vivo than in vitro.Conclusion 2.5×104 RMA-E7 cells could form tumor in syngeneic mice.E7 protein level was much higher in in vivo than in in vitro cultured RMA-E7 cells,which might cause immune tolerance.