【作者】 邵乐南；

【导师】 赵怡芳；

【作者基本信息】 武汉大学 ， 口腔颌面外科， 2002， 硕士

【摘要】 目的 体外观察腺病毒载体介导的单纯疱疹病毒胸苷激酶基因及羟基无环鸟苷系统(ADV-HSV-Tk／GCV)对人腺样囊性癌细胞(ACC-2及ACC-M)的杀伤作用。方法 分别将人腺样囊性癌细胞细胞株(ACC-2及ACC-M)用腺病毒构建的绿色荧光蛋白按1：10，1：20，1：30转染，在12～36hr内用荧光显微镜观察细胞的转染率。MTT法观察ADV-HSV-TK在不同感染复数MOI(5～60)下以及不同浓度GCV处理时对ACC-2及ACC-M细胞的杀伤作用以及旁观者效应。结果体外研究发现，ACC-2及ACC-M均能够被腺病毒-绿色荧光蛋白转染。最小感染复数为10-20，转染率与感染复数呈正相关。在感染复数为5，GCV为25μg／ml时能够发挥细胞杀伤效应且有剂量依赖性。MTT法检测发现：ACC-2及ACC-M经ADV-HSV-Tk处理后，当GCV为25μg／ml或高于该浓度时，细胞抑制率在38％-66％间；当GCV单独作用时则无明显作用。转染ADV-HSV-Tk的细胞与未转染细胞按不同比例混合作用结果，提示了“旁观者效应”的存在，且转染细胞的比例越高，被杀伤的细胞比例越大。结论 腺病毒-绿色荧光蛋白(Ad-GFP)能够有效地对腺样囊性癌细胞进行体外转染，且有感染复数(MOI)和时间依赖性。腺病毒载体介导的单纯疱疹病毒胸苷激酶基因及羟基无环鸟苷系统(ADV-HSV-Tk／GCV)系统能够在低感染复数的情况下抑制腺样囊性癌细胞，然而，其细胞杀伤效率及“旁观者效应”仍有待提高。更多还原

【Abstract】 Objective To observe the killing effect of Adenovirus vectors encoding herpes simplex virus thymidine kinase gene/ganciclovir(ADV-HSV-Tk/GCV) system on human Adenoid Cystic Carcinoma Cells(ACC-2 and ACC-M) in vitro. Methods Both ACC-2 and ACC-M cell lines were transduced with the ADV-GFP at the proportion of 1:10,1:20,1:30 for 12 to 36 hour. Next, the adenoid cystic carcinoma cell lines which exposed to ADV-HSV-TK at different M.O.I(5 to 60) were treated with ganciclovir (GCV) in different concentration, while the two cell lines were transferred at an M.O.I of 100 and subsequently mixed with non- transferred cells at varying proportion, the cell killing and also the "bystander effect" were detected with MTT assay. Results It was demonstrated that both ACC-2 and ACC-M could be transduced with AD-GFP in vitro. The minimal multiplicity of infection (MOI;for the purpose of this study the number of vector genomes per target cell) was 10 to 20,and the gene transduction efficiency was dependent on multiplicity of infection. The system could present the killing activity to both ACC-2 and ACC-M cells in a dose-dependent manner at lower concentration of MOI(5) and GCV(25ug/ml ).Detection of MTT showed that when they were exposed to ADV-HSV-Tk, the growth of Acc-2 and Acc-M cells were inhibited significantly by GCV when its concentration was equal to or over 25ug/ml, and the rate of inhibition was 38% to 66% respectively, while the same cell lines were not or only slightly inhibited by GCV of any concentration the otherwise. The transferred ADV-HSV-Tk cells mixed with none-transferred cells in different proportion revealed the existence of "bystander effect", the more the transferred cells, the less the living cells existence. Conclusion ACC can be transferred effectively by Ad-GFP in a dose and time dependent manner . The growth of ACC-2 and Acc-M can be inhibited by the ADV-HSV-Tk/GCV system at a low MOI in vitro. However, the system is still to be modified to enhance the cell killing efficiency and the "bystander effect".更多还原