逆转录病毒载体介导的CD自杀基因对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞的杀伤作用

发布时间：2018-05-27 12:19

本文选题：卡波济肉瘤相关疱疹病毒(KSHV) + 肿瘤转化基因K12　；参考：《南京医科大学》2005年硕士论文

【摘要】：本研究旨在探索胞嘧啶脱氨酶(cytosine deaminase,CD)自杀基因联合5-氟胞嘧啶(5-fluorocytosine,5-FC)(cD/5-FC系统)对卡波济肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus,KsHV)肿瘤转化基因K12诱导NIH3T3转化细胞的杀伤作用。首先分别构建含KSHV K12基因和CD自杀基因Fcy∶∶Fur的重组逆转录病毒表达质粒LZRS-K12和pLXSN-FF,并将重组质粒LZRS-K12单独或与pLXSN-FF一起稳定转染MH3T3细胞,获得稳定表达卡波济蛋白(kaposin)的N/K12和N/K12/FF细胞。然后通过普通倒置显微镜、透射电子显微镜观察和软琼脂集落形成实验以及裸鼠成瘤实验验证K12基因能够体外诱导细胞转化、体内诱导肿瘤的形成。结果显示,K12基因转染后的NIH3T3细胞与对照细胞相比其生长特性和形态结构均发生明显改变,表现为失去生长接触抑制而呈多层生长、出现异型核及生长依赖性降低而能够在软琼脂培养基上形成细胞集落。动物实验结果表明,K12转化的NIH3T3细胞接种至裸鼠颈背部皮下2～3w后均可在接种部位形成肿瘤。瘤组织经石蜡切片后HE染色,镜下可见血管丰富,瘤细胞呈梭形,排列紊乱,核分裂旺盛,伴有异型核和病理性核分裂。5-FC使用后,采用MTT法、流式细胞仪和免疫组化(IHC)染色等方法分别检测细胞存活率、凋亡率及凋亡相关蛋白的表达。结果显示,随着5-FC作用浓度的提高,N/K12/FF细胞存活率明显下降。流式细胞仪
[Abstract]:The aim of this study was to investigate the killing effect of cytosine deaminase (CDCD) suicide gene combined with 5-fluorocytosine 5-fluorocytosine (5-fluorocytosine 5-FC system) on K12 induced NIH3T3 transformation cells induced by Kaposiella sarcoma-associated herpesvirusKsHV. Firstly, the recombinant retroviral expression plasmids LZRS-K12 and pLXSN-FFF containing KSHV K12 gene and CD suicide gene Fcy::Fur were constructed, and the recombinant LZRS-K12 was stably transfected into MH3T3 cells either alone or together with pLXSN-FF to obtain N/K12 and N/K12/FF cells stably expressing kaposin. Then the normal inverted microscope, transmission electron microscope, soft Agar colony formation test and nude mice tumorigenesis experiment proved that K12 gene can induce cell transformation in vitro and tumor formation in vivo. The results showed that the growth characteristics and morphological structure of the NIH3T3 cells transfected with K12 gene were obviously changed compared with the control cells. The abnormal nuclei and growth dependence were decreased, and cell colonies could be formed on soft Agar medium. The results of animal experiments showed that the transformed NIH3T3 cells could form tumor at the inoculation site after inoculation to the subcutaneous of the neck and back of nude mice for 3 weeks. The tumor tissue was stained with HE after paraffin section. The tumor cells were fusiform, disordered, mitotic and accompanied with abnormal nucleus and pathological mitosis. MTT method was used. The survival rate, apoptosis rate and the expression of apoptosis-related protein were detected by flow cytometry and immunohistochemical staining. The results showed that the survival rate of N- / K12 / FF cells decreased with the increase of 5-FC concentration. Flow cytometry
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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