CD/5-FC对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞的杀伤作用  

The Killing Effect of CD/5-FC Administration on NIH3T3 Cell Transformed with KSHV Tumor-transforming Gene K12

作　　者：唐桂霞[1] 卢春[1] 

机构地区：[1]南京医科大学微生物学与免疫学系,南京210029

出　　处：《病毒学报》2005年第3期188-197,共10页Chinese Journal of Virology

基　　金：国家自然科学基金项目(30100160和30271179)

摘　　要：采用分子克隆技术构建含卡波济肉瘤相关疱疹病毒(Kaposi′ssarcoma-associatedherpesvirus,KSHV)转化基因K12和含胞嘧啶脱氨酶(CD)基因重组逆转录病毒表达质粒,分别命名为LZRS-K12和pLXSN-Fcy∷Fur。将LZRS-K12质粒转染NIH3T3细胞系,诱导细胞转化获得具有肿瘤细胞生物学特性的N/K12细胞,进一步将pLXSN-Fcy∷Fur质粒转染N/K12细胞,获得含CD基因的N/K12/FF细胞。加入5-氟胞嘧啶(5-FC)后,采用MTT法、流式细胞仪和免疫组化(IHC)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,N/K12/FF细胞在使用5-FC后存活率明显下降。流式细胞仪检测结果显示,5-FC作用后的N/K12/FF细胞的凋亡率与作用前相比增加了4.6%。与此同时,经5-FC作用后的N/K12和N/K12/FF细胞的生长周期发生了明显变化,均表现为G0-G1期细胞比率降低,S和G2-M期细胞比率增高。5-FC使得N/K12细胞的增殖主要阻滞在G2-M期,而N/K12/FF细胞的增殖主要阻滞在S期。IHC检测结果表明,N/K12细胞在5-FC使用前后Fas和Fas-L表达改变均不明显;而N/K12/FF细胞在5-FC使用后的Fas和Fas-L表达水平均明显高于5-FC使用前的水平。提示,CD/5-FC对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞具有杀伤作用;凋亡有可能介导了部分杀伤过程;Fas和Fas-L有可能部分参与凋亡的诱导。Two recombinant retrovirus expressing plasmids containing tumor-transforming gene K12 of Kaposis sarcoma-associated herpesvirus(KSHV)and cytosine deaminase(CD)gene named LZRS-K12 and pLSXN-FcyFur respectively were constructed with molecular cloning technology.Mouse fibroblast cell line NIH3T3 was firstly transfected with plasmid LZRS-K12 and the transformed cell was named N/K12 cell,implicating the existence of the biological characteristics of tumor cell.Further,the N/K12 cell was transfected with plasmid pLXSN-FcyFur to obtain the stably transfected cell containing CD gene,named N/K12/FF cell.Thiazolyl(MTT)assay,flow cytometric analysis,and immunohistochemical staining(IHC)were used respectively to test the survival rate,the percentage of the apoptotic cells,and the expression of apoptotic-associated protein of N/K12/FF cell after treated with prodrug 5-fluorocytosine(5-FC).The results of MTT assay showed that the survival rate of N/K12/FF cell declined significantly after treated with 5-FC.Flow cytometric analysis indicated that the apoptotic rate of N/K12/FF cell increased 4.6% after being treated with 5-FC as compared with the control.Meanwhile,the growth cycles of the N/K12 and the N/K12/FF cell changed evidently after being treated with 5-FC.Both of which showed that the rate of G0-G1 phase cells decreased,while the rate of S and G2-M phase cells increased.5-FC made the proliferation of N/K12 cell inhibited mainly at the G2-M phase,while N/K12/FF cell inhibited mainly at the S phase.IHC assay indicated that whether N/K12 cell was treated with 5-FC or not,neither Fas nor Fas-L expression changed strikingly;while after N/K12/FF cell being treated with 5-FC,both Fas and Fas-L expression level increased significantly as compared with the control.These results suggested that CD/5-FC may kill the transformed NIH3T3 cell induced by KSHV tumorigenic gene K12;the apoptosis might play a part role in the killing mechanism;Fas and Fas-L might participate partially in the induction of apoptosis.

关 键 词：卡波济肉瘤相关疱疹病毒 肿瘤转化基因K12 CD/5-FC系统 细胞凋亡 卡波济肉瘤 杀伤作用 基因治疗 

分 类 号：R730.26[医药卫生—肿瘤]