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

采用分子克隆技术构建含卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma—associated herpesvirus，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。

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

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.