HSV1-TK-medicated apoptosis and cell killing effect on human epidermoid carcinoma MEC-1 cells / 实用口腔医学杂志

ABSTRACT

Objective:To estimate the effect of HSV1-TK/GCV suicide gene therapy on human salivary gland epidermoid carcinoma cell line MEC-1. Methods: Expression vector G 1NAtK containing HSV-TK cDNA was transfected into MEC-1 cells.After transfection, the cells were selected by G418 for two weeks. The integrated gene and mRNA were detected with PCR and RT-PCR. The cytotoxicity and bystander effect were estimated by MTT and typan blue exclusion assay. The morphological changes after GCV treatment were observed with HE and 33258 stain and in situ cell apoptosis detection kit. Results: The 404 bp DNA fragment was amplified through PCR and RT-PCR in the transfected cells respectively. TK positive clone was named MEC-1/TK. The sensitivity of MEC-1/TK to GCV was 1 000 times more than that of parent MEC-1 cells.More than 90% of MEC-1 cells were killed by 10 ?g/ml of GCV when only 10% of MEC-/TK cells were present. The morphological changes included shrinking,detaching and floating of the cells. Some of the cells showed nucleus condensation and breakage of nucleus. A lot of cells showed nucleus positive in in situ apoptosis detection. Conclusion: HSV1-TK/GCV can confer MEC-1/TK cell killing efficiently. MEC-1/TK also has strong bystander effects. HSV1-TK/GCV system confers its effect, in part, by inducing apoptosis in TK positive cells.