[In vitro killing effect of mutant thymidine kinase mediated by lentiviral vector on T lymphocytes].
OBJECTIVE
To explore the killing effect of the mutant herpes simplex virus thymidine kinase (HSV-sr39tk) and its wild-type (HSV-tk) mediated by lentiviral vector on T lymphocytes in vitro and compare T cell survival rate after GCV or ACV treatment.


METHODS
The three-plasmid lentiviral vector system including packaging plasmid DeltaNRF, envelope plasmid VSV-G and vector plasmid (pTK151 + HSV-sr39tk or pTK151 + HSV-tk) were cotransfected into human embryonic kidney 293T cells using modified calcium phosphate precipitation methods. The packaged virus was harvested 72 h later. The survival of T cells expressing HSV-sr39tk or HSV-tk was measured by MTT assay after 4 day-culture against a gradient of GCV or ACV concentrations.


RESULTS
The three plasmids were effectively cotransfected and a high titre of lentivirus was obtained (2 x 10(6) IU/ml). 39tk(+) T cell survival rates declined promptly when the prodrug GCV/ACV concentrations increased from 0 micromol/L to 10 micromol/L. The T cell survival rates in GCV group declined from (96.04 +/- 3.23)% to (36.76 +/- 4.38)% while in ACV group from (97.31 +/- 4.61)% to (43.75 +/- 8.99)%. However, when GCV/ACV concentrations were more than 10 micromol/L, further decline of 39tk(+) T cell survival rates became unobvious. The growth rate of 39tk(+) T cell exposed to GCV or ACV was obviously lower than that in un-transfected T cells (P < 0.05). Tk(+) T cells were sensitive to GCV (P < 0.05), but not to ACV (P > 0.05). There was a significant difference in killing effects between 39tk(+) T cell + GCV group and tk(+) T cell + GCV group (P < 0.05).


CONCLUSIONS
The lentiviral vectors containing HSV-sr39tk gene could infect T lymphocytes effectively and stably without affecting the proliferation of the transduced cell. In contrast to HSV-tk gene, T cells infected HSV-sr39tk were more sensitive not only to GCV but also to ACV.
Xu
Kai-lin
KL
Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Pan
Xiu-ying
XY
Yang
Yu-juan
YJ
Lu
Qun-xian
QX
Li
Zhen-yu
ZY
He
Xu-peng
XP
D000212:Acyclovir / Q000494:pharmacology; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015774:Ganciclovir / Q000494:pharmacology; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D013937:Thymidine Kinase; D015774:Ganciclovir; D000212:Acyclovir