[Effective and stable in vitro expression of human coagulation factor VIII by retrovirus-based plasmid vector coupled with polyamidoamine dendrimer].
OBJECTIVE
To demonstrate the effectiveness of a retrovirus-based plasmid vector coupled with nanometer material-polyamidoamine (PAMAM) dendrimer in stable gene expression of FVIII in vitro and to study the cytotoxicity of PAMAM.


METHODS
The retrovirus-based plasmid vector pLNC-FVIII BD was generated by cloning a B-domain-deleted (760aa - 1639aa) FVIII cDNA (FVIIIBD cDNA) into retroviral vector pLNCX. The complex that contained PAMAM and pLNC-FVIII BD transfer FVIII BD cDNA into NIH3T3 cell line. In day 2, 5, 10, 15, 30 after transferring, the antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of FVIII BD mRNA. Inhibitory percentage of cell vitality was used for cytotoxicity of PAMAM.


RESULTS
Human FVIII was expressed for 30 days by transfected cells. The mean procoagulant activity of secreted FVIII in these 30 days was 0.929 U/ml, and the FVIII antigen was 0.188 micro g/ml by 10(6) cells in 24 hours, respectively. The level of FVIII didn't significantly decreased during these days. Inhibitory percent of cell vitality was only 5.32%.


CONCLUSIONS
PAMAM could effectively transfer pLNC-FVIII BD into NIH3T3 cells and FVIII could be stably and effectively expressed by the transfected cells. Cytotoxicity of PAMAM was low.
Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Wen-ying
WY
Kang
Hong-li
HL
Wang
Xue-feng
XF
Wang
Hong
H
Wang
Cong-Zhu
CZ
Wang
Qi-hua
QH
Fu
Qiu-lan
QL
Ding
Wen-man
WM
Wu
Yi
Y
Fang
Bao-hua
BH
Duan
D000818:Animals; D050091:Dendrimers; D005169:Factor VIII / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D011073:Polyamines / Q000494:pharmacology*; D012190:Retroviridae / Q000235:genetics*
D050091:Dendrimers; C104700:PAMAM Starburst; D011073:Polyamines; D005169:Factor VIII