[In vitro transduction of human osteoblast cell populations with retroviral vectors].
OBJECTIVE
The involvement of cytokines in degeneration and inflammation of human tissue is well established. Interleukin-1 (IL-1) is a major agent in the pathophysiology of periarticular bone resorption in rheumatoid arthritis and in osteoporosis. Because the use of recombinant cytokines and growth factors is limited due to their short half lives, techniques are needed to get a permanent release of these therapeutic proteins. The rational of this study was to show that retroviral transduction of human osteoblastic cells is possible in vitro using the marker gene LacZ and the potentially therapeutic gene encoding for human interleukin-1 receptor antagonist protein (IL-1Ra). Different transduction techniques were combined to improve the rate of transduction in vitro.


METHODS
Osteoblastic cells were isolated from human spongious bone and cultured in vitro. The beta-galactosidase (LacZ) gene and the cDNA of IL-1Ra were introduced into the isolated cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1Ra was measured quantitatively by ELISA.


RESULTS
The transfer of retroviral IL-1Ra led to IL-1Ra expression of 8614 to 10,089 pg IRAP/50,000 cells/48 h. By combining different techniques to improve transduction, the X-gal staining established a rate of transduction of 60%.


CONCLUSIONS
Our results demonstrate that retroviral transduction of human osteobalstic cells is possible in vitro, and leads to high levels of the synthesized transgene product. The rate of retroviral transduction can be accelerated in vitro.
Orthopädische Klinik der Heinrich-Heine-Universität Düsesldorf. axel.baltzer@uni-duesseldorf.de
A W
AW
Baltzer
J D
JD
Whalen
T
T
Muzzonegro
H I
HI
Georgescu
P D
PD
Robbins
C H
CH
Evans
D002460:Cell Line; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007763:Lac Operon / Q000235:genetics; D010006:Osteoblasts / Q000276:immunology*; D012190:Retroviridae / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection
C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D012795:Sialoglycoproteins
In vitro Transduktion humaner osteoblastärer Zellpopulationen mit retroviralen Vektoren.