[Construction of the stable expression system of NIH3T3 fibroblast with pcDNA3.1(-)-hTGFbeta3 and the study on the proliferation of the system fibroblasts].

OBJECTIVE

To construct and identify the stable expression system of NIH3T3 fibroblast with eukaryotic expression vector of human transforming growth factor beta3 (pcDNA3.1 (-)/TGFbeta3). So as to investigate the proliferation of NIH3T3 fibroblasts transfected with hTGFbeta3 gene stably.

METHODS

The stable transfection of NIH3T3 fibroblasts with recombinant plasmid expressing hTGFbeta3 was established by using LipofectamineTM2000 and G418 selection. The mRNA and protein expression of TGFbeta3 were detected by the RT-PCR and Western blot method, respectively. Microscope and MTT were adopted to examine the proliferation of the stable expression system of fibroblasts with hTGFbeta3.

RESULTS

After G418 selection, RT-PCR and Western blot analysis, 7 out of 10 cell lines transfected with pcDNA3.1 (-)/TGFbeta3 expressed with very high level of TGFbeta3, as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low level. The stable transfection of NIH3T3 fibroblasts growth slowed down significantly (P < 0.05).

CONCLUSION

The stable expression system of NIH3T3 fibroblast with hTGFbeta3 were constructed successfully. The TGFbeta3 gene could inhibit the proliferation of NIH3T3 fibroblasts in vitro.