Experimental study on the in vitro transfection of thiolated N-alkylated chitosan bearing coexpression plusmid pIRES-hVEGFl2lcDNA/hBMP4

Abstract

Objective The recombinant coexpression plasmid of pIRES-hVEGFl2lcDNA/hBMP4 was transfected into rat bone marrow mesenchymal stem cells (MSCs) mediated by thiolated N-alkylated chitosan (TACS),and the feasibility of using TACS as a gene vector in bone tissue engineering was inves-tigated. Methods The TACS-pDNA nanoparticles were prepared by complex coacervation. The morpholo-gy and size of nanoparticles were observed under the transmission electronic microscopy and nanoparticle size analyzer. MSCs were isolated and cultivated from the bone marrow of rats. TACS bearing pIRES-hVEGFl2lcDNA/hBMP4 was transfected into the third generation of MSCs. Meanwhile, chitosan (experi-mental control) group,liposome (positive control) group and naked pDNA (negative control) group were set up. Effect of the nanoparticles on cell viability was illustrated by MTT assay. At the 3rd and 4th day af-ter transfection,the total RNA and total protein were extracted from MSCs respectively. The target gene ex-pression was detected by RT-PCR and Western blot. Results TACS could protect the encapsulated pDNA effectively. Cell viability of TACS group (73.18±6.56)% was significantly higher than positive control group (45.92±24.93)% (P<0.01). HVEGF121 and hBMP4 were expressed in transfected MSCs detec-ted by RT-PCR and Western blot except the negative control group. The target protein expression in TACS group was lower than in positive control group (P<0.05), but significantly higher than in experimental control group (P<0.01). Conclusion The coexpression plasmid was transfected into MSCs successfully by TACS. TACS has low cytotoxicity and its transfection efficiency was obviously higher than non-modified chitosan. Key words: Chitosan; Mesenchymal stem cells; Vascular endothelial growth factor; Bone morphogenetic protein; Transfection