Chitosan Mediated ENOS Gene Transfer On Prevention Of Restenosis Of Balloon Injuried Rabbit Carotid Artery

Gene therapy is one of the important approaches for cardiovascular deseases. Generally, gene vectors can be divided into two categories :viral vectors and nonviral vectors. Adenovirus is one of the most commonly used viral vector for its high efficient infectivity. However, adenoviral vector has its own defects: (1)the replication deficient adenovirus may reverse to the pathogenic wild type virus, (2)adenoviral vector is cytotoxingenic and affects the viability and function of the target cells. (3) adenoviral vector generally stimulate the immuno-response of host and the neutralizing antibody of adenovirus may interfere with the efficiency and efficacy of in invo gene transfer, which limited the repeated application of the vector. (4)the complicated procedures need prolonged time to construct vector and is very expensive. (5)may have untoward effects to the distal organs when systemically used. Taken into consideration of these disadvantages of adenoviral vectors, its application in practise has been greatly limited, comparatively, these highlights the non-viral vectors for their easily being obtained, free proinflamatory effects and immunpgenic effects, good bio-compatibility and being biodegradable and of safety, additionally, can be recurrently administrated if necessary.Currently there are many kinds of materials available as non-viral vectors such as cationic liposome, cationic lipid and polymers. The commonly used liposome has high in vitro transfection efficiency, but has systematic toxicity in in vivo gene transfer. This paper focused on the biodegradable polymers chitosan as the non-viral vector material. Chitosan is relatively non-toxic and of good bio-compatibility. Nonviral vectors made it possible that the DNA can be given as a drug and is considered as a candidate to substitute viral vectors in gene transfer.In the present study, we firstly amplified the plasmid pCDNA3-eNOS which included the target gene encoding epithelial nitric oxide synthase(eNOS) and constructed the transfering plasmid pAdTrack-CMV-eNOS with shuttle plasmid pAdTrack-CMV. Thereafter we formed the adenoviral frame plasmid pAd-CMV-eNOS-CMV-GFP through intra-bacteria recombination and become infectious adenoviral vector(Ad-eNOS) by assembly in HEK293 cell line. All the resultant plasmid and adenoviral vectors are confirmed by restrictive endonuclease digest and agarose gel electrophoresis.Chitosan of 70kDa molecule weight were purified by re-precipitation methods and dissolved in 1% acetic acid aqueous solution. Sterilization were performed by filtered through 0.2um filter. Chitosan reacted with pAdTrack-CMV-eNOS encoded the epithelial nitric oxide synthase (eNOS) through eletrostatic mechanism and formed chitosan/plasmid complexes (CPC-eNOS) according to the ratio of nitrogen in chitosan backbone to the phosphate of plasmid DNA and validated the formation of CPC-eNOS by agarose gel electrophoresis as well as transmission electron microscopy.We cultivated primary aorta vascular smooth muscle cells (VSMCs) of male SD rats. With employment of FCM and western blot techniques we investigated the possibility of gene tranfer mediated by CPC-eNOS into VSMCs and the possible factors such as pH value, N/P ratio as well as the serum levels in the DMEM which may influence the transfection efficiency. The results indicates that CPC-eNOS can mediate in vitro gene transfer into the cultured VSMCs of SD rats. The pH value of the transfection medium and the N/P ratio of the resultant CPC-eNOS from 70kDa chitosan are the primary factors which exert influences upon the transfection efficiency. The transfection efficiency increased with the N/P ratio increased and the pH increased. But the transfection efficiency declined when pH value is above 7.0 and the N/P ratio is above 6. The concentration of serum can stimulate the proliferation and increased the viability of VSMCs, which resulted in the increased transfection efficiency of CPC-eNOS with the serum levels increased in the range from 0.5% to 10%. The transfection efficiency decrease...