Experimental Study Of Chito-oligosaccharide As Gene Deliver Carrier

The drug/gene delivery system based on biodegradable polymer macromolecular has been one of the hot topics of the biological materials research in recent years. As natural biodegradable polymer and oligosaccharides, COS (also known as Oligochitosan, chitosanoligosaccharide, chitooligosaccharide, chitosan oligomers) are the product from degradation of chitosan. COS is expected to be an ideal gene delivery vector as it shows lots of avantage including various origin, stable physical and chemical characteristics,variety of biological activity and good biocompatibility. Based on above, the two diffent experimental studies of the COS as a gene delivery vector in this thesis were carried out as below:(1)Study on oligochitosan nanoparticles for intracellular EGFR DNAzyme deliveryAs a new tool of gene silencing, one of the most important problems of DNAzyme (DRz) is how to enter the target cells efficiently. Therefore, it would be meaningful in gene therapy application to find a kind of in vivo gene delivey material with good compatibility and stability. In this section, EGFR DRz(epidermal growth factor DNAzyme)that targeting to EGFR mRNA were designed firstly. Then COS-EGFR DRz complexes were synthesized by means of electrostatic adsorption, meanwhile, particle sizes were charactered by zeta potential meter. And the results of confocal laser microscopy showed that the EGFR DRz were delivered into Hela cells by COS successfully, and the distribution of EGFR DRz were in both cytoplasm and nucleus. The data of flow cytometry showed that there were no statistically significant difference in transfection efficiency between liposome-EGFR DRz (90.7%) and COS-EGFR DRz (88.7%). The results of semi-quantitative RT-PCR confirmed that EGFR mRNA in Hela cell could be targeting cleaved by COS-EGFR DRz and the EGFR mRNA expression were decreased. The cell cycle analysis in Hela cells tansfected with COS-EGFR DRz revealed an arrestment in the G0/G1 phase. And the cell apoptosis rate induced by COS-EGFR DRz was 19.3%, which was higher than the rates induced by liposome-EGFR DRz (13.0%). These results demonstrated that COS compared with liposomes possesseed similar transfection efficiency and lower cytotoxicity. It is expected to be a potential effective DNAzyme delivery vector.(2) Study of transformation efficency by using ultrasound combined with COS-plasmid DNA complex in plant cellsA rapid and effective transformation system was built by using ultrasound combined with COS-plasmid DNA complex in Arabidopsis suspension cells and callus suspension cells of rice. The COS-plasmid DNA complex were prepared by means of electrostatic adsorption as well. The optimal concentration of COS was choosed to be 0.2mg/ml according to both size and binding rate given by Zeta particle size analyzer.And the optimized ultrasonic intensity (180w) and ultrasonic time (15s) were obtained after 72h plant cell culture. The results of confocal laser microscopy showed that COS-plasmid DNA complex entered the Arabidopsis or rice suspension cells and GFP protein were expressed successfully during the process of ultrasonic transformation, as COS could protect the degradation of DNA mediated by the ribozyme. It is expected to provide a simple and effective technical support for genetic transformation.