Preparation Of Different PH PEG Chitosan-based PACE-shRNA Nanoparticles And Transfection To Rat Aortic Endothelial Cells

PartⅠStudy of Preparation and characteristics of chitosan nanoparticles loaded with geneObjective: To prepare different pH chitosan nanoparticles (CS-NP) and PEG/CS-NP carrying plasmid and study the characteristics of PEG/CS-NP and its capability of combinability and protection to plasmid.METHODS: The different pH CS-NP was prepared with ion exchange method, ACE-shRNA to CS-NP was conneted by adsorption of static electricity; Preparation of different pH PEG-chitosan nanoparticles plasmid and its diameter, the diametric distribution range and its form was measured by puff gold scanning electron microscope; The PEG/CS-NP's and different pH PEG/CS-NP's capability of combinability and protection to plasmid was studied by agarose gel electrophoresis;The protection of PEG/CS-NP to plasmid was observed by DnaseⅠ.RESULTS: The PEG/CS-NP was showed to be well-distributed spheroidal particle by SEM ,average diameter was 5nm;Agarose gel electrophoresis showed that DNA was absorbed by PEG/CS-NP effectually;The protection of different pH PEG/CS-NP to plasmid were different ,when pH<7 the combination of PEG/CS-NP to plasmid were 100%; PEG/CS-NP can protect the plasmid by DnaseⅠ.CONCLUSION: Small diameter and well-distributed PEG/CS-NP can be prepared with ion exchange method, and PEG/CS-NP can combine and protect plasmid effectually.PartⅡOptimization of the transfection condition of tranfecting pACE -shRNA to rat aortic endothelial cells.Objective: To research the transfection of CS-NP/DNA and PEG/ CS-NP/DNA to rat aortic endothelial cells.METHODS: The rat Aortic endothelial cells(RAEC) were obtained from thoracic aortas and cultured by tissue explant method. The morphology of RAEC was studied by phase contramicroscope. Its molecular markers were observed byⅧfactor immunocytochemistry. Passage 3～4 cells were used in experiment, the transfection to RAEC we used 40ul,60ul,80ul,100ul,150ul of CS-NP/DNA and CS-NP /DNA modificated by PEG 4000 and 5000. RESULTS: The efficiency of transfection of CS-NP/DNA was (26.0±3.9)% by flow cytometry; the highest efficiency of transfection of PEG/ CS-NP/DNA was 100ul(63.4±4.0)%, which was higher than other groups(p<0.05); No significant difference between PEG 4000 group and PEG 5000 group was found(P>0.05); the result of MTT showed that the ratio of cells survival of PEG/ SC-NP/DNA was (96.0±1.4) % and the ratio of cells survival of CS-NP/DNA was (93.0±2.5) %.CONCLUSION: Optimized transfection conditions, PEG/ SC-NP could transfect pACE-shRNA to rat aorta vascular endothelial cells with high effect and cells viability.PartⅢSturdy on the inhibition of ACE mRNA in rat aortic endothelial cells by PEG/SC-NP/DNAObjective: Sturdy on the inhibition of ACE mRNA in rat aortic endothelial cells by PEG/ SC-NP/DNA.METHODS: Experiment was divided into three groups: Blank control group; naked plasmid group;PEG/ SC-NP/DNA group; the level of ACE mRNA(24h,48h,72h) was assayed by RT-PCR.RESULTS: The level of ACE mRNA of transfected by PEG/ SC-NP/DNA was decreased by(14.7±5.9)%(24h)(,53.6±5.4)%(48h)(,60.1±2.1)%(72h).When 48h the level of ACE mRNA of transfected by naked plasmid was decreased by(0.6±0.3)%, which were differet with PEG/ SC-NP/DNA(p<0.05).CONCLUSION: Transfection of PEG/ SC-NP/DNA to rat aorta vascular endothelial cells could initiate sequence post-transcriptional gene silencing and inhibit expression of ACE mRNA.