The Preparation Of Chitosan Nanoparticle Loaded With Human Telomerase ASODN-t And Its Transfection To HepG-2 Cell

Objective:To prepare appropriate chitosan nanoparticles by optimizing theexperimental condition. To load it with plasmid and study its loadingcapability and protection ability to plasmid.To transfect it toHepG-2 cell and evaluate the possibility and efficiency of chitosannanoparticle as a vector in gene transference compared to cationicliposomes. And to load it with telomerase antisensenucleic ascids. Totransfect it to HepG-2 cell compared to the transfection of cationicliposomes. Then to examine their apoptosis and to compare theirapoptotic rate.Methods:To prepare the chitosan nanoparticle with cocervation process;Tomeasure its diamerter,the diametric distribution range and its form byscanning electron microscope and laser atomic examining apparatus.Theenhanced green fluorescent protein as the reporter gene is adsorbed to thechitosan nanoparticle by electrostatic forces; The potential of adsorbingDNA on nanoparticles was analyzed by agarose gel electrophoresis andspectrophotometer. To study the release process of pDNA fromchitosan nanoparticle adsorbing DNA by spectrophotometer.To check itsprotection ability to DNA by DNase I digestive experiment. Totransfect it to HepG-2 cell and evaluate the possibility and efficiency ofchitosan nanoparticle as a vector in gene transference compared tocationic liposomes by fluorescence microscope. And to load it withtelomerase antisensenucleic ascids, then to transfect it to HepG-2 cellcompared to the transfection of cationic liposomes. To research the growth condition and apoptosis by microscope orelectron microscope. To examine apoptotic ladder by agarose gelelectrophoresis. To examine the apoptotic rate of the transfectedHepG-2 cell by flow cytometer, and to compare their apoptotic rate.Todraw the growth curve of HepG-2 cell by MTT method. To examinetelomerase activity by TRAP-ELISA method.Results:1.Chitosan nanoparticles is showed to be spherical particlesdistributed evenly, the least nanoparticles is 36nm in diameter, d(0.5)is95nm,by scanning electron microscope and laser atomic examiningapparatus.2.Agarose gel electrophoresis showed that the enhanced greenfluorescent protein was adsorbed to the chitosan nanoparticleeffectively.The encapsulation efficiency of nanoparticles-DNA mixed bydifferent ratio(nanoparticle: plasmid) is 100% (50: 10), 100% (50:25), 100% (50: 50), (92±2.3)% (50: 75), (69±2.2)% (50: 100)by the examining of fluorescence microscope.3.The experiment of the release of pDNA showed that its prosesscombined with a outbreak release phase and a slow release phase.During the outbreak release phase(early 1-72h), the amount of pDNAreleased can reach (75.4±1.8)% of the whole compared to the slowrelease phase(72-168h) with only (94.6±1.4)%.4. DNase I digestive experiment showed:there is no apparent DNAstrip when we use no nanoparticles or nanoparticles-DNA accounting tothe ratio of 5:50,while there is an apparent DNA strip when we usenanoparticles-DNA accounting to the ratio of 50:50,100:50. And itshowed that chitosan nanoparticles can protect DNA well at such ratio. 5. By fluorescence microscope,it was found that its transfectionefficiency(58.6±2.6)% is higer than that of cationic liposomes(45.5±2.5)%when we use 30ul chitosan nanoparticle which has combinedwith pE-GFP-C1gene.The difference has statistical meaning.6. Electron microscope showed that the HepG-2 cell transfected bytelomerase antisensenucleic ascids (induced by chitosan nanoparticles orcationic liposomes) have apoptotic cell.7. Agarose gel electrophoresis showed that both of the HepG-2 celltransfected by telomerase antisensenucleic ascids (induced by chitosannanoparticles or cationic liposomes) have a apoptotic ladder.8. Flow cytometer showed that the apoptotic rate induced bychitosan nanoparticles is higher than that induced by cationic liposomes.The difference has statistical meaning.9. MTT method showed that the HepG-2 cell transfected bytelomerase antisensenucleic ascids (induced by chitosan nanoparticles orcationic liposomes) growed slowly, and the HepG-2 cell induced bychitosan nanoparticles growed more slowly than the HepG-2 cellinduced by cationic liposomes. The difference has statistical meaning.10. TRAP-ELISA method showed that the telomerase activity ofboth of the HepG-2 cell transfected by telomerase antisensenucleicascids (induced by chitosan nanoparticles or cationic liposomes) wasinhabited apparently, and the optical density (A) was 0.11±0.016,0.17±0.018. The difference has statistical meaning.Conclusions:Chitosan nanoparticles can be effectively loaded with plasmid andcan protect the plasmid from been digested. HepG-2 cell is transfectedsuccessfully by the chitosan nanoparticles,and its transfection efficiency is higer than that of cationic liposomes. The transfection of telomeraseantisensenucleic ascids can effectively inhibit the growth and promoteapoptosis of HepG-2 cell,and the apoptotic rate induced by chitosannanoparticles is higher than that induced by cationic liposomes.