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Preparation and Performance Examination of Chitosan-PGenesil-TGF-β1Nanoparticles

Author: DuZuoLian
Tutor: DengCunLiang
School: Luzhou Medical College
Course: Internal Medicine
Keywords: Hepatic fibrosis TGF-β1 RNA interference Gene carrier Chitosan nanoparticles
CLC: R575.2
Type: Master's thesis
Year: 2013
Downloads: 16
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Abstract


Objective: To construct and identify eukaryotic expression plasmids containingshort hairpin RNA(shRNA)that target at rat TGF-β1gene.To prepare chitosannanoparticles carrying recombinant plasmid PGenesil-TGF-β1and investigatetheir structural and function characteristics. This study provides a powerful toolfor exploring a promising gene therapy way of hepatic fibrosis.Methods:1.Two pairs of shRNAs that target at rat TGF-β1gene were designed,and no-isogeny gene segment HK was selected as a negative control. Afterannealed, the inserts were separately ligated into the linearized pGenesil-1.1plasmids. The eukaryotic expression plasmids pGenesil-TGF-β1-A,pGenesil-TGF-β1-B and pGenesil-HK were constructed, and then identifiedusing restriction enzyme identification and sequencing analysis.2.Thechitosan-PGenesil-TGF-β1nanoparticles were prepared by a complexcoacervation process. The morphology and particle diameter of nanoparticleswere observed under transmission electron microscope; the binding of pDNAwas evaluated by agarose gel electrophoresis analysis and the protective effectof nanoparticles to pDNA against Deoxyribonuclease was analyzed by DNase Idigestion experiment;the encapsulating rates were determined using fullspectrum spectrophotometer. Results:1.By restriction enzyme identificationand sequencing,the DNA fragments were inserted correctly in pGenesil-1.1 vectors as designed, respectively.2.The prepared chitosan-PGenesil-TGF-β1-Ananoparticles and chitosan-PGenesil-TGF-β1-B nanoparticles were mainlyspherical and the diameter distribution was100-200nm,with an averagediameter of （127.37±19.75）nm; the agarose gel electrophoresis analysisconfirmed that the nanoparticles could combined plasmids through electrostaticinteractions,and DNase I digestion experiment demonstrated the nanoparticlescould protect plasmids effectively from nuclease degradation;the encapsulationrates of two chitosan-PGenesil-TGF-β1nanoparticles were （94.38±0.45）%and （93.19±3.21）%,which were all more than90%.Conclusions:Wesuccessfully constructed2eukaryotic expression recombinant plasmidspGenesil-1.1-shRNA targeted to rat TGF-β1gene.Chitosan nanoparticleprepared by complex coacervation method own high encapsulation with adiameter distribution of100-200nm, which could combine the pDNA efficientlyrate and protect the pDNA from degradation against nucleases, and it will laythe foundation for further research on gene treatment of liver fibrosis.

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Cirrhosis
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