Research On The Preparation Of A New Particle Used In Gene Gun-Hydroxyapatite

Many human disease is caused by genetic defects, if there is a way of repairing defective genes on the level of gene, it will generate revolutionary impacts on traditional treatment of disease. In current gene therapy, gene delivery system can be divided into two categories:viral vectors and non-viral vectors. Viral vectors have potential risk in clinical application; and the transfection efficiency of non-viral vector is low. Compared with other technology, gene gun technology is a purely physical gene-transfer technology which have obvious advantages:simple,transfer genes rapidly,safe, etc.This series of advantages bring gene gun technology a promising prospect. However, the gold or wolfram particles used in gene gun system can not be metabolize by human life, so may have security issues. Hydroxyapatite has good biocompatibility,can be metabolize and provide no harm to cells. Consequently, in this study, HAP is selected as gene vector in replace of gold and the feasibility of HAP using in gene gun system was evaluated. The research work include the following three parts:Part one:Preparation of HAP partcles through stability reagent H-protection methordObjective:This work was directed at preparing HAP partcles with average size,good mono-dispersion,specific surface properties and specific size range.Methods:HAP was prepared through stability reagent H-protection methord, its properties were characterized by granularity testing,potential analysis and morphology mensuration, and also the influence of different reaction conditions on the surface properties of HAP is studied.Results:Through Ca/P mensuration,TEM,granularity and potential testing, the HAP partcles we prepared is mono-dispersed with nanoscale and good stabilization.Conclusion:1,HAP which was prepared through stability reagent H-protection methord is needle-like with good dispersion, the particle size is small.2,Dosage of stable reagent H,temperature,the dropping rate of Ca(OH)2, ultrasonic and so on have a certain influence on the size and potential of HAP. Part two:The preparation of pEGFP-C3 plasmid DNA and research on adsorption of DNA to HAP Objective:To obtain pEGFP-C3 plasmid DNA with fine purity and concentration that consistent with gene transfer requirements, and also the plasmid DNA should have a good load on the HAP particles.Methods:PEGFP-C3 plasmid DNA was extracted by alkaline lysis method. Its concentration and purity was determined by agarose gel electrophoresis and ultraviolet absorption. The adsorption capacity of DNA to HAP was determinated under different operating conditions by method of ultraviolet absorption.Results:1,The result of agarose gel electrophoresis showed that the DNA we prepared is just pEGFP-C3 plasmid DNA, and its purity is high, can meet the requirments of gene transfection.2,Different adding ordors of reagents and the regulation of spermidine's pH have no influence on the adsorption capacity of DNA to HAP to some exent.Conclusion:1,Spermidine can improve the adsorption capacity of DNA to HAP to some extent.2,If only ensuring the certain reaction system of HAP,DNA and spermidine, DNA can be loaded to HAP excellently. Part three:Research on HAP mediated in vitro gene transfection to RSC96 cellsObjective:To investigate the capability of HAP as a carrier for in vitro gene transfection.Methods:Select pEGFP-C3 plasmid DNA as target gene, conduct gene transfection under different gene vector and different apparatus parameters, then compare its transfection efficiency with traditional gene-gun technology.Results:1,A great lot of green fluorescence was found when select gold as gene vector.2,The transfection efficiency is relative low when gene transfection is conducted by vector HAP.Conclusion:1,The transfection efficiency is high when selected gold as gene vector.2,HAP can mediate plasmid DNA into target cells as vector under certain work conditions, and the gene can then express green fluorescence protein. All this showed that HAP is competent to replace gold to conduct gene transfection, but its efficiency is relative low compared with gold.