The Study Of Chitosan-pIRES2-EGFP-hBMP2-his Nanoparticles Synthesis And The Gene Transfection Efficiency In MG63Cells

BackgroundIn the field of oral medicine, implant denture fully possesses the advantages which can not be matched against with the traditional denture. It is the best way to repair the lack of dentition defect at present.The stability of dental implant in early time and the good formation of osseointegration are critical to the success of the implant. Osseointegration is a directly ordered kind of structure and function connection which is formed between the implant and bone surface. Therefore, the surface treatment of implants is a hot spot of academic research all the time. But previous work mostly focused on the physical and chemical processing of material surface, lack of the related biology research in implant surrounding bone cells. The nanoscale gene vector was introduced onto implants surface, then directly enhance the start-up and transduction of osteogenesis signal by gene activation function of vector system.It can speed up the osteogenesis and bone integration, and this research is still a blank in the domestic and overseas.Bone morphogenetic protein has the ability of inducing osteogenetic so that it plays an important role in repairing bone defects. The related research suggests that the BMP family has21members, namely BMP1-BMP21. In the seven gene members of hBMP which have been cloned, the bone formation induction activity of hBMP2is thestrongest. hBMP2is a kind of low molecular weight glycoprotein, belonging to the TGF family. As a kind of protein with bone induction activity, it can promote the deposition of calcium and phosphorus in the titanium surface and accelerate combination of implant-bone interface.When the target gene is selected, it is extremely important to establish a safe and effective vector system in gene therapy. The traditional gene therapy using slow virus vector and liposomes vector has been widely studied, but all of them have not been able to apply to clinical because the uncertainty of biological security. Chitosan is a kind of biodegradable nanoparticles with cationic vector material and is the one and only in the nature. It is not only cheap, good biocompatibility,biodegradable and can adhesion mucous membrane but also with a wide range of content. In weak acid solution, chitosan can positively charged because the glucosamine base in the molecule has been protonized, and then work with the negatively charged DNA by electrostatic interactions condense into polymer composites, thereby make the structure of DNA change into the particles with relative density of nano-scale. So it is especially suitable for entrap the bioactive molecules such as proteins, peptides, vaccines, gene and so on. Now, instead of liposomes and virus, chitosan is a promising vector acting on drug, protein, gene and some other macromolecular complexes.This project can build the pIRES2-EGFP-hBMP2-His plasmid in vitro which can coexpress the EGFP and hBMP2-His gene in eukaryotic cells. This research use the chitosan to package the plasmid by desolvation method, to form a nano-sized complex and control its size, detection of its associated indicators, try to achieve the best condition of transfecting MG63cells, and then to generate hBMP2in succession by target plasmids programmed into the target cells. It can accelerate the function expression of osteoblast in implant surface, accelerate the bone formation, even to guide bone augmentation and format a titanium implant surface of gene activation. Therefore, this research has important significance for the study of implant surrounding rapid bone formation and implant stability in early time. Chapter one:The construction,amplification, purification and determination of the plasmid pIRES2-EGFP-hBMP2-HisObjectiveTo construct the plasmid pIRES2-EGFP-hBMP2-His in eukaryotic cells which can express EGFP and hBMP2-His, and complete its amplification, purification and determination, for further to provide experimental basis for the research of constructing nano composites applying that plasmid and chitosan nanoparticles and transfecting MG63cells.MethodsTo amplify the hBMP2-His Gene fragment by PCR, and to introduce restriction sites of BamH I and EcoR/in the primers, large fragment of pIRES2-EGFP plasmid and hBMP2-His cleavage products of the target gene were recovered respectively. The plasmid pIRES2-EGFP large fragment was connected together with the fragment of target gene hBMP2-His. The ligated product was transformed into competent bacteria DH5a, the plasmid was amplified by shaking. Using a plasmid extraction kit, we extracted and purified the plasmids, and then performed determination and sequencing of restriction enzyme digestion for the plasmids.ResultsTo synthesize pIRES2-EGFP-hBMP2-His plasmid successfully, and complete the identification, amplification and purification. BamH I and EcoR I restriction enzyme digestion results are correct, the sequencing result turn out to be correct. So as to provide a basis for the follow-up study.Chapter two:The synthesis and charaterization of chitosan/pIRES2-EGFP-hBMP2-His nanoparticlesObjective:To explore the synthesis condition of nano complexes in weak acid solution with five different molecular weight and degrees of deacetylation at different N/P ratios, caculate the plasmid DNA loading efficiency, to detect the particle size and zeta potential,and to discuss the best conditions of chitosan as gene vector mediated gene transfection.Methods:With the gel permeation chromatography (GPC) and the acid-base titration(ABT), the determination results of the molecular weight of five kinds of chitosan by gel permeation chromatography range from17KDa to379KDa respectively, and the determination results of the degree of deacetylation acid-base titration method are from79%to97.37%. According to the different molecular weight and deacetylation degree. Chitosans will be divided into five groups of A, B, C, D, E as mediated gene transfection carrier, using solvent method at different N/P ratio (molar ratio of amino content in chitosan and the phosphate content in plasmid) to synthesize chitosan/pIRES2-EGFP-hBMP2-His nanocomposites. The gene encapsulating ability of chitosans were evaluated by the agarose gel electrophoresis and fluorescence spectrophotometry while the size and the potential were analyzed by the zeta potential analyzer. In addition, the shape of nanopaticles were observed by atomic force microscope(AFM)Results1.0.6%agarose gel retardation electrophoresis experiments confirmed that chitosan has wrapped effect on pIRES2-EGFP-hBMP2-His plasmid. When the N/P ratio is1,chitosan was not able to completely wrapped plasmid.While the N/P ratio is equal or greater than3, chitosan could wrapped the plasmid DNA totally.2.The result of fluorescence spectrophotometry shows that the average loading efficiency reaches (96.03±0.25)%(X±S, n=3) in group C when the N/P ratio is5.3.The average size of synthetic chitosan/pIRES2-EGFP-hBMP2-His nano complexes is range from111.7nm to3214.2nm, and the average zeta potential of nanospheres is from4.93mV to16.79mV. The particle size decreases as the N/P ratio increases, and zeta potential increases while increasing N/P ratio and showssteady state. The morphology of the nanoparticles was observed under the atomic force microscope and it turned to be spherical.4.1n the detection of the nanopaticles’size, we found that in each group there exist two peaks of the size distribution. Its main peak shows the smaller particle size but with the larger number. But the number of the larger size nanospheres is smaller. Test results also indicate that along with the increase of N/P ratio, probability of the second high peak will increase. So the number of the nanopaticles with lager size will increase.5.According to the measurement results of the nanoparticles’size and zeta potential, we can conclude that when the N/P ratio is5in Group C,the nanospheres have the suitable grain size and the appropriate distribution so that it may obtain higher transfection efficiency.Chapter three:Transfection efficiency of chitosan meadited pIRES2-EGFP-hBMP2-His plasmid in MG63cellsObjectiveTo explore the transfection efficiency of chitosan-plasmid in MG63cells at different N/P ratios.MethodsAfter the recovery and subculture, the MG63cells were seeded in12-well plates. Select group C chitosan (molecular weight of77.8KDa, degree of deacetylation97.37%) as the experimental group, and make chitosan/pERES2-EGFP-hBMP2-His nanoparticles according to the N/P ratio (chitosan amine/plasmid ratio of phosphate groups in DNA) in3,5,7,10to transfect MG63cells. After48hours of transfection, we detect the percentage of the cells expressing green fluorescent protein by flow cytometry and measure the transfection efficiency. And we set a control group with liposomes and a blank control group.Results1.In the experimental group, positive transfected cells can be observed in transfection wells in different N/P ratios. And MG63cells expressing green fluorescent protein can also be observed under the inverted fluorescence microscope. But as the N/P ratios increase, the increasing trend in transfection efficiency cannot be seen. On the contrary, the mean transfection efficiency reached (12.31±0.76)%(X+S, n=3) in the cell transfection well which the N/P ratio is5, which is the highest. And the transfection efficiency decreases with the N/P ratio increases when N/P>5.2.1n the control group which is only added with blank plasmid, MG63cells expressing green fluorescent protein can hardly be observed under the fluorescence microscope after48hours of transfection. The results measured by flow cytometry show that the transfection efficiency is essentially0. There were significant differences (P<0.05) by LSD-t compared with the experimental group.3.In the control group with liposomes, apparently positive expressed cells can be observed under the fluorescence microscope after48hours of transfection. The results measured by flow cytometry show that the transfection efficiency is (30.36±3.32)%(X±S, n=3)ConclusionsChitosan can successfully wrap the target gene and compress pIRES2-EGFP-hBMP2-His plasmid into the particle with diameter of several hundred nanometers which is suitable for transfecting MG63cells with solvent method. N/P ratio is a vital factor which influences chitosan-mediated gene transfection and this may be associated with the endocytosis of the nanocomposites and the release of genes after the endocytosis. Chitosan-mediated gene transfection efficiency was significantly lower than the ones of liposomes, and we presumed that compared with liposome through recognition system of lipoprotein receptor on cell membrane surface, the gene’s endocytosis process mediated by chitosan requires the endocytosis of cells. As cell membrane is poorly water-soluble and will be dissolved in the acidic environment, this characteristic may have certain influence on physiological metabolism of cells.