| Biomineralization refers to the process in which organisms regulate the formation of inorganic mineral structure through their own metabolism,with the participation of biological macromolecules,organic matrix,inorganic salt ion transport and other factors.it is a reaction in which cell function realizes or responds to environmental changes.Bio-induced mineralization is a mineralization phenomenon caused by the interaction between the environment and the life activities of the body.the occurrence of this kind of mineralization is not strictly controlled by cytogenetics,and the change of environmental factors is the main trigger factor.With the interpretation of the mechanism of biomineralization,the study of induced mineralization is more bionic mineralization,that is,the use of bionic strategies to transform exogenous artificial materials into biomaterials,or the use of organisms to create or induce spontaneous mineral structures with special structures and functions,which not only help to weaken the heterogeneous immune response of materials,but also actively participate in the chemical and biological regulation of life.Biomineralization based on gene therapy not only does not cause immune rejection of life,but also is expected to provide a new strategy for the treatment of cancer without drugs.In this paper,based on gene editing technology and gene delivery system,an eukaryotic expression plasmid capable of expressing human calcium binding protein Calbindin-D28 k in vitro was constructed for the purpose of bioinduced mineralization,and a method for the preparation of nano-scale gene delivery vector with high transfection efficiency and low cytotoxicity was explored.We reported an eukaryotic expression plasmid of human calcium binding protein Calbindin-D28 k.Using human embryonic kidney HEK-293 T cell line as template,the target fragment with molecular weight similar to the theoretical sequence was obtained by total RNA extraction,reverse transcription and PCR amplification.The concentration of PCR gel recovery products and the value of A260/A280 were measured by ultra-micro spectrophotometer to evaluate the quality of the target gene.The blank plasmid vector inactivated by dephosphorylated enzyme and the target gene inactivated by enzyme digestion were ligated at3:1 with the ratio of A260/A280 close to 1.8.the plasmid after 12 hours of ligation reaction was transformed,and the extracted plasmid was amplified by single clone.Then the strains that successfully connected the target gene into the vector plasmid were screened by agarose gel electrophoresis and gene sequencing.The results showed that the eukaryotic expression plasmid containing the target gene was successfully prepared.In order to transfect the prepared plasmids into eukaryotic cells,we prepared nanopolyethyleneimine(PEI)microspheres,which is the gold standard of cationic non-viral gene transfection reagents,and the reaction mode was emulsion cross-linking method.The effects of oil-water ratio,cross-linking agent dosage and cross-linking reaction time on the product microspheres were explored,and the optimal experimental scheme was determined.Through the characterization of physical and chemical properties,it was found that the particle size range of the nano-PEI microspheres prepared under the optimal conditions was about 72.8 ±28.4 nm,and the spheroidization and dispersion were good.Biosafety evaluation showed that the nanoPEI microspheres had certain cytotoxicity and showed a certain toxicity accumulation with the extension of time.In order to solve the cytotoxicity of the above-mentioned PEI microspheres,we prepared nano-PEI-FA microspheres based on chemical grafting method to activate the carboxyl group of folic acid(FA)and amide reaction with the amine group of PEI.Based on the layer-by-layer self-assembly method,we adsorbed negatively charged hyaluronic acid(HA)on the surface of positively charged PEI microspheres to prepare nano-PEI-HA microspheres.Through the characterization of its morphology,particle size,Zeta potential,Fourier transform infrared absorption spectrum(FTIR)and other physical and chemical properties,it is confirmed that the formation process of the microspheres is consistent with the theoretical design,and the particle size is about 300 nm,which can meet the needs of subsequent experiments.Biocompatibility evaluation confirmed that the two modification methods effectively reduced the cytotoxicity of PEI,which may be related to the reduction of the surface charge of the microspheres.The ability of microspheres to bind to DNA and the transfection efficiency were further evaluated.The ability of nanocomposite microspheres to bind to DNA can be further evaluated by gel block assay.The transfection efficiency was verified by transfection experiments.In summary,the eukaryotic expression plasmid which can express human calcium binding protein Calbindin-D28 k in vitro was constructed based on gene editing technology,and the plasmid and strain were of good quality.PEI-FA nanoparticles and PEI-HA microspheres with good biocompatibility and transfection efficiency were prepared based on chemical grafting and layer-by-layer self-assembly technique,respectively.The eukaryotic expression plasmids of Calbindin-D28 k can be transfected into eukaryotic cells.The construction method of eukaryotic expression plasmid in vitro,the eukaryotic expression plasmid of human calcium binding protein Calbindin-D28 k,the preparation methods of nano-PEI-HA microspheres and PEI-HA microspheres can be more fully studied and applied in related fields.However,there is still a long way to go to realize the biomineralization of eukaryotic cells by gene therapy.In the follow-up,gene transfection vectors with intelligent response can be designed,or other eukaryotic expression plasmids with biomineralization function can be constructed to do more in-depth research on bio-induced mineralization. |
