Ch-GNPs/?6 Compounds On The Proliferation And Differentiation Of Mouse Embryos Osteoblast Precursor Cells MC3T3-E1

ObjectiveGold nanoparticles with chitosan as the carrier,with integrin alpha v beta 6plasmid,formed the Ch-GNPs/beta 6 compounds on mouse embryos osteoblast precursor cells MC3T3-E1 proliferation and differentiation.MethodsThe Ch-GNPs were conjugated with the pc DNA-?6 through a coacervation process.The size of Ch-GNPs and surface characterization of Ti was performed using UV-vis spectroscopy,TEM(Transmission electron microscopy).The DNA conjugation and transfection capacity of Ch-GNPs were simultaneously confirmed by agarose gel electrophoresis,b-galactosidase staining,and immunoblotting.In order to study the Ch-GNPs/?6 effects on osteoblast proliferation and differentiation,set up the blank group,Ch-GNPs/Lac Z as control group,and Ch-GNPs/?6 as experimental group.Through the cell adhesion,cytoskeleton form by CLSM,MTT,and analyzed of ALP activity,COL-?and OCN content of experiment,research Ch GNPs/?6effects on osteoblast proliferation and differentiation.Results1.Ch-GNPs solution is wine red,measured in the ultraviolet-visible spectrophotometer meter maximum absorption peak at 520 nm,this is typical of Ch-GNPs surface visible absorption peak.Ch-GNPs was observed under transmission electron microscopy(TEM)dispersed spherical particles,the average particle size of about 10 to 20 nm.2.There was not DNA belt observed in the adding sample hole of Agarose gel electrophoresis.because of there is electrostatic force attraction between Ch-GNPsand pc DNA-?6,DNA can not migration is still located in adding sample hole of gel,suggests that the Ch-GNPs and pc DNA – ?6 has the strong combination.Western blot results show that the ratio in 2:1of Ch-GNPs/Lac Z,?-gal showed high level expression.While the GNPs/Lac Z,as an effective transfection molecules,galactose glucoside enzyme dyeing experiments showed obvious blue in MC3T3-E1 cells.3.The results of the proliferation and differentiation of MC3T3-E1 showed:adhesion ability of MC3T3-E1 in Ch-GNPs/?6 groups significantly higher than that of blank group and the CH-GNPs/lac Z group.From the cytoskeleton form,Ch-GNPs/?6group has better spreading,longer filaments and tabular pseudopodia,bunchy fibre arrangement direction more consistent,MTT method is used to test cell proliferation,results show that the Ch-GNPs/?6 groups have the optimal performance of proliferation,particularly in the third days multiplied,shows the increasing trend of best;4.In the determination of ALP activity and COL-I,results show that Ch-GNPs/?6 group,the secretion ALP by MC3T3-E1 cells is higher than the other two groups,Among 2 to 4 day,the activity of ALP increased several times.Ch-GNPs/?6group alkaline phosphatase(ALP)activity and collagen type ?(COL-I)content in the early days of osteoblast differentiation have significantly higher performance,and osteocalcin(OCN)is at its peak in the late differentiation.Conclusion1.The chitosan and the acid chloride alloy through the simple condensation process can form Ch-GNPs compounds.The Ch-GNPs has the right granularity can be swallowed in cells,it can be used as the carrier of gene transfer.2.Ch-GNPs and pc DNA-?6 formed the Ch-GNPs/?6,when the ratio between2:1 can have a strong ability of combining ability and transfection.3.Ch-GNPs/?6 can promote the osteoblast adhesion,is advantageous to the proliferation of osteoblast.Ch-GNPs/?6 also can regulate cytoskeleton,have activation ALP activity,at the same time improve the COL – I?OCN content,promote the proliferation and differentiation of osteoblast.