Osteoblast-vascular Endothelial Cell Co-culture System In The Expression Of IGF-1and The Influence Of The Vascular Endothelial Cell Biology Research

Objective: In recent years, bone tissue engineering has attracted widespreadattention. Osteoblasts-endothelial cells co-culture system is important in orderto bone tissue engineering research. The study start with the establishment ofco-culture system, and then investigate the effect of co-culture system oninsulin growth factor1(IGF-1) and on the biological characteristics of vascularendothelial cells. Methods: The experiment consisted of three parts. In the firstpart, MG63and VECs cells were subcultured to the fifth-generation backup.After established MG63-VECs co-culture systems, the best ratio of the twotypes of cells when they were cultured were determined through observing cellmorphology by an inverted microscope. In the second part, the best trainedproportion to establish MG63-VECs trained system in the first part asexperimental group, MG63and VECs training alone as control group,respectively in the12h,24h and36h and48h clear liquid extraction group,using the method of ELISA test was carried out on the experimental group andcontrol group all time points IGF-1expression value detection;Reuse transitChambers will VECs and MG63and indirect trained by the best proportion, theexperiment is divided into four groups, trained in group A, the trained group B,MG63group, VECs group, respectively in the12h,24h,36h and48h to RNA extraction of groups of each point in time, then use fluorescent quantitativePCR to detect the expression of IGF-1mRNA.Co-culture system in the thirdpart to observe the influence on vascular endothelial cell biologicalcharacteristics, on the one hand, by observation of DIi dyeing tracer method,will be DIi VECs dyeing, with MG63directly trained as the experimental group,according to1:1VECs DIi dyeing after training for the control group alone,96hours after the fluorescent microscope in the two groups of tube type;On theother hand through the rat tail collagen experimental observation.Use of rat tailcollagen preparation of three-dimensional culture, the experimental group ofMG63and VECs cell co-culture, the control of the VECs cells develop along.72h after they were observed under an inverted microscope tube type.More thanthe original experimental data analysis using SPSS13.0software, the data withmean±standard deviation (x±s), said two sample mean compare withindependent sample t-test;Multiple samples of two compared with analysis ofvariance (ANOVA) first, then use this comparison of SNK method ofcomparing two diverse,p<0.05and p<0.01showed significant difference and isvery significant. Results: The first part, MG63training to fifth generationobserved under inverted microscope, cell shape is irregular, triangle andpolygon.us assumes the circular or elliptic, the paving stone samplegrowth;Directly trained according to1:1MG63and VECs cells in culture twokinds of cells growth is most good, markings cell growth, the vascularendothelial cells in nodular growth, growth of osteoblast nodules in endothelial cells.Co-culture group in the second part, the ELISA results showed that IGF-1expression values than the control group increasedsubstantially(P<0.05).Fluorescence quantitative PCR results showed that12h-48h trained group A and group MG63IGF-1mRNA expression values overtime is on the rise, trained A group of IGF-1mRNA expression values in theMG63group increased significantly (P<0.05);Trained group B and VECs groupof IGF-1mRNA expression values appear unstable trend over time, within12hand36h is on the rise,24h and48h a downward trend;Trained A group ofIGF-1mRNA expression each time point were significantly higher than thosetrained group B raised (P<0.01).The third part, type tube in vitro experiment,DIi tracer method96h fluorescence microscope experimental class tubes VECspattern structure, control group upper VECs cells did not see an obvious patternstructure formation.Rat Tail glue test72h were observed under an invertedmicroscope, mature class vascular cavity structure control group; Experimentalsmall cell necrosis, not seen an obvious sample structure formation in the lumen.Conclusion:1, directly trained MG63and VECs according to1:1training intwo kinds of cells to grow the most good, cell markings, including VECs showsnodular growth, OB cell growth around the nodules in vascular endothelialcells.2, in the training system of MG63-VECs were the expression of IGF-1value superior to the two separate training;Indirect trained under the conditionof trained A group of IGF-1mRNA expression is significantly higher thantrained group B.3, through the cultivation system of tube type experiment proves that under the participation of osteoblast endothelial cells are more likelyto form a tube type is verified in the bone tissue vascularization OB not onlybuild the basic structure of bone tissue is a type of pipe induced cell function,participated in the VECs migration and tube formation.