Study The Biological Effection Of Chitosan Membrane And MG 63 Which Transfected With Bone Morphogenetic Protein Eukaryotic Expression Vector-2,7

Background:Bone defect is a kind of common,multiple and treatment complicated diseases, which due to trauma, inflammation,cancer and other acquired factors and congenital malformations, involving explicit and fuctional part, not only to patients with serious abnormal appearance and functional disorder, but also become a major challenge for clinicians. Recently, with the development of materials science and biology, tissue engineering has been become a new branch of science and develop rapidly because of high technique, and engineered bone has become a research hotspot.Objective:Chitosan materials can have good biocompatibility with seed cells became a key factor for application in bone tissue engineering. In this study, culture transfected cells with chitosan membranes and investigate biocompatibility which transfected cell on the membranes through cell proliferation and adhesion experiment.Methods:1 Construction bone morphogenetic protein-2,7 eukaryotic expression vector:1.1 Construction of eukaryotic expression vector:construct BMP-2/pcDNA3.1(-), BMP-7/pcDNA3.1(-)expression vector especially and analysis through sequencing, PCR and Restriction Enzyme.1.2 The constructed plasmid were used to transfect MG 63 cells, which are mediated by lipofectamine. To detecte the expression of hBMP-2, hBMP-7 in MG 63 by methods of RT-PCR, Western blot and Immunocytochemical stain.2 The proliferation anddifferentiation experiment that cells on the chitosan materials:2.1 MTS proliferation test:the membrane and cells were cultured in 96 well plate, have MG 63, MG 63/CS; transfected BMP-2, transfected BMP-2/CS; transfected BMP-7, transfected BMP-7/CS, MTS proliferation test were assayed at 24h,48h,72h, 96h.2.2 SEM obserbations:the membrane and cells were cultured in 24 well plate, have BMP-2/pcDNA3.1(-)transfected MG 63+CS and BMP-7/pcDNA3.1(-)transfected MG 63+CS, cultured 24h,72h,120h, colleted samples for investigated by SEM.Results:1 Construction of eukaryotic expression vector:the resutlts of sequencing, PCR and Restriction Enzyme show that BMP-2/pcDNA3.1(-) and BMP-7/pcDNA3.1(-) expression vector were constructed successfully.2 Transfection and expression:RT-PCR results showed that the mRNA of purpose gene was expressed in transfected cells, Western blot and Immunocytochemical stain results indicated that target protein BMP-2 B and MP-7were detected in transtected cells.3 MTS proliferation test:the growth MTS at 24h,48,72h,96h showed that cell grew and prolifated on chitosan surpace along with time lapsed. Compared with the control group, trsafected cells were no significant difference in proliferation (p>0.05), indicated that transfected MG 63 cells on chitosan membrane materials not have the significant change in vitro.4 SEM observation:It is showed that transfected cells could adhesion,grow and prolifetate on chitosan membrane materials at 24h,72h,120h.Conclusion:The construction of two eukaryotic vectors which coulod express BMP-2,BMP-7 in MG 63 were successful.Chitosan membrane materials have acceptable biocompatibility and profit for transfected MG 63 of adhesion and prolifetion, and the materials is expected to become the alternative materials for defect repair and recondtruction.