| [Objective]Different reasons caused by various types jaw bone defect in clinical are very common, and now commonly used repair methods such as distraction osteogenesis, autogenous bone,allograft, such as by donor shortage, often donor lesions, secondary injury, potential effects, infection of the factors such as check and improved urgently. Jaw bone defect repair problems have been is clinically difficult problem to solve. Tissue engineering has emerged and development for bone defect repair brought the opportunity, its ultimate objective will function cells and biodegradable3d biological scaffold materials in the joint training in vitro, built into the active tissue or organs, and then implanted in our bodies, alternative damage organization, restore its form, structure and function. At present bone tissue engineering method of building simple seed cells multi-purpose vaccination, although can osteogenesis, but there are vascularized bone tissue engineering slowly, and the new bone growth slow growth not stable.Human bone marrow Mesenchymal stem cells (hBMSCs) has the potential of multiplex differentiation, in the right conditions can not only differentiation for fat cells, bone cells, cartilage cells, myocardial cells, neurons, into muscle cells, tendons cells and astrocytes, etc. At present about induction pure hBMSCs osteoblast differentiation to achieved considerable progress, among them with BMP2, BMP4, BMP6and the function of the BMP7the most representative, can cause a variety of cell proliferation, differentiation and apoptosis, participate in tissue regeneration and to build (remodeling), in the cell division and migration of many kinds of life activities play a regulatory role. However hBMSCs induction pure osteoblast differentiation to existing osteogenesis cycle is long, the efficiency is low, the cells lining the aging shortcomings.In recent years, researchers found that endothelial cells can secrete BMP (Bone morphogenetic protein), promote Bone of osteoblast differentiation and stimulate and the progenitor cells secrete vascular endothelial growth factor (VEGF), and VEGF in angiogenesis and forming process plays an important role, can promote the endothelial cell proliferation. But now the vascular endothelial cells of bone marrow mesenchymal stem cells lining the disintegration of the specific mechanism are not clear still, still lack the gene level validation of bone marrow vascular endothelial cells lining the mesenchymal stem cells differentiate role. Smad protein family is the new cells found in recent years signaling protein, directly involved in TGF-B super family members into growth factors(Transforming growth, TGF-beta), BMP (bone morphogenetic protein), BMPs in the present people, a total of nine Smad family members. Among them, Smadl,2,3,5,8, the regulation of the receptor for Smad (receptor-regulated-Smad, R-Smad), Smad-4for common accidentally ligands Smad (common-partner-Smad, Co-Smad), Smad-6,7for suppression Smad (inhibitory Smad,]-Smad). BMPR-I make Smad-1,5,8, at the end of the ser (SSXSmotif) phosphorylation, then two or1R-Smad and a Smad-four to three different source polymer or different source dimers R-Smad-Co-Smad form into the nuclear, action at the osteoblast specific transcription factors Cbf a1, Osx, etc on the genetic sequence of lift its expression, and strengthen the AKP osteoblast and OC expression. In order to promote the osteoblast differentiation and mature.Runt related gene2(Runx2) transcription factors:Runx2, say again Cbfal, or NMP2, or AML3, is Drosophila Runt protein homologous protein. Now known, in the development of Runx2bone in the process of mesenchymal cells in the condensed, mesenchymal stem cells to the osteoblast differentiation, cartilage cells of the fat DaHua, bones, and other steps into the blood vessels, such as vascular endothelial growth factor (vascular endothelia growth factor, VEGF) expression regulation Runx2tissue specific to the participation to promote bone into the blood vessels. The osteoblast and mast cartilage cells are Runx2expression, the osteoblast differentiation and cartilage cells of all need it in the mature, Runx2is adjusting cartilage cells eventually mature key factor.The experiment proved Runx2defects in mice, due to the bone and cartilage were ruled out in the film bone, the bone structure no bone formation, the mice in soon after the birth of death. Runx2mutation is congenital cranial collarbone dysplasia pathogenic genetic basis. Runx2don't play directly osteogenetic gene expression regulatory role, it must be combined with a variety of protein, in different stage or within cells play different roles, has the characteristics of space and time. So, Runx2bone is called the central adjustment factor. At least12kinds of Runx2isomers was found, of which Runx2-Ⅰ role in early stage of osteoblast differentiation and membrane in ossification process, Runx2-Ⅱ role of osteoblasts in the mature and in cartilage ossification process, the function of other isomers is not clear.Therefore, the subject focuses on human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs) in the joint training system in a human bone marrow Mesenchymal stem cells (human bone marrow Mesenchymal stem cells, hBMSCs), the shape of the growth, cell differentiation and Smad-1genes and gene expression Runx2, the effects of the validation from gene level of bone marrow vascular endothelial cells lining the Mesenchymal stem cells differentiate role. For umbilical vein endothelial cells and bone marrow mesenchymal stem cells in the joint training as the bone tissue engineering seed cells provide theoretical foundation.[Method]1We extracted a volunteer's bone marrow fluid and isolated the bone marrow mononuclear cells by the way of density gradient centrifugation. And we purified the hBMSCs by its characteristic of adhesion to the plastic bottom. In order to identify the hBMSCs, We cultured hBMSCs to passage to the third generation and then we detected CD34, CD29, and CD44's surface antigen expression by flow cytometry.2We observed the morphological changes by phase contrast microscope. The co-culture of the third generation hBMSCs and HUVECs was established in the rate of1:1, and DMEM with10percent FBS were used as the medium of the joint culture system.3The separate cultured hBMSCs and hUVECs as a negative control group.We observed the morphological changes by phase contrast microscope in4th,6th,8th,10th day, and counted the number of each hBMSCs group by count plate; we detected alkaline phosphatase and osteocalin in three culture groups at4th,6th,8th,10th day. And we make a statistical analysis about the test value with software SPSS17.0.4We detected the expression of smad-1and Runx2gene in hBMSCs group and co-culture group at4th,6th,8th,10th day. At last, we make a statistical analysis about the test value with software SPSS17.0.[Result](1)We make an analysis and identification on third-generation hMSCs's cell phenotype By flow cytometry, the expression of CD34is negative and the expression of CD29,CD44are positive; We can get higher purity hBMSCs by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.(2) new tyres bovine serum separation of the training of the human bone marrow mesenchymal stem cells into thin spindle, cells for the typical fibroblasts kind, swirling stick a wall growth, a tiny cells, the cart before the horse a microscope, visible within24h after inoculation a few individual nucleus cell wall stick, stick wall cells in a circle, have little cytoplasmic projections. The original generations to cultivate4to5days until the growth of visible cells; The third generation of bone marrow stromal stem cells form a single, into a spindle, a spiral shape distribution, have apparent polarity, no cell overlapping phenomena. HBMSCs in four to six days, a logarithmic growth,8-10days later growth into platform stage, the quantity change is not obvious. HUVECs about2-3days or visible single cell growth, the polygons in shape, cobble Mosaic of arrangement, borders, the cytoplasm is rich, the nucleus a round or oval, accidentally see dual-core,5d confluent, first generation to the fourth generation of growth speed,2-3days can be passage.;(3)The amount of alkaline phosphatase in each group gradually increased with time, and the co-culture group's ALP is the highest at all time; The ALP of Mesenchymal stem cells group and umbilical vein endothelial cells group did not change; The comparisons between co-culture group and other groups were statistically significant(P<0.05). The osteocalcin detected in each group was gradually increased with time. The highest osteocalcin of co-culture group is detected at8th day. The comparisons between co-culture group and other groups were statistically significant(P<0.05).(4)The volume of co-culture group's Smad-1and Runx2gene was gradually increased with time, and the co-culture group's gene expression is the highest at all time; The expression of Smad-1,Runx2in Mesenchymal stem cells group and umbilical vein endothelial cells group did not change;The comparisons between co-culture group and other groups were statistically significant(P<0.05). The osteocalcin detected in each group was gradually increased with time. The highest osteocalcin of co-culture group is detected at10th day. The comparisons between co-culture group and other groups were statistically significant(P<0.05).[Conclusion]1We can get higher purity hBMSCs cultured with FBS by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.2It shows good compatibility about the co-culture of Mesenchymal stem cells and umbilical vein endothelial cells. Umbilical vein endothelial cells can promote bone marrow Mesenchymal stem cells proliferation.3The joint training group Smad-1, Runx2gene detects the quantity is increasing extended over time, and the other group contrast the time the joint training group Smad-1, Runx2gene expression were higher;8days Smad joint training group-1, Runx2gene detects the highest amount; The joint training group and bone marrow stromal stem cell group is statistically significant between significance (P<0.05). |
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