| [Objective and background] Bone defect which were caused by various types of congenital or acquired reasons is a common disease in plastic surgery. The rehabilitation of maxillary defects has been a difficult problem in clinical. The current and new treatment of this disease is constructing tissue engineered bone in vitro.Bone marrow Mesenchymal stem cells have the potential of multiline differentiation and are the early development of the mesoderm cells. They can not only differentiate into mesoderm from the same Mesenchymal cells, but also break mesoderm boundaries, differentiate into mesodermal tissue, such as fat cells, bone cells, cartilage cells, cardiac cells, nerve cells, muscle cells, tendon cells and astrocytes, etc. Currently, we have make great progress in the study that induce pure BMSCs to osteoblastic differentiation.However, this way exist some problems, such as a long cycle into bone, low efficiency to formation bone, cells easy to aging and other shortcomings.Researchers found VECs have the ability of secreting bone morphogenetic protein, stimulating osteoblasts and their precursor cells to secrete vascular endothelial growth factor when it promotes osteoblast differentiation, and the vascular endothelial growth factor play a very important role in the process of angiogenesis and the formation of vascular. It can promote endothelial cells proliferation and angiogenesis. These studies have shown that endothelial cells can support bone marrow Mesenchymal stem cells to change into bone. However, there is still lack of research from the level of genes in vascular endothelial cells of bone marrow mesenchymal stem cells into osteoblasts differentiation with specific gene signaling mechanism.Our research silented hUVECs'BMP-2genes expression with RNAi technology.We used the normal hUVECs,hUVECs with BMP-2gene silented and hBMSCs constructing co-cultue system,Then we analysis influence of each part in training system hUVECs hBMSCs Bmi-1and Runx2genes expression with fluorescence quantitative PCR technology;we confirmed the influence of the hUVECs to hBMSCs in proliferation and bone induction mechanism promotion; discusseed the hUVECs' BMP-2factors on hBMSCs Bmi-1and Runx2genes expression;clear BMP-2is hUVECs regulation hBMSCs Bmi-1and the main factors Runx2gene expression; provide the data and the theoretical basis for For hVECs and hBMSCs co-culture system ueing in bone tissue engineering seed cells research. This study can provide the data and theoretical basis of aplling hBMSCs and hUVECs to the joint co-culture system in bone tissue engineering seed cells research.[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 MSCs by its characteristic of adhesion to the plastic bottom. In order to identify the MSCs, We cultured MSCs to passage to the third generation and then we detected CD34, CD29, and CD44's surface antigen expression by flow cytometry.2. The hUVECs which were ordered were cultured in vitro, were identified by immunohistochemical staining method. Cells were cultured to the third generation,then the expression of BMP-2protein was verifid in4th,6th,8th,10th day by western blot method.3. We design four BMP-2gene interfering sequences and decorate it into plasmid Using the method of optimized liposome,we transfect cells with preconstructed interference gene sequence.Then we observed the effect of transfection by fluorescence microscope, detected the expression of BMP-2protein by western blot method, and identied RNA silent effect of hUVECs cells'BMP-2.4. The co-culture of the third generation hMSCs and HUVECs was established in the rate of1:1, and DMEM with10percent FBS were used as the medium of the co-culture system. The co-culture of the third generation hMSCs and HUVECs with RNAi treatment was established in the rate of1:1, and DMEM with10percent FBS were used as the medium of the co-culture RNAi system. The 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 make a statistical analysis about the test value with software SPSS17.0.5. Alkaline phosphatase and osteocalin were detected in hBMSCs group, co-culture group and co-culture rnai group at4th,6th,8th,10th day. And we make a statistical analysis about the test value with software SPSS17.0.6.The expression of Bmp-2were detected in hUVECs group, co-culture group and co-culture rnai group at4th,6th,8th,10th day by western blot method.4The expression of Bmil and Runx2gene were detected in hBMSCs group, co-culture group and co-culture rnai group at4th,6th,8th,10th day by fluorescence quantitative PCR method. And we make a statistical analysis about the test value with software SPSS17.0.[Result] l.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.The hBMSCs,which are cultured with FBS, are elongated spindle and small. The Primary generation cells grow into groups at4to5days. The third generation of bone marrow Mesenchymal stem cells forms a single, and into the spindle, vortex-like distribution, there is no cell overlap. The hBMSCs grow in logarithmic at4-6days, and into platform at8-10days. HUVECs grow as monolayer, polygonal shape, cobblestone-like arrangement. The hBMSCs have clear boundary and rich cell slurry,nuclei were round or oval. Occasionally.they show dual-core and confluent at5th. From first generation to fourth generation, the hBMSCs grow faster,2-3days can be passage.3. The target cells is affirmed as hUVECs by Immunohistochemical staining.Protein test shows they can normally express BMP-2protein.4.The cells BMP-2protein expression is decreased obviously after no.2plasmid transfected. The design of the shRNA interference plasmid is effective to silence hUVECs cells BMP-2protein expression. The best tranfection conditions is3ug Plasmid, l0ul liposomes and tranfect6hours, the efficiency is about60%5.The form change results of hBMSCs in Culture system are shown that the hBMSCs in co-culture group has a certain osteoblast differentiation performance at Each time point, The co-culture rnai group showed weaker osteoblast differentiation performance,there was not a obviously osteoblast differentiation performance in hBMSCs group.6.The number of hBMSCs in each group gradually increased with time, and the co-culture group's cell number was the highest at all time; the co-culture rnai group was lower than co-culture group;the hBMSCs group was lowest.The comparisons between all groups were statistically significant(P<0.01).7. Western-blot test shows that Bmp-2expression of hUVECs in the co-culture group was higher than its'in the hUVECs group.8. The amount of alkaline phosphatase in co-culture group and co-culture RNAi group gradually increased with time, The amount of alkaline phosphatase in hBMSCs group had a small changes at each time point。 The co-culture group's ALP was the highest at each time point。; the co-culture rnai group was lower than co-culture group;the hBMSCs group was lowest.The comparisons between all groups were statistically significant(P<0.01).9.The amount of osteocalcin in co-culture group and co-culture RNAi group gradually increased with time, The amount of alkaline phosphatase in hBMSCs group had a small changes at each time point。 The co-culture group's osteocalcin was the highest at each time point。; the co-culture rnai group was lower than co-culture group;the hBMSCs group was lowest.The comparisons between all groups were statistically significant(P<0.01).10.The expression of Bmi-1gene in co-culture group and co-culture RNAi group gradually increased with time, The expression of Bmi-1gene in hBMSCs group had a small changes at each time point。 The expression of Bmi-1gene in co-culture group was the highest at each time point。; the co-culture rnai group was lower than co-culture group; the hBMSCs group was lowest.The comparisons between the hBMSCs group and other groups were statistically significant(P<0.01), but The comparison between the co-culture group and co-culture RNAi group was not statistically significant(P>0.05).11.The expression of Runx-2gene in co-culture group and co-culture RNAi group gradually increased with time, The expression of Runx-2gene in hBMSCs group had a small changes at each time point。The expression of Runx-2gene in co-culture group was the highest at each time point。; the co-culture rnai group was lower than co-culture group; the hBMSCs group was lowest.The comparisons between the all groups and other groups were statistically significant(P<0.01).[Conclusion]1.We can get higher purity hBMSCs cultured with FBS by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.2.hUVECs can normally express BMP-2protein. Four plasmids constructed are all correct and No.2have the best effect.The result is up to standard of RNA interference requirements.3.1t shows good compatibility about the co-culture of hBMSCs and hUVECs. hUVECs can promote hBMSCs multiplication.4. Alkaline phosphatase and osteocalcin which were secreted by hBMSCs increased in the co-culture system and hBMSCs's Osteogenic Differentiation was speed up.5.hUVECs can enhance Bmi-1expression of hBMSCs in co-culture system, this is closely related to hBMSCs multiplication.6. Bmi-1gene expression of hBMSCs increased in the co-culture system, but this Signaling pathways is not regulated by Bmp2.7.hUVECs can enhance Bmi-1expression of hBMSCs in co-culture system, this is closely related to hBMSCs's Osteogenic Differentiation.8.hUVECs can enhance Runx2expression of hBMSCs in co-culture system by Bmp2,this improve that Bmp2/Runx2Signaling pathways exist in co-culture system.but Bmp2is not only one factor in Runx2pathways. |
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