| [Study background and Objective]Study background:The repair therapy of bone defect result from trauma, infectation,necrosis and bone neoplasms is always a tough problem of orthopedics. In order to resolve this issue, many scientists find that human bone marrow Mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (hUVECs) co-culture maybe stimulate the proliferation and differentiation of hBMSCs and accelerate the bone healing. In recent years, researchers found vascular endothelial cells(VECs) have the ability of secreting bone morphogenetic protein, wich is the best bone induced factor all of the world. The effect of secreting BMP of hUVECs whether is a key role in hBMSCs and hUVECs co-culture? According to the specific inhibition of BMP-2gene by siRNA, we designed siRNA targeting BMP-2and gain small hairpin loop RNA, and transfected in hUVECs. To demonstrate the role of BMP-2in hUVECs by observate the expression of BMP-2gene in h UVECs after inhibition. So, we can provide a new knowledge and best therapy for tissue engineering and regeneration Medicine.[Objective]:(1) To construct hBMSCs and/or hUVECs co-culture system isolated from the bone marrow of hunman in vitro.(2) According to the guidance of FuNeng company in GuangZhou, we designed siRNA targeting BMP-2and gain small hairpin loop RNA, and transfected in hUVECs.(3) To detect the expression of BMP-2mRNA and protein after transfection by special shRNA on hUVECs. (4) To observe the inhibition effects of shRNA targeting BMP-2on the proliferation, adhesion and migration of cultivation of the hUVECs and hBMSCs co-culture.[Methods](1) We extracted5millilitre of 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) We were selected hUVECs of ATCC(hUVEC, Sciencell company, catalog number:8000) as the source of hVECs. To observe the morphosis of all kinds of cells in complete medium(500millilitre of basic fluid, blood serum, growth factor and double antigen) at37centigrade after the third generation of passage in5percent saturated humidity incubator. The third generation of passage of hBMSCs, hUVECs and interferential hUVECs were to establish the system of cultivation of the co-culture in the6wells of23plates.(3) BMP-2mRNA sequence of hUVECs was retrieved in nucleotide library of NCBI. BMP-2specific shRNA(shRNAl, shRNA2, shRNA3and shRNA4) was designed and chosed on Dharmacon siDESIGN Center. And further, four kinds of shRNA were synthesized the plasmid vector of marked eGFP. In addition, the gene sequence of random was synthesized to negative control. The gene sequence of interference was transfected in hUVECs by pure method. To observe the transfection effects by fluorescence microscope, to detect the expression of BMP-2by SDS-PAGE, Bradford and Western Blot.(4) The experiment were divided into three groups, I group:Cultivation of hBMSCs; II group:Cultivation of the co-culture after RNAi; IIIgroup:Cultivation of hBMSCs and hUVECs co-culture;. 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 detected alkaline phosphatase and osteocalin in three culture groups(I, III and IV group) at4th,6th,8th,10th day. We detected the expression of BMP-2gene in hUVECs groups(II, III and IV group) at4th,6th,8th,10th day. At last, we make a statistical analysis about the test value with software SPSS17.0.[Results](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) The results of DNA plasmid sequence were shown that the second plasmid sequence is the best inhibition effects for BMP-2. To measure the expression of BMP-2by Western Blot, the level is similar to the second plasmid.(3) After transfection by pure plasmid on hUVECs at48hours, the transfection efficiency is60precent by fluorescence microscope.(4) The amount of alkaline phosphatase in each group gradually increased with time, and the co-culture groups 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<0.01). 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<0.01).(5) IVgroup comparison of IIIgroup revealed the ALP and osteocalin of hBMSCs is lower by cell growth curve. And the proliferation, adhesion and migration of cultivation of hBMSCs co-culture at IVgroup is better than I group. Moreover, the level of ALP and osteocalin is also higher than I group.[Conclusions](1) One BMP-2specific shRNA plasmid vector was successful synthesized in vitro through MessageMuterTM shRNAi Production Kit. It can supply stable RNAi technique for following experiments.(2) Small hairpin RNA targeting BMP-2of hUVECs efficiently and specifically inhibits the expression of BMP-2.(3) To determine the osteoblast differentiation status of the hBMSCs is down-regulation after RNAi in the co-culture(IVgroup). Meanwhile, IVgroup also reveal low ALP and osteocalin level.(4) The results of study is also suggested that shRNA targeting BMP-2of hUVECs maybe inhibit the osteoblast differentiation status of the hBMSCs in cultivation of the co-culture. |
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