| BackgroundBone defect caused by such reasons of the trauma, tumor and joint reoperated is the common problem of bone surgery. Autogenous bone grafts are limited for clinical application which have to face secondary surgery, source shortage, wound infection and so on.At the same time, because of the immune rejection, spread of diseases and ethics, the bone allograft is restricted in clinical application. The construction of active artificial bone materials provides direction for the bone defect repair, and seed cells and biological activity factor are important factor for the active artificial bone materials.The requirements of seed cell for the research of active artificial bone materials mainly:(1) suitable for clinical application, that is, wide range of materials, simple, small trauma.(2) there is no immune rejection after induction of cell transplantation into the body.(3) the potential of differentiation, in vitro amplification to reach the required number of cells to keep its ossification phenotype, in vivo can expand proliferation under the action of the induced factor. As is known to all, stem cells have the potential of multidirectional differentiation, which could differentiated to osteoblast, cartilage cells undenr special culture condition in vitro. Stem cells include organization stem cells and embryonic stem cells, embryonic stem cells have to faces a lot of ethics and safety problems, and the culture condition is very strict in vitro, so its application is limited. In comparison, tissue stem cells may be the best sources of seed cells foe bone tissue engineering especially the clinical application in the near future. At present,it is more clear that the tissue stem cells can differentiated to osteoblast mainly is bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells have much differentiation potential,which could differentiate into fiber cell, osteoblast and other cells.BMSCs make the attention of people for its value-added ability, wide range of materials, convenient and area a little injury.Biological activity factors play an important role in promoting bone repair process, bone defect healing is the process of bone tissue regeneration completely, the histological process accompany with complex molecular biology adjustment mechanism, a large number of active factors play different role in different bone healing stage, and the activity factors which promote the new bone formation and local reascularization are particularly important. Bone Morphogenetic Proteins (BMPs) are studied deeply as the biological factor for influence of new bone formation, which is the main factor to promote osteogenesis and play a decisive role on the differentiation of osteoblast. There are many Members in the family of BMPs,and approximately40kinds of BMP protein have been successfully separated. BMP2is the multifunctional cell factor of super family of transforming growth factor β, which could promote cell proliferation in bone damage local or ectopic and improve its alkaline phosphatase activity, and also it is only induced signal molecules for bone formation separately in the ectopic, consequently,BMP2has potential application value in the clinical treatment for the joint diseases, especially fracture and bone defect.Researchs show that BMP2is the strongest factor for osteoinduction in the family of BMP, a large number of experiments have confirmed that BMP2can promote bone formation directly or through the carrier local application. At the same time, bone vascularization is also a key link in the bone tissue engineering, which role is throughout the bone regeneration repair process. Adequately blood supply is not only beneficial to seed cell survival, proliferation and differentiation, and can also accelerate the formation of new bone, so as to speed up the bone defect repair. There are many biological factors for promoting vascularization, vascular endothelial growth factor (VEGF) is recognized as the strongest effect, which is one of the most specific factor to promote angiogenesis. VEGF has direct and indirect control effect in angiogenesis, on the one hand, it can stimulate the endothelial cell proliferation and migration, on the other hand, it can increase the permeability of blood vessels, which can make a lot of plasma protein bleeding blood vessels outside, which is important for the reconstruction of local extracellular matrix to angiogenesis. VEGF can also stimulate osteoblast differentiation proliferation, accelerate bone formation and remodeling. The VEGF165is a kind of secretory growth factor, widely occurring in a variety of tissues and cells, which not only promote angiogenesis activity, and its expression product is soluble, the expression is easy to reach target cells after secreting from cell because of strong diffusivity, and its biological activities can be performed enough.Since the discovery of BMP2and VEGF165are all involved in bone formation, the utilization of biological activity factor separately in local work has to face the shortage of half the period, easily washed away from body fluids or dilution problem, it is difficult to keep local effective treatment concentration. The development of gene technology provides us some effective means, gene therapy may be the most promising mean to maintain effective concentration of local biological activity factor. Currently the carrier of gene therapy are mainly divided into the non-viral carrier and the viral carrier, the lentiviral vector is used widely as the viral carrier. Lentivirus is a kind of immune deficiency virus, which is named from chronic infection caused by these viruses.The lentiviral vector is also a kind of retrovirus vector, which can infect splinter cell, but also can infect the cell division, the lentiviral vector can infect most not splinter cell, and with efficient integration and stability to express purpose gene in the target cell ability, so it has been widely applied in all kinds of gene therapy experimental research. Some scientists speculate that BMP2combined with VEGF165relative to a single gene will made more effective effect of bone regeneration, this speculation is worth trusted? it will be involved whether if VEGF165gene and BMP2gene co-transfection with stem cells can be used in bone defect repair, therefore, It is a great significance for assessing the role of BMP2and VEGF165in the rat bone marrow mesenchymal stem cells induced osteogenic regeneration.Objective1、Master the technique of lentivirus transfection to apply in the future other researchs based on Sprague Dawley (SD) rat bone marrow-derived mesenchymal stem cells (BMSCs) lentivirally co-transfected with hBMP2and hVEGF165.2、Investigate the role of BMP2and VEGF165genes in the rat bone marrow mesenchymal stem cells induced osteogenic regeneration in vitro.Materials and Methods1、BMP2and VEGF165cDNAs were amplified from Cyagen Biosciences by polymerase chain reaction (PCR) using the primers. Then BMP2and BMP2-VEGF165cDNAs were subcloned into the pLV.EX2d-EF1A expression lentivector respectively. Positive clones were selected by restriction endonuclease enzyme, lentivector were identified by PCR. Gene sequencing and comparison with the Genebank.2、293FT producer cells were co-transfected with pLV/helper Packaging Plasmid Mix and expression lentivector (containing BMP2or BMP2-VEGF165) plasmid by using Lipofectamine TM2000. Thus, the lentivirus containing BMP2cDNA (Lv-BMP2-EGFP-Neo) and lentivirus containing BMP2-VEGF165cDNA (Lv-BMP2-VEGF165-EGFP-Neo) were obtained, and detect the virus titer.3、Rat MSCs (obtained from Cyagen Biosciences)of passage5in culture were infected with Lv-BMP2-EGFP-Neo and Lv-BMP2-VEGF165-EGFP-Neo. The efficiency of lentiviral gene transfer in MSCs was quantitatively determined by the fraction of fluorescent cells with fluorescent microscopy at2days after transfection. When grew to fusion at80%, the two cells were screened with G418at various concentration of0.5mg/ml,1.0mg/ml,1.5mg/ml.4、Total RNA was extracted using the TRIzol method from each cell sample and the cDNA was synthesized from total RNA. Gene-specific primers for VEGF165, BMP2and GAPDH were designed as the primer set to detect the relative mRNA expression. MSCs were collected respectively from the2groups described above at5days after transfection or untransfected cells as control. QPCR was carried out using Syber Green Realtime PCR Master Mix. To correct for differences in both RNA quality and quantity among samples, the data were normalized to those for GAPDH.5、SD rat bone mesenchymal stem cells were infected with Lv-BMP2-EGFP-Neo and Lv-BMP2-VEGF165-EGFP-Neo virus and incubated for48hours. Transfection cells as experimental group and the untransfection group as control. The separated proteins of three groups were then transferred to a PVDF transfer membrane. Membranes were blocked for1hour at room temperature in Tris-buffered saline with casein. Antibodies against BMP2or VEGF165were incubated on the membrane The PVDF transfer membrane was washed with tris-buffered saline, and was developed using an enhanced chemiluminescence detection system and exposed to Fiuorchem HD2,observed and took pictures.6、SD MSC/BMP2-VEGF165and SD MSC/BMP2as experimental group and the untransfection group as control.The cells of each group were analyzed3d,7d and14d after cell seeding for osteogenic differentiation. Alkaline phosphatase (ALP) activities in the cells were determined by using the ALP Detection Kit.ALP and alizarin red staining were performed in each group at14days after osteogenic differentiation using osteogenic differentiation medium. The photos were be observed under the microscope.Results:1. Lentiviral expression vectors carrying BMP2and BMP2/VEGF165were successfully constructed. It is demonstrated that the constructions were correct with PCR identification and sequencing compared with Genebank.2. Large green fluorescences were observed because of fluorescence excitation after that lentiviral expression vectors carrying BMP2and BMP2/VEGF165transfected293T cells. The lentiviral titer were7.45x107TU/ml and6.45x107TU/ml respectively.3. The efficiency of lentiviral gene transfer in MSCs was quantitatively determined by the fraction of fluorescent cells with fluorescent microscopy at2days after transfection. Lv-BMP2and Lv-BMP2-VEGF165transfection rate were about85%and80%respectively, and the fluorescences were strong.4. The mRNA expression level of both BMP2and VEGF165genes in Control group were undetectable. VEGF165was immeasurable in BMP2group, however was measurable in BMP2+VEGF165group. For BMP2expression, not only in BMP2group but also in BMP2+VEGF165group can be detected. Moreover, no significant difference of BMP2expression was detected between BMP2+VEGF165group and BMP group (P>0.05).5. Western blot analysis indicated that the SD-MSCs which were transfected with Lv-BMP2-VEGF165or Lv-BMP2secrete large quantities of BMP-2, but those non-transfected secrete low quantities. On the other hand, all of the groups, SD-MSCs transfected or non-transfected with Lv-BMP2-VEGF165or Lv-BMP2, secrete large quantities of VEGF.6. The positive stain area in BMP2group was the largest of the three groups at14days after transfection. And the positive stain area in BMP2+VEGF165group was larger than that in control group, although it wasn’t as large as BMP2groups. Both of alizarin red staining and ALP staining was negative in the control group. The ALP activity was enhanced after14days inducing of osteogenic differentiation in all groups compared to that after3d and7d’s inducing (P<0.01). In BMP group, the ALP activity was further stimulated compared to that in BMP2+VEGF groups (P<0.01).conclusions:1. Overexpression of VEGF inhibits BMP2MSC differentiation and osteogenesis in vitro.2. Whether or not local VEGF gene therapy will effect bone regeneration in vivo needs to be further investigated. |
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