| There are two systems of hematopoiesis and matrix in the bone marrow cavity. The matrix contains fibroblast, desmacyte, adipocyte, macrophage, endotheliocyte and so on. Bone marrow stromal stem cells, a kind of multi-potential stem cells are also called the mesenchymal stem cells (MSCs). Bone marrow stromal cells (MSCs) are nonhematopoietic multipotent stem-like cells that adhere to culture dishes. They are capable of clonal expansion in culture, MSCs share characteristics with other multipotent stem cells such as neural stem cells, hematopoietic stem cells because they possess the ability to self-renew and can differentiate not only into osteoblasts, chondrocytes, neurons, and skeletal muscle cells but also into vascular endothelial cells and cardiomyocytes. It has been became a kind of ideal cell in tissue and cell repair for its merits asfollowed:1. Bone marrow is an altenrative source of stem cells which are well suited for clinical application because they are easily obtained from patients and because autologous transplantation,which obviates immunologicin compatibilities is possible.2. Of the various Progenitor cells that exist within bone marrow, mesenchymal stem cells (MSC) are particularly attractive for clinical use because they are easily isolated, can be expanded in culture,and call be genetically manipulated using currently available molecular techniques.3. MSCs are adult stem cells, which has no ethics questions. According to the metrits above, the MSCs will have wide perspective and practicability on the treatment of degenerative diseases, defects of tissues or organs and genetic defect diseasesLymphedema is a problem of tissue fluid imbalance often due to defects in lymphatic uptake and/or transport. Primary lymphedema results from developmental defects of the lymphatic system with fewer vessels leading to insufficiencies in transport, whereas the more common secondary lymphedema arises as a consequence of surgical, malignant, inflammatory, or traumatic disruption of the lymphatics. Although secondary lymphedema is a common clinical condition, treatment for this disabling condition remains limited and largely ineffective. The rebuild of circulatory structures is a good choice to treat lymphedema, but the way how to rebuild lymphatic vessels effectively is still under study.Recent molecular studies have begun to elucidate the basis for lymphangiogenesis which has been shown to be stimulated by various cytokines, including those in the vascular endothelial growth factor (VEGF) family:VEGF-A, VEGF-B, VEGF-C, VEGF-D, Orfvirus-encoded VEGF-E, and the placenta growth factor. These ligands bind to VEGF receptors (VEGFR)-1, VEGFR-2, and VEGFR-3with partially overlapping receptor specificities. In traditionally, the prototype VEGF binds VEGF receptor (VEGFR)-1and VEGFR-2and is angiogenic, whereas VEGF-C, which binds to VEGFR-2and VEGFR-3, is either angiogenic or lymphangiogenic in different assays, and the affinity of VEGF-C toward their receptors is regulated by proteolytic processing. The affinity of themature, proteolytically processed form of VEGF-C is40times higher for VEGFR-3than for VEGFR-2. Furthermore, in adults VEGFR-3is largely absent from blood vessel endothelia and remains predominantly expressed in the lymphatic endothelium. Targeted gene inactivation and transgene approaches in mice have also revealed the critical roles of VEGF-C in lymphatic vessel function Adenoviral expression of VEGF-C can induce lymphangiogenesis in the skin of normal mice. Thus, we induced the MSCs into endothelial cell with VEGF-C156s and labeled MSCs in vitro. We transplanted MSCs into the model of secondary lymphedema and observed the treatment effect and the migratation, development of MSCs in vivo.Rheumatoid arheritis is a kind of chronic, autoimmuitic arthrosis. Its pathological characteristic involves proliferation of synovial membrane vascular and infiltration of inflammatory cells, which result in the damage of synovium, cartilage, and the destruction of articulation. There have no special treatment without definite pathogenesis of rheumatoid arthritis. At present, the clinical methods conclude:1. Non-drug Therapy,2.drug therapy,3.Hematopoietic Stem Cell Transplantation (HSCT),4. Gene therapy,5. Surgical operation. The present methods mainly try to control the progression and regulate the immune response of human body. There have no more effective methods to cure the happened damage of cartilage.Bone morphogenetic protein belongs to a member of the transforming growth factor-β superfamily. Urist et al1965first isolated from the extract of the demineralized bone matrix to obtain a protein having activity, this protein has the undifferentiated mesenchymal cells differentiate into bone cells, and thus can be syntheic immunogenic proteinformation of bone tissue calcification ability, so named for the bone morphogenetic protein. In vivo and in vitro experiments show that BMPs regulating differentiation into osteoblasts and chondrocytes induced ectopic bone formation, promote fracture healing, and its control different morphological characteristics of mammalian skeletal formationfunction. With further research, the researchers found that the biological role of the BMPs are not limited to the development and formation of bone, involving almost all the systems of the body, including the cardiovascular, gastrointestinal, respiratory, urogenital, body and embryonic development of the nervous system,growth and differentiation. Currently, BMPs have confirmed that over40family members. BMP-6was originally isolated from a mouse placenta cDNA library obtained, and in accordance with the Xenopus Zodiac similar, was named as growth associated pale gene (Vgr-1). In1990Celeste et al were cloned from human placenta and fetal brain cDNA homologous to Vgr-1, based on its homology with BMP family named hBMP-6. The study shows that the BMP-6can activate mature cartilage cells, inducing mesenchymal cell differentiation into chondrocytes directions chondrogenic differentiation of an autocrine stimulation factor. Induced by BMP-6, or as target gene integration into MSCs induced differentiation of research at home and abroad is still small.Based on the above background, we intend to create a rheumatoid arthritis rat model, isolated and cultured in vitro amplification of bone marrow mesenchymal stem cells using an adenovirus vector hBMP-6-mediated gene transfection of MSCs and injected into the model of intra-articularinduced double therapeutic effect of anti-inflammatory and cartilage repair. ObjectiveTo build adenovirus vector carrying hBMP-6target genes; transfected bone marrow mesenchymal stem cells to differentiate into chondrocytes in arthritis model in rats and the secretion of the target gene protein for bone marrow mesenchymal stem cells in clinical treatment class rheumaticarthritis to provide new ideas and experimental evidence.Methods1. Bone marrow stem cells were isolated, cultured, identified:Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described, and flow cytometry to detect the surface markers:CD29, CD90, CD34and CD45. CD29and CD90positive of CD14and CD34negative cells were measured for the MSCs.2. The experimental study of Ad-hBMP-6adenovirus vector construction and bone marrow mesenchymal stem cells transfected:many reagents of this part of the experiment involved, the method is also more complex, and the technology sector of the population is difficult. The first thing to do is the adenovirus vector pDc strains resurrection, activation, formation the competent after digestion, identification, preservation, and other series process. Also apply the12-15passage HEK293cells resurrection, incubation, lipid plasmid transfection, in addition to the drugs, and other procedures, the first generation of toxic species involved in the next experiment. Application digested conversion means to achieve the build carry hBMP-6shuttle plasmid pDC316purpose, and later involved in adenovirus hBMP-6vector and restructuring. The recombinant adenovirus hBMP-6protein after immunization group, ELASA detected, amplified, concentrated, restructuring the detection of virus titration titer of7.94×1010IU/ml reach the standard of composite applications, ultimately proves our successconstructed Ad-hBMP-6adenoviral vector, containing BMP-6gene and the third step of the research work to provide effective tools on the molecular level.3. The theroputic study after transfection of bone marrow mesenchymal stem cells in the treatment of rheumatoid arthritis model in rats:we selected16 male Wistar rats modeling, strengthen the immune14d applications arthritis index evaluation method (arthritis index, AI) score, AI rated>2means successful. During the experiment, we observed rats living state, changes in body weight, model joint swelling degree. MSCs treatment group compared to the control group, accurate and detailed record of the measured data collated for statistical processing. MCSs different times after transplantation, the ankle after take decalcified, paraffin-embedded sections, line immunohistochemical staining, synovial mesenchymal stem cells in the bone marrow transplant adenovirus transfection damage to the CIA model rats, to evaluate the therapeutic effect.Results1. We successfully isolated from rat bone marrow bone marrow mesenchymal stem cells, the cultured and passaged identified, confirmed the mesenchymal stem cells in the bone marrow. Under certain conditions, to induce differentiation, making it having osteoblast differentiation potential of stem cells into chondrocytes.2. With the adenovirus AdMaxTM packaging systems, we successfully constructed a recombinant adenovirus carrying BMP-6gene hBMP-6carrier confirmed by restriction enzyme digestion and sequence analysis of recombinant plasmid was constructed correctly, without missing, and then by the insertion frameshift and other procedures are consistent withrequirements. And a variety of advanced technology using PCR, immunohistochemistry and ELISA confirmed gene hBMP-6the Ad-hBMP-6adenovirus vector was constructed successfully.3. We successfully constructed an adenovirus vector carrying the target gene BMP-6, and successfully transfected mesenchymal stem cells in the bone marrow.4. After transfection of bone marrow mesenchymal stem cells for the treatment of rheumatoid arthritis model in rats, and achieved the expected results.Conclusion1. We successfully isolated the mesenchymal stem cells in the bone marrow, cultured and passaged verified as mesenchymal stem cells in the bone marrow and stem cells induced differentiation potential to differentiate into chondrocytes.2. To successfully adopted adenovirus AdMaxTM, constructed a recombinant adenovirus carrying hBMP-6gene, digestion, sequence analysis, and other means restructuring adenovirus Ad-hBMP-6, building has been tested properly, without missing, after insertion, frameshiftand other procedures that are in line with the requirements, using PCR, immunohistochemistry and ELISA advanced technical methods, confirmed the adenoviral vector Ad-hBMP-6containing hBMP-6gene was successfully constructed.3. We carry the target gene hBMP-6adenovirus vector transfected the marrow mesenchymal stem cells, in vitro experiments showed that stem cells can differentiate into chondrocytes.4. hBMP-6gene adenovirus vector transfection of bone marrow-derived mesenchymal stem cells for the treatment of rheumatoid arthritis model in rats, to obtain a good therapeutic effect.5. This study provides experimental evidence of bone marrow mesenchymal stem cells in the clinical treatment of rheumatoid arthritis.6. The experimental results show that this experimental design, reasonable technical route, the advanced methods used to achieve the desired experimental research purposes.Bring New Ideas1. The first successful application the adenovirus AdMaxTM packaging systems and building a recombinant adenoviral vector carrying hBMP-6gene and transfected rat bone marrow mesenchymal stem cells generated to carry the target gene hBMP-6bone marrow mesenchymal stem cells.2. First application hBMP-6gene modified rat bone marrow mesenchymal stem cells in the treatment of collagen-mediated rat model of rheumatoid arthritis, and achieve meaningful efficacy for bone marrow mesenchymal stem cells to treat rheumatoid arthritisthe research opens up new ways and ideas.3. Lymphology, cellular and molecular biology, virology, immunology research a variety of advanced technology applied in the course of the experiment, a wide level of technology involved, difficult, a lot of the research process is to borrow the conventional methods can not complete the intended purpose, many methods and steps need to change the path, dose, time to get results. For example, the shuttle plasmid pDC316-hBMP-6’s build, recombinant adenovirus Determination of rat bone marrow mesenchymal stem cell infection efficiency. These improvements in technology and methods of change, in fact many ways is the study of innovation in technology, methods, and also from eating a lot of bitterness, making the five years courses extended to seven years. But I also obtain a valuable experience and lessons. |
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