| Background: Massive bone defects caused by severe trauma,infection,tumor resection and bone diseases are very common in clinic work and are a great challenge to reconstructive surgery. Bone tissue engineering is a perfect method to treat massive bone defect prior to other methods,such as bone autotransplantation,bone allotransplantation and prosthetic replacement. Tissue engineering combines knowledge from the biological sciences with the materials and engineering sciences to quantify structure-function relationships in normal and pathological tissues, to develop new approaches to repair tissues, and to develop replacements for tissues. It involves three main elements: seed cells,scaffold and tissue reconstruction technique .Up to now,it is considered that bone marrow derived mesenchymal stem cells(MSCs) is one of the best seed cell sources of bone tissue engineering. Human mesenchymal stem cells are thought to be multipotent cells, they have potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, marrow stroma, etc. Although no specific symbol is detected in expressed surface antigens,MSCs comprise a single phenotypic population (95 and 98% homogeneous at passages 1 and 2, respectively). That is positive for SH3, SH4, CD105, Stro-1, CD29, CD44; and is negative for CD34,CD45,CD14. Many investigators gain good results when they use MSCs as seed cells in bone tissue engineering to repair bone defect of animal model by autotransplantation or allotransplantation into immunodeficiency animals. But the number of human MSCs is very few and gradually decreases with age;it is also hard to expand in vitro. Therefore, autotransplantation of MSCs is limited by the number of MSCs. Allogeneic MSCs application is not limited by their number;and it maybe another seed cell source of bone tissue engineering. However, allogeneic MSCs grafting results almost invariably in graft rejection,unless immunosuppressive therapy is given to control the alloimmune response. Rejection occurs because of allelic differences between graft and host at polymorphic loci. That give rise to histocompatibility antigens. On the other hand, although MSCs express major histocompatibility complex(MHC) class I and adhesion molecules including VCAM-1, ICAM-1, and LFA-3; they express negligible levels of MHC class II and do not express B7-1, B7-2, CD40, or CD40L. These features are responsible for their low immunogenicity. Previous studies indicate that MSCs fail to elicit a proliferative response of allogeneic lymphocytes. MSCs lead to reduction in proliferative activity when they add into a mixed lymphocyte reaction;and this effect depends on the dose of MSCs.In order to avoid shortcomings which go with autotransplantation of MSCs,we try to explore the possibility of using gene modified MSCs as allogeneic seed cells of bone tissue engineering. Therefore,we must overcome the graft rejection along with allotransplantation. As we know,there is no method that can induce donor specific,complete and permanent immune tolerance. Among numerous studies of immune tolerance,CTLA4 is one of the most important focus points. CTLA4 can bond B7 molecule on APC competitively with CD28 and block the B7-CD28 costimulatory pathway. That inhibits T cell full-activation, induces T cell anergy, apoptosis or clone deletion, results in immune tolerance. CTLA4Ig, a fusion protein of CTLA4 extracellular domain and partial segment of IgG-Fc,has the same funtion with CTLA4. Therefore,we design to modify MSCs with CTLA4Ig gene and screen out transfected cells(MSCs-C),then explore the possibility of using MSCs-C as allogeneic seed cells of bone tissue engineering. Furthermore,MSCs have a limited life-span and gradually lose their stem cell properties during culture in vitro,and this feature is not very suitable for mono-clonal screen. For this reason,if we want to screen out mono-clone of gene modified MSCs,we should extent its life-span at first. Some researcher reported that ectopic expression of human telomerase revers...
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