| Background: Allogeneic bone marrow transplantation(allo-BMT) offers a cure for the patients with malignant and nonmalignant diseases. However, graft-versus-host disease(GVHD) interferes with the development of allo-BMT because of its high incidence and high mortality.Traditional methods to prevent and cure GVHD include systemic immunosuppression and T-cell depletion from graft. Systemic immunosuppression increases susceptibility to infection and heoplasia. As to T-cell depletion, which weaks the action of graft-versus- leukemia (GVL). Studies have shown that the mature T-cells have a central role in the occurrence and development of GVHD,but two signals are required for T-cell activation.An antigen-specific signal is delivered by T-cell receptor binding of peptide presented by class I or class â…¡ molecules. Additional costimulatory signals are also required and are antigen nonspecific .Hence,the lacks or blockades of costimulatory signals will result in the occurrences of T-cell anergy, apoptosis or clone depletion,etc.As a result,immune tolerance has been induced. Among so many costimulatory signals,B7/CD28 pathway has been proved that it is the most effective and definite.In view of this,interfering with B7/CD28 pathway to induce specific immune tolerance will be a new method to prevent GVHD.Bone marrow stromal cells(BMSCs), an important components of hematopoietic inductive microenvironment (HIM), have many actions on the habitation, proliferation, differentiation, growth and maturation of hematopoietic cells.On the other hand,HIM damage resulted fromforedisposal is one of important causes that lead to hematopoietic damage,poor immunity,and even transplantation failure. Aims: To investigate the possibility of BMSCs,which was genetically modified by adenovirus to express CTLA4Ig to induce antigen-specific T cells immune toleronce,search for the molecular mechanisms related with immune tolerance, and observe the possibility of the modified BMSCs to enhance hematopoiesis. Methods: The cultured BMSCs were transfected by recombinant adenovirus encoding CTLA4IgcDNA(AdCTLA4Ig)(MOI=50). The. transcription of CTLA4Ig-gene was assessed by RT-PCR, we .used immunohistochemical techniques to confirm the expression and the location of CTLA4Ig in the transfected BMSCs,To ascertain the time phase of CTLA4Ig expression,we used Dot-ELISA to detect it in the culture.medium at different time points.The biological activity of the purified CTLA4Ig was tested by mixed lymphocyte reaction (MLR) through MTT, followed by IL-2 quantitation with ELISA in the MLR system.We measured the value of cpm from the mononuclear cells(MNC) marked by 3H-TdR after having adhered to the BMSCs to evaluate the change of adhesion ability because of transfection.On the other hand,we compared the function of supporting CD34+ cells to proliferate through the numbers of cells and colony forming cell (CFC).Results: RT-PCR can detect the transcription of CTLA4Ig-gene in the transfected group and in the passage number two.Immunocy tochemistry indicated that the rate of CTLA4Ig expression in the transtected BMSCs (MOT=50) was as high as 85%,and the expression located in cytoplasm of BMSCs disseminatedly.Dot-ELISA could detected the expressed CTA4Ig in the culture medium at 12h and even in culture medium of the passage number l after transfection.The level of CTLA4Ig in culture medium was at peak at 72h.MLR tested that CTLA4Ig had significant inhibition on lymphocyte proliferation.Morever,the inhibition effect was concentration-dependent within a certain range.The change of IL-2 level in the MLR system had a similar tendency.Morever,the level of IL-2 was correlated with the number of lymphocyte positively (r=0.8522) .Compared with the control group,the transfected groups had significant differences in IL-2 level and the number of lymphocyte (p<0.05).But their abilities to support CD34+ cells to preliferate and adhesion abilities were not significantly different (p>0.05).Conclusion: 1.Ad-CTLA4Ig could transfect BMSCs...
|
PDF Full Text Request