The Mechanism And Effect Of Bone Marrow Mesenchymal Stem Cells In Transplant Arteriosclerosis

BACKGROUD: The common histomorphological feature of chronic transplant dysfunction(CTD) in solid organ transplants is the development of transplant arteriosclerosis(TA). TA is characterized by vascular lesions in the graft that consist of concentric myointimal proliferation resulting in the development of an occlusive neointima in the arterial structures of the graft, this progressive blood vessel occlusion could lead to graft ischemic tissue damage and disruptive fibrosis, and has therefore generally been accepted as the main cause of progressive deterioration in graft function(CTD). It has been recently identified that the cells of neointima originate from the recipient, which more than 95% of the cells in neointima was made up of vascular smooth muscle cells, many kinds of cells can contribute to intimal hyperplasia and differentiate to smooth muscle cell. Bone marrow cells involve in intimal hyperplasia depending on the extent of damage in media smooth muscle cells, only when the media smooth muscle cells suffer from severe damage, bone marrow cells would be mobilizated in the intimal and take part in the process of vascular repair. Bone marrow mesenchymal stem cells have the ability of multipotency differentiation, which are involved in repair of many kinds of tissues. Though bone marrow mesenchymal stem cells are inducible to differentiate to smooth muscle cells and endothelial cells in vitro, it is uncertain yet whether bone marrow mesenchymal stem cell plays a role in transplant arteriosclerosis. So in this study we firstly investigated the effect of bone marrow mesenchymal stem cells contribute to transplant arteriosclerosis and then promotion on bone marrow mesenchymal stem cells involved in transplant arteriosclerosis by inducing a great quantity media smooth muscle cells apoptosis. Our research can help to elucidate mechanisms involved in transplant arteriosclerosis and to provide effective therapeutic strategies of both theorical and application significance. OBJECTIVE: To construct bone marrow mesenchymal stem cell which can express EGFP stablely, to identify the bone marrow mesenchymal stem cells involved in transplant arteriosclerosis, to construct lentiviral vector carrying P53 drived by SM22αpromoter, to figure out the effect of LV-SM22α-P53 vector-induced vascular smooth muscle cells apoptosis on proliferation and migration of bone marrow mesenchymal stem cells in vitro and on transplant arteriosclerosis in vivo.Methods: Primary culture of endothelial cells from rat aorta by ordinary vascular ring method, Collagenase digestion method and improved vascular ring method, purification of endothelial cells by limiting dilution assay, primary culture of smooth muscle cells from rat aorta by tissue pieces method. SM22αpromoter was amplified by PCR technology, to construct lentiviral vector driven by the promoter, infection of vascular smooth muscle cells and endothelial cells in vitro by the lentivral vector. Primary culture mesenchymal stem cells from rat bone marrow, identification of mesenchymal stem cells by cell surface marker detected by flow cytometry and the ablility of differentiation in vitro. To construct BMMSC-EGFP that mesenchymal stem cells which can express EGFP stablely by lentivirus carrying EGFP infection. To establish rat aorta transplant model, infusion BMMSC-EGFP to recipient rat through tail vein, transplant arteriosclerosis detection by histological section, observing EGFP-expressing cells in graft by fluorescence mircoscope. Constructing lentiviral P53 driven by SM22αpromoter, inducing smooth muscle cells apoptosis in vitro by the lentiviral vector infection, and detection of apoptosis by Hoechst33258 staining and flow cytometry by Annextin V-FITC/PI staining, collected and concentrated the supernatant of cell apoptosis, to measure the proliferation effect of cell apoptosis supernatant on mesenchymal stem cells by CCK-8, to evaluate the migration effect of cell apoptosis supernatant on mesenchymal stem cells by transwell chamber. Aorta graft infection ex vivo by lentiviral P53 driven by SM22αpromoter, to detect apoptosis of media smooth muscle cells by TUNEL method, to observ the EGFP-expressing cells in neointima by fluorescence microscope. Results: Endothelial cells cultured by improved vascular ring method had best performance. Limiting dilution assay can purify the primary endothelial cells from rat aorta, vascular smooth muscle cells were cultured successfully by the method of tissue pieces method. SM22αpromoter was identified by sequencing, the lentiviral vector which driven by SM22αpromoter was constructed successfully, it can infect vascular smooth muscle cells specifically at high efficiay in vitro. The bone marrow mesenchymal stem cells via total bone marrow adherence method were of high purity, identified by flow cytometry detecting cell surface marker, it can differentiate to adipocyte, vascular smooth muscle cell, and osteocyte in vitro, it was necessary to keep pH value stable to long-term culture in vitro. The lentiviral vector infected bone marrow mesenchymal stem cells stably at high efficiency, which was strengthened by application of polybrene. Typical transplant arteriosclerosis was observed in graft by mircoscope, EGFP-expressing cells which arranged multilayers in neointima located near lumina. LV-SM22α-P53 vector was constructed successfully, vascular smooth muscle cells can be inducing apoptosis in the condition of apoptosis stimuli reagent existence after lentivirus infection, the concentrated cell apoptosis supernatant can promote the proliferation and migration of bone marrow mesenchymal stem cell. Infection of lentiviral P53 drived by SM22αpromoter leaded to mass apoptosis in media smooth muscle cells, and more EGFP-expressing cells can be found in neointima by fluorescence microscope.Conclusion: Bone marrow mesenchymal stem cells involved in transplant arteriosclerosis is allied to media smooth muscle cells apoptosis, inducing media smooth muscle cells apoptosis can promote mesenchymal stem cells involved in transplant arteriosclerosis, it may be ascribed to apoptosis of smooth muscle cell can promote migration and proliferation of bone marrow mesenchymal stem cells.