| Pathological responses of the structure and function of blood-brain barrier (BBB ) can occur in the brain ischemia caused by middle cerebral artery occlusion ( MCAO). Inflammatory cells and some enzymatic toxic substances in the circulation enter the brain resulting in the neurons' functional deficits, which causes some clinical symptoms such as hemiplegia hemianopia and partial sense disorders. It has been reported previously that adult mesenchymal stem cells (MSC) could enter the brain of rats and differentiated into neurons to heal the injury of central nervous system when they " heard" the signal from the injured neurons in the brain. Mesenchymal stem cells are a homogenous population of non- hematopoietic stem cells that have diverse potential for differentiation and neural specific markers can be detected on MSC after inducement in vivo and in vitro. Because of the convenient obtainment isolation culture and comparably lesser immunocompetence, MSC can be used for the treatment as donors of cell transplantation in the appropriate condition.Although there is now evidence that mesenchymal stem cells lie in the receptor ' s brain tissues and differentiate in the right way after systemic transplan-tion, it is not in the full conviction that how MSC can cross the blood-brain barrier and transmigrate into the cerebral tissue. So in the meantime, we also focus our mind on the molecular mechanism of interaction of MSC with human brain microvascular endothelial cells ( HBMEC) , the in vitro model of BBB. It will provide the experimental evidence and theoretical basement for the control of MSC across BBB to heal the neural injury.In the inflammatory diseases, the transmigration of capillary endothelium includes rolling adhesion and migration, which is the result of recognizing andinteraction of adhesion molecules, and the firm adhesion by integrina4/VCAM-l signaling is more pivotal than others. It is reported that both lymphocytes and neural precursor cells express mtegrina4 when they cross BBB in the study of multiple sclerosis. The tight binding of integrina4 with VCAM-1 mediates FAK/ Paxillin phosphorilation and cytoskeletal rearrangement, which facilitate adhesion on endothelial cells and enlargement of lymphocytes or neural precursor cells. So, there is certain theoretical and practical foundation to analyze expres-sional alterations in the gene and protein level of integrina4/VCAM-l caused by the interaction of MSC and HBMEC.This research is aimed at two aspects including in vivo and in vitro experiments. In vivo experiment: systemic venous transplanting mesenchymal stem cells of adult rats into cerebral ischemia animal model to heal neural injury. In vitro experiment; analyzing the molecular mechanism of integrina4/VCAM-1 pathway caused by the interaction of MSC and HBMEC. Whereby, to elucidate the mechanism of mesenchymal stem cells crossing the blood-brain barrier and to accumulate evidence for therapeutic benefit of MSC to ischemic cerebral tissue.Methods1. In vivo experiments1. 1 Isolation culture and identification of MSCa. Obtainment of bone marrow cells from male adult rats under aseptic conditions , isolation, culure and propagation in vitro.b. Immunocytochemistry identification of the expressions of CD45 and CD90.1.2 Suture occlusion MCA model.1. 3 Preparation of brain tissue sections.a. Transplantation of MSC introduced by tail vein injection into the MCAO model.b. Preparation of ice sections after brain perfusion and fixation. 1.4 Preparation of Cy3-labelled Y chromosome DNA probe.a. To obtain the genomic DNA from male rats and to generate the PCRproduct (459bp) by using primer set to regions of the SRY gene ( " sex-determining region" on Y chromosome) of rats.b. Establishment of recombinant plasmid pUCmT-SRY459.c. To obtain the PCR product (305bp) from the recombinant plasmid pUC-mT-SRY459 as the DNA probe and to fluorescence label the probe using Cy3-dCTP.1.5 Triple-color fluorescent dyeing of cerebral cells.a. Fl... |
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