The Mechanism Of Peripheral T Lymphocyte Transmigrating The Blood-brain Barrier In Alzheimer's Disease

Alzheimer's disease( AD)is a disease seriously affecting the senior people's living conditions. According to the statistics, There are about 3-4 million people with AD in China. There are no effective prevention and treatment method for AD because its pathology and mechanism is unknown.There are a lot of theories about AD. The theory of D. J. Selkoe in Harvard believed that the disturbance in the production and elimination of amyloid - B protein (AB) is the forepart and center tache of AD. The elimination of AB is related with immune respond.The purposes of this experiment is to probe into the capability of the peripheral immune cells of AD transmigrating blood - brain barrier, and to find out that MIP - la was abnormally expressed in T lymphocyte of AD patient. Further we investigated its function in the transmigrating process. We are trying to do some basic work to investigate the molecular mechanism about the T lymphocyte transmigrating BBB and to find out the interfering method.Methods1 The blood - brain barrier( BBB) : Place 2 x 106HBMEC in Transwell culture membrane, cultured 3 days before use. HBMEC was donated by K. S. Kim of Johns Hopkins Medical University.2 Selected 31 AD patients and 25 age - matched healthy contrast. Collected their blood samples. Separated the peripheral blood mononuclear cell(PBMC). Counted the cell density and the CD4 + CD8 + CD14+ CD19+ rate by flow cy-tometer. Added the PBMC onto the BBB model. Cultured them in 5% CO237℃ for 20 hours. Counted the cell under the well and analysis the subset on flow cytometer. Calculated the transmigration rate of respective subsets.3 Examined the ZO - 1 and F - actin change of HBMEC adhered by T lymphocyte of AD and its model cell; 6T - CEM.3. 1 Cultured HBMEC on the cover slice to confluent. Added T lymphocyte of AD patient and age - matched healthy contrast separated by immunofluorescence magnetic beads on the transwell. Cultured at 37℃, 100% moist for 20 hours. Examined ZO -1 at different time by immunofluorence method.3. 2 Cultured HBMEC on the cover slice to confluent. Added 6T - CEM on the transwell. Cultured at 37℃ and took out the cover slices at hour 4 8 and hour 20. Examined ZO - 1 by immunofluorence method.3. 3 Cultured HBMEC on the cover slice to 60 -70% . Added T lymphocyte of AD patient and age - matched healthy contrast separated by immunofluorescence magnetic beads on the transwell. Cultured at 37℃ , 100% moist for 20 hours. Examined the F - actin by immunofluorence method dyed with rhodamin- phallodin.3. 4 Cultured HBMEC on the cover slice to 60 -70% . Added 6T - CEM on the transwell. Cultured at 37℃ and took out the cover slices at hour 4 8 and hour 20. Examine the F - actin by immunofluorence method dyed with rhodamin- phallodin.4 Investigated the mechanism of T lymphocyte transmigrating BBB.4. 1 Extracted RNA from the T lymphocytes of AD and age ?matched contrast separated with immunofluorescence magnetic beads. Investigated the abnormally expressed gene by BiostarH140s RNA chip. Validated its expression in AD patients and age - matched contrast by semi quantity RT - PCR.4. 2 Investigated the function of MIP - la in the transmigrating process 4. 2. 1 Examed the MIP - la expression in 6T - CEM cells, which is a cell model of T cell of AD, by RT - PCR.4.2.2 Separated the T lymphocyte of AD by immunofluorescence magnetic beads. Added the T cells of AD and its model cell, 6T - CEM cells onto the BBB model. Different quantity' s antibody of MIP - la was added in some wells together with T cells. After 20 hours'culture, counted the transmigrated cells.Compared the transmigration rate between the T cells treated with different quantity' s MIP - la antibody.4. 2.3 Cultured HBMEC on cover slices to confluent. Then added T lymphocyte of AD and 6T - CEM cells. For two slices, MIP - la antibody was added at the same time. Took out the cover slices after 20 hours' culture. Investigated the ZO - 1 of HBMEC by immunofluorescence.4.2.4 Cultured HB...