| To treat brain diseases, protecting "Brain Neurovascular unit" is proved to be more effective than only protecting single cells.The better way means doing with all the structures and function cells of brain tissue. "Brain Neurovascular unit" is a compomers constructed by micrangium (their endothelium-ground mass-astrocytic foot processes are complex and. to be an integral part), astrocytic processes around blood vessels and neurons and axons supported by them. So, it's significant to establish the in vitro model of "Brain Neurovascular unit" by co-cultured primary 3 cells.Objective1,To build "Brain Neurovascular Unit" constructed by 3 kinds of brain cells of microvascular endothelial cells, rats brain astrocytes. and brain neuronvitro test model, and to used for the research of cerebrovascular disease and drug screening.2,To observe of the Chinese herbal drugs for protecte the oxygen injury effec on the "Brain Neurovascular unit" Gregory freeze-dried ischemic by use "Brain Neurovascular Unit" in vitro test model, providing the main pharmacodynamic basis for traditional chinese drug to bring new ideas.Material and method1,By reference of foreign methods and improved, original generation culture rats 3 kinds of brain cells of microvascular endothelial cells, brain astrocytes. the method is as follows. (1) Separating brain microvascular endothelial cells form SD neonatal rats within two weeks, and they were purified by digestion and passage, and were identified by immuneocytochemistry of their specific protein factorⅧ. (2)Separating brain astrocytes form newborn SD rat within 1-2 d,and the hybrid cells were removed by differential adhesion and shaking culture.what's more, purity was identified by immunocyto-chemistry for specific glial fibrillary acidic protein (GFAP). (3)Separating cortical neurons of the newborn SD rat within 1d, to inhibit the growth of hybrid cells by Ara-C inhibitors and purified neurons were identified by immunohistochemistry with the neuron-specific enolase NSE enzyme compounds.,2,After research the condition of three cells in co-culture, the "Brain Neurovascular unit" model was established. Choose the Transwell of diameter with 12 mm, microholes 0.4 mm, microporous membrane area 1.12 mm2 (12 holes, No.3401. Corning) small room or diameter 24 mm, micro holes 0.4μm, microporous membrane area of Transwell (3.0 mm2 3412 six holes, No.3, Corning) plant cells, respectively on orifice plate bottom first vaccination has purification of neurons,2d growth on the lateral film after inoculated tension after nestin) purification astrocyte,2d within membrane after inoculation after the pool of nestin) purification vascular endothelial cells, co-culture under the same conditions for 7 days. the growth morphology of cells was observed by Inverted Microscope,and the accurate connection among models was observed by Transmission Electron Microscopy,what's more, permeability was tested by sodium fluoresce (FLU) and horseradish peroxidase (HRP).Efflux of y-Glutamyl Transpeptidase y-GT) as well as cell membrane resistance (TEER)of models were also be tested.3,Apoplexy models are on the condition of OGD, the protective effect by Catalpol and puerarin freeze-dried powder injection was tested to "brain vascular cells", Westerblot detected protein expression of erythropoietin (EPO) and its receptor (EPOR) and vascular endothelial cell growth factor (VEGF) (P<0.05), axonal growth protein (GAP-43), myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1). interleukin-1 (IL-1), metal substrate enzyme 9 (MMP-9) (P<0.05), nuclear factor (NF-KB) P65 and (IκB-α) protein and neuron repair related proteins (TGF-β1) (P< 0.05). Inadition, ELISA detected nerve growth factorβ(NGFβ) and inflammatory cytokines tumor necrosis factor (TNF-α) (P<0.05) and so on.ResuLts1,It is successed to culture brain cells of the three original generation respectively. The successfully isolated primary brain endothelial cells were cultured to three generations, and the cytoplasm of positive cells were into brown by immunocytochemistry, and calculated purity of 95%; The successfully isolated primary brain astrocytes were cultured to three generations, and calculated purity of 90%; The successfully isolated primary brain neurons were culturedand calculated purity of 85%; 2,The test showed three kinds of brain cells grow well together, and cultivate the role of promote each other growth. Viadetecting the following index appraisal proof "cranial nerve vascular unit in vitro model successfully created". Fluorescent element sodium (making F) and horseradish peroxidase (HRP) permeability reached 4632±168.79; Cell membrane resistance (across TEER) value is 268.67±4.16(Qcm2); Cell discharge system gamma-glutamine gamma GT activity for155.33±0.80. Related indexes accord with model standards. Transmission electron microscopy shows endothelial cells are present precision connection.successfully isolated primary astrocytes were cultured to three generations after handing by differential adhesion and vibratile shake flask method, the cytoplasm of positive cells were into brown by immunocytochemistry, and calculated purity of 95%, in line with experiments.3,Catalpol and puerarin freeze-dried powder injection displays activity in "brain vascular cells" which is conditional on OGD. Inflammatory cytokines tumor necrosis factor (TNF-α), intercellular adhesion molecule-1 (ICAM-1), interleukin 1(IL-1). the metal substrate enzyme 9 (MMP-9)of were reduced by it. Nuclear factor (NF-κB) P65 and nerve repair related proteins (TGF-β1) and apoptotic proteins(casepse-3) expression, increased nuclear factor (IκB-α) protein expression were inhibited by it. However, brain microvascular endothelial erythropoietin and its receptor (EPO, EPOR). vascular endothelial growth factor (VEGF) protein expression were promoted。Conclusions1,Through the separation and the original generation training SD rats cerebral cortex breasts endothelial, star oligodendrocytes, and neurons, successfully established three kinds of brain cells common training body. The testing by the relevant index,3 kinds of brain cells common training body with "brain vascular cells" between the main function and cell related information, prove the model can be used as "the brain blood vessels in vitro model experiment unit"2,Catalpol and puerarin freeze-dried powder injection displays remarkable activity in vitro model unit of "Brain Neurovascular unit" which is conditional on OGD. |
PDF Full Text Request