Experimental Study On Culture Supernatant Of Ad-HGF Infected RMMC Of Rat CAEC

Background Cerebral artery endothelial cells (Cerebral artery endothelial cell, CAEC) is the main component of the blood brain barrier and has an active secretion, synthesis, metabolism, and immune function, may produce a variety of cytokines and bioactive substances, and involved in the regulation of the body physical activity, a variety of physiological and pathological factors of target cells.HGF (Hepatocyte growth factor, HGF) is a separate angiogenic factors, more and more research suggests it has neurotrophic promote neurogenesis, angiogenesis, axons, reducing scar formation and promote proliferation and differentiation of ventricular subventricular zone (Subventricular zone, SVZ) neural stem cell. TGF-(31 is an important factor in the regulation of blood-brain barrier integrity, by effect of interaction between the two cells, the intracellular environment and differ primarily active factor, particularly for the regulation of glial HGF-TGF-β1 playing a balanced important role. Ad-HGF RMMC infected culture supernatant is constructed by genetic engineering techniques Ad-HGF transfected cell culture supernatant obtained RMMC。We conducted Ad-HGF transfection activity of rat meningeal cells cultured mesothelial cells (rat meningeal mesothelial cells, RMMCs) resulting supernatant were expected to clear infected with culture supernatant infected by Ad-HGF RMMC the role of double effect has angiogenic and neuroprotective provide an experimental basis, thus further application of Ad-HGF treatment of senile ischemic cerebrovascular disease after subarachnoid provide experimental clinical and theoretical basis.Objective Constructed by genetic engineering techniques Ad-HGF transfected cell culture supernatant role rat meningeal mesothelial cells (rat meningeal mesothelial cells, RMMCs) obtained in vitro in primary cultured rat brain SD artery endothelial cells (Cerebral artery endothelial cell, CAEC). CAEC proliferation and migration affect the ability to observe the supernatant after effects. And then through research and overall research cell, forward-reverse engineer by molecular biology techniques,, to detect the expression of TGF-β1 in CAEC, and analyze the role of HGF-TGF-(31 and related activity factor in CAEC newborn, nerve regeneration, attempt to reveal the role of molecular regulation and neuroprotective mechanisms TGF-β1 factor signaling in promoting angiogenesis in the brain.Methods We removed under sterile conditions born 1-2d SD rat brain, the use of mechanical pipetting, light digestion method combined with tissue culture in primary cultured rat cerebral artery endothelial cells, After three generations pass through inverted phase contrast microscope to observe cell morphology, cell ordinary optical microscope HE staining factor Ⅶ related antigen immunofluorescence staining cells were identified in three areas; Ad-HGF infection detected LMC culture supernatants on cell proliferation by MTT assay; The cells detected on cell migration through Transwells law; by immunoblotting (western blotting, WB) assay CAEC in TGF-β1 protein expression.Results1.We observed CAEC under the ordinary optical microscope,they spread in nonmol polygonal-shaped。CAEC grew as a net after 96 hours, after 8-10 days primary culture, the cells exhibited "cobble" pattern, they became 90% confluent and displayed uniformly。2. After primary cell culture 8-10 d, the cells covered the bottom line digestion and passage by passage 3rd generation cell for the identification, HE staining, cells were short spindle or polygonal, ranging in size, abundant cytoplasm, thin dyed reddish colored uniform。3. Under inverted fluorescence microscope, there were pass aged cell nucleus in the cytoplasm surrounding the yellow-green fluorescence, factor Ⅷ antigen positive, the positive rate of 90%, a clear boundary between the cytoplasm and the nucleus, whereas control cells without staining (negative), indicating that the cultured cells to rat cerebral artery endothelial cells。4. We observed a lot of films are crystal violet staining of CAEC Transwells cell migration assay PVPE;randomly selected 100 times the number of cell migration perspective by comparing the experimental group, the difference was significant。5. MTT assay infected with Ad-HGF LMC culture supernatants of different lengths of time to act on CAEC Enzyme-linked immunosorbent assay OD570nm absorbance measured at each hole, the experimental group, the difference was significant。6.The same point in time, infection with Ad-HGF increase LMC culture supernatant and exogenous HGF concentration, CAEC in TGF-β1 mean optical density decreases, TGF-β1 mean optical density values showed a certain amount of relevance, the difference significant。Conclusions1.Mechanical pipetting, trypsin digestion method combined with tissue culture method can successfully obtain high purity cerebral artery endothelial cells in rats。2.CAEC promote proliferation and the effect of migration in rat cerebral artery endothelial cell。3. Culture supernatant of Ad-HGF infected LMC of rat CAEC and HGF bring inhibition of TGF-β1 protein expression。...