| ObjectivePart1:To establish the method of isolating and culturing highly purified primary rat brain microvascular pericytes.Part2:To explore the role of melatonin in H2O2induced brain microvascular pericytes apoptosis.Part3:To explore the underlying mechanism of melatonin in protecting brain microvascular pericytes against H2O2-induced apoptosis.MethodsPart1:The brains of ten3-weeks old Wistar rats were recieved by decapitation. Meninges and white matter were carefully removed and gray matter was minced into approximately lmm3fragments. After twice enzymatic digestion and a33%continous Percoll gradient centrifugation, micro-vessel fragments were incubated in35mm dish plates. Brain microvascular pericytes were cultured in DMEM supplemented with10%FBS at37℃with a humidified atmosphere of5%CO2/95%air. After2days, the medium was changed with a new one. The grow course and morphology of brain microvascular pericytes were observed with inverted microscope. And, the pericytes were identified by immunocytochemical method for the immunostaining of NG2, α-SMA, vWF and GFAP. MTT assay was performed to examine the cell growth curve.Part2:MTT assay and trypan blue exclusion assay were carried out to measure the cell viability of cultured pericytes after treatment with different concentrations of H2O2respectively. Intracellular reactive oxygen species (ROS) was measured by using a Reactive Oxygen Species Assay Kit. To distinguish the types of cell death induced by H2O2treatment in brain microvascular pericytes, TdT-mediated dUTP nick-end labeling (TUN EL) assay was used to identify apoptosis. The protective effect of melatonin and anti-oxidant GSH was also measured.Part3:To determine whether caspase-3activation is involved in H2O2-induced apoptosis, Caspase-3activity was measured by using a Caspase-3activity detection kit. The expression of apoptosis-relating proteins Bax and Bcl-2were detected by western blotting.ResultsPart1:Brain microvascular pericytes can grow out of the microvessel fragments after 5-6days. Cultured rat brain microvascular pericytes were irregular in shape and formed multilayers in some areas of the culture dishes. These pericytes can reach confluency after8-10days. The pericytes have long cell process, larger cell body and round cell nucleus. Immunostaining results indicated that brain microvascular pericytes express NG2and a-smooth muscle actin, while markers of astrocytes (glial fibrillary acidic protein, GFAP) and endothelial cells (factor Ⅷ-related antigen/von Willebrand factor) are negative. The purity of brain pericytes is up to96%. The peak point of grow shown at10th cultured day.Part2:H2O2treatment elicited a dose-dependent cytotoxicity in rat brain pericytes. The treatment of pericytes with0.05mM,0.1mM, and0.2mM H2O2caused approximately12%,25%, and28%cell damage, respectively, at2h (P<0.05). The cells treated with0.5mM H2O2experienced an approximately38%decrease in viability at2h (P<0.01). Even more significant damage was found in the1mM H2O2group, as with a78%decrease in viability at2h (P<0.001). A similar pattern of cell death was also observed using a trypan blue exclusion assay. After3h pre-incubation with melatonin and the antioxidants GSH, intracellular ROS levels were decreased approximately by27%and36%respectively. TUNEL assay results indicated that pericyte death was mediated by apoptosis. Pre-incubation with0.1mM melatonin significantly reduced cell apoptosis, and the percentage of apoptotic cells was reduced to approximately half that of the H2O2-treated group that did not receive melatonin pretreatment. Pretreatment with GSH (0.5mM) also attenuated H2O2-induced apoptotic cell death.Part3:Compared with the controls, the activity of Caspase-3was increased by2.24-fold (P<0.05) in the brain pericytes treated with0.5mM H2O2for2h. However, incubation with melatonin (0.1mM) prior to H2O2treatment reduced the caspase-3activity by19%compared with H2O2-treated cells(P<0.01). Similar results were obtained with the antioxidant glutathione (GSH,0.5mM). The expression of the anti-apoptotic protein Bcl-2was reduced by approximately50%in cells that were treated with0.5mM H2O2for2h (P<0.05) compared with the controls. A3-fold increase in Bcl-2levels (P<0.01) was detected after pre-incubation with melatonin (0.1mM), in comparison to the H2O2-treated group. In contrast, Bax expression levels were not affected:both treated and control groups showed lower levels of Bax expression.ConclusionPart1:Highly purified primary rat brain microvascular pericytes were successfully isolated through this method. Part2:Melatonin was able to protect pericytes from H2O2-induced apoptotic cell death.Part3:Melatonin prevents H2O2-induced apoptosis in cultured brain pericytes by inhibiting caspase-3activation and by up-regulating the expression of Bcl-2. |
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