褪黑素对H₂O₂诱导的大鼠脑微血管周细胞损伤的影响

【作者】 鹿文葆；

【导师】 修瑞娟；

【作者基本信息】 北京协和医学院 ， 生物医学工程， 2012， 博士

【摘要】 目的第一部分：探讨获取高纯度大鼠原代脑微血管周细胞的分离和培养方法,并对其鉴定。第二部分：探讨褪黑素在过氧化氢(H2O2)诱导的大鼠脑微血管周细胞损伤中的保护作用。第三部分：探讨褪黑素抑制H2O2诱导的大鼠脑微血管周细胞凋亡的保护机制。方法第一部分：选取10只3周龄Wistar大鼠,取大脑,去除白质,剪碎成大约1mm3大小,通过两次酶消化、20%BSA离心和33%连续密度Percoll梯度离心获得纯度较高的大脑微血管片段,接种于35mm培养皿中,加入含10%FBS的DMEM培养基,37℃、5%CO2细胞孵箱中培养,2天后更换新鲜培养基；采用倒置显微镜观察周细胞生长状况及其形态；免疫细胞化学法检测NG2、α-SMA、vWF及GFAP表达,鉴定分离的细胞及其纯度；MTT法检测绘制其增殖曲线。第二部分：应用MTT试验和台盼蓝拒染试验检测不同浓度H2O2处理后脑微血管周细胞的细胞活力；应用胞内反应性氧簇检测试剂盒检测褪黑素及抗化剂GSH对胞内活性氧聚集的影响；采用TUNEL法确定细胞死亡类型；检测褪黑素及GSH对H202诱导大鼠脑微血管周细胞损伤的保护作用。第三部分：检测褪黑素及抗氧化剂GSH对H2O2诱导的脑微血管周细胞调亡过程中关键蛋白Caspase-3的活性影响；应用Western blot法检测凋亡相关蛋白Bax和Bcl-2的表达,观察褪黑素在H2O2诱导脑微血管周细胞凋亡中的作用。结果第一部分：培养5天左右可见周细胞从贴壁的脑微血管片段周围长出；培养的周细胞成不规则外形,在某些区域可重叠生长；8～10天可形成汇合；脑周细胞拥有长的细胞突触、较大的细胞体及圆形的细胞核；免疫细胞化学染色结果显示脑微血管周细胞NG2和α-SMA表达阳性,并可同时表达这两种标志物,而对vWF和GFAP两种蛋白的免疫反应呈阴性,排除了内皮细胞和星形胶质细胞的存在；培养的周细胞纯度达96%以上；在培养到第10天左右增殖至顶峰。第二部分：MTT结果显示,H202处理脑微血管周细胞可引起剂量依赖的细胞毒性作用；使用0.05mM、0.1mM和0.2mM H202作用脑微血管周细胞2h可分别导致大约12%、25%和28%的细胞死亡(P<0.05),当使用0.5mM H202处理时可诱导大约38%的细胞死亡(P<0.01)；而当使用1mM H202处理时会诱导大部分(78%)周细胞死亡；应用台盼蓝拒染试验也得到了一致的结果；胞内活性氧检测结果显示使用褪黑素或抗氧化剂GSH预孵育可分别降低胞内活性氧大约27%或36%。；TUNEL实验结果表明,H202诱导的脑周细胞死亡是由凋亡介导的；与H202处理组相比,褪黑素(0.1mM)预处理可减少一半数量的H202诱导的TUNEL-阳性细胞；抗氧化剂GSH(0.5mM)也可降低H202诱导的周细胞凋亡。第三部分：H2O2(0.5mM)处理脑周细胞可显著增加Caspase-3活性,与对照相比增加约2.24倍(P<0.05),应用褪黑素(0.1mM)或GSH(0.5mM)预孵育后可减弱H202引起的Caspase-3活性的增加,与H202-处理组相比降低约19%(P<0.01)；Western blot结果显示,与对照组相比,H202(0.5mM)处理周细胞(2h)可显著降低抗凋亡蛋白Bcl-2的表达,下降约50%(P<0.05)；与H202处理组相比,使用褪黑素(0.1mM)预处理可上调Bcl-2的表达,大约增加3倍(P<0.01)；但是,对促凋亡蛋白Bax的影响很小,发现对照组与处理组均表达很低。结论第一部分：该方法可成功获得纯度较高的原代大鼠脑微血管周细胞。第二部分：褪黑素可抑制H202-诱导的脑微血管周细胞凋亡,验证了其抗凋亡作用。第三部分：褪黑素可通过降低Caspase-3活性及上调抗凋亡蛋白Bcl-2的表达来发挥抗凋亡作用。更多还原

【Abstract】 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.更多还原