【作者】 程岗；

【导师】 章翔； 高大宽；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 一、建立氧糖剥夺/复氧离体神经元模型目的原代培养获得纯化的小鼠脑皮层神经元,在细胞水平建立一种可靠的、简便易行的离体神经元氧糖剥夺/复氧(OGD/R)模型,为研究神经元缺血性损伤机制及进行药物筛选奠定基础。方法选择14-15 d Balb/c胎鼠作为大脑皮层神经元的来源,采用酶消化法获得脑皮层神经元。首先在含20%胎牛血清的DMEM中,于37℃、5%CO2孵箱中体外培养,24 h后换为含有神经元培养添加剂B27 (2%)的无血清DMEM继续培养,以促进神经元的分化及抑制神经胶质细胞的增生。倒置显微镜观察细胞形态。10 d时采用免疫荧光染色法进行β-tubulin染色,鉴定神经元纯度。体外培养10 d左右即可用于OGD/R试验。OGD 4 h后复氧20 h,然后进行细胞活力测定。采用台盼蓝(TB)染色检测细胞存活率,通过检测培养液中乳酸脱氢酶(LDH)的漏出率评估细胞膜通透性改变,反映细胞的损伤程度,从病理生理学角度阐明神经元损伤的状况。结果神经元体外培养10 d,光镜下可见成熟神经元特征,如细胞核饱满、清晰、透亮,核仁明显,核膜清晰可见,胞体呈多形性,胞质透亮,细胞具有折光性,自胞体伸出较多突起,神经元突起间相互联系,形成复杂的网络结构。β-tubulin免疫荧光染色细胞阳性率达70%。TB染色结果显示,OGD 4 h可引起明显的神经元死亡,且稳定性较好。LDH漏出率结果与TB染色结果相一致。结论采用酶消化法可分离获得小鼠大脑皮层神经元,B27不但可诱导神经元体外分化,还可有效抑制胶质细胞的增生。OGD 4 h/R 20 h可引起明显的神经元损伤,死亡率达50%,且较稳定,适宜作为脑缺血再灌注损伤的体外研究对照模型。二、短暂性OGD/R致神经元损伤时白藜芦醇(Res)的保护作用及对基质金属蛋白酶9(MMP-9)表达的影响目的在体外验证Res对短暂性OGD/R神经元损伤的保护作用,进一步在细胞水平探讨Res对脑缺血再灌注损伤的保护作用机制,即其对原代皮层神经元MMP-9表达的影响。方法以小鼠大脑皮层原代神经元OGD 4 h/R 20 h模型为研究对照。Res溶于DMSO,储液浓度为0.1 M。实验时用PBS将储液稀释到所需浓度后加入培养基,终浓度分别为2.5μM、5μM和10μM,对照组加入等体积DMSO(0.1%),治疗时间从缺氧开始,直至试验结束。TB染色法计算细胞存活率,LDH漏出率评估细胞的损伤程度。提取培养细胞总蛋白,采用Western blot分析过氧化物增殖活化剂受体(PPAR)α、γ和MMP-9的蛋白表达,提取培养细胞总RNA,采用反转录聚合酶链反应(RT-PCR)检测MMP-9 mRNA水平。结果TB染色结果显示,OGD 4 h/R 20 h可引起约50%神经元死亡,0.1% DMSO并没有加重神经元的损害。而Res干预治疗可减少这种条件下神经元的死亡,而且这种保护作用具有明显的剂量依赖效应,5μM、10μM的Res对于离体神经元OGD/R损伤具有良好保护作用,没有发现明显的副作用。Western blot和RT-PCR结果显示,正常神经元MMP-9的表达水平很低,PPAR-α和PPAR-γ的表达水平也很低。OGD 4 h/R 20 h可显著提高神经元MMP-9的转录和翻译,同时也明显激活PPAR-α和PPAR-γ的表达。加用Res后,MMP-9的转录和翻译被明显抑制,同时,PPAR-α和PPAR-γ的表达水平进一步上调,而且Res的上述作用随其浓度的增加而增强。结论Res可以抑制OGD/R模型神经元MMP-9的转录和翻译,同时激活PPAR-α和PPAR-γ的表达,而且Res的上述作用随其浓度的增加而增强。三、Res对OGD/R神经元损伤的保护作用机理探讨目的体外研究探讨Res对OGD/R神经元损伤的保护作用机理。方法以小鼠大脑皮层原代神经元OGD 4 h/R 20 h模型为研究对照。将神经元分成不同的治疗组,分别加入不同的药物进行治疗。药物分别溶于DMSO制成母液,-20℃保存。然后按照不同的剂量和组合加入培养基。包括Res(10μM)、选择性PPAR-γ激动剂troglitazone(5μM)、选择性PPAR-α激动剂wy14643(5μM)、选择性PPAR-γ抑制剂GW9662(10μM)和选择性PPAR-α抑制剂MK886(10μM)。TB染色法计算细胞存活率,LDH漏出率评估细胞的损伤程度。提取培养细胞总蛋白,采用Western blot分析PPAR-α、PPAR-γ和MMP-9的蛋白表达,提取培养细胞总RNA,采用RT-PCR检测MMP-9 mRNA含量。结果TB染色结果显示,OGD 4 h/R 20 h可引起约50%神经元死亡,0.1% DMSO并没有加重神经元的损害。Res、troglitazone和wy14643干预治疗可减少这种条件下神经元的死亡。Western blot和RT-PCR结果显示,Res、troglitazone和和wy14643都能不同程度的抑制OGD/R条件下增高的MMP-9的表达。但是加入GW9662或MK886与上述激动剂共培养后,troglitazone和wy14643对MMP-9的抑制作用及对神经元的保护作用被不同程度的阻断。Res对MMP-9的抑制作用及对神经元的保护也被MK886部分阻断,但是GW9662对Res的上述作用基本没有影响。结论Res对MMP-9的抑制作用及对神经元的保护作用与选择性激活PPAR-α有关,阻断PPAR-α的激活可以部分影响Res对MMP-9和神经元的生理作用。虽然Res和troglitazone都能激活PPAR-γ的表达,但是对PPAR-γ的下游靶点产生的生理作用并不完全一致,这可能与PPARs的结构复杂性有关。更多还原

【Abstract】 1. Establishment of neuron oxygen glucose deprivation/reoxygenation (OGD/R) model in vitroObjective To obtain pure mouse cerebral cortex neurons by primary culture, and establish a reliable and easy-conducted OGD/R neuron model in vitro to provide necessary theory for the researches of mechanisms of neuron ischemia and treatment.Methods Primary cortex neurons were obtained from embryo (14-15 d) Balb/C mice by the enzyme digestion method. Neurons were firstly cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 20% fetal bovine serum at 37℃in 5% CO2 atmosphere. After 24 h the medium was replaced by DMEM with 2% B27 supplement to facilitate differentiation of neurons and minimize glial growth. Morphology of neurons was observed under microscope. After 10 days of culturing in vitro, immunofluorescence staining ofβ-tubulin was used to identify the purity of neurons. Experiments of OGD/R were performed on cultures at 10 d in vitro. The time of OGD was 4 h followed by 20 h reoxygenation. Then typan Blue (TB) staining was used to detect the cell viability, and lactate dehydrogenase (LDH) leakage ratio in the culture medium was used to evaluate the changes of permeability of cell membrane.Results After 10 days of culture in vitro, the primary cortex neurons showed typical morphologic characters of mature neurons. The neuronal nucleus was large and clear with round or elliptical outline. The nucleolus and the membrane of nucleus were easily found. There were pleomorphic neurons in the medium, and the cytoplasm was bright with many dendrites and axons striking out from the cellular body, which connected each other to form a complicated reticular formation. The percent ofβ-tubulin positive neurons was more than 70%. The results of TB staining showed that 4 h OGD could induce neuron death significantly. The results of LDH leakage ratio were consistent with the results of TB staining.Conclusion Primary cortex neurons could be successfully obtained from the embryo (14-15 d) Balb/C mice by the enzyme digestion method. B27 Supplement not only facilitated differentiation of neurons, but also minimized glial growth. Neuron ischemia reperfusion model induced by OGD 4 h/R 20 h showed moderate and stable damage to cell viability, and was suitable for control research of cerebral ischemia reperfusion injury in vitro.2. Protective effects of Resveratrol (Res) against neuronal injury induced by OGD/R in vitro and effect on matrix metalloproteinase (MMP)-9Objective To confirm the protective effects of Res against neuronal injury induced by OGD/R in vitro, and further research the mechanisms of neuroprotection of Res in cell level, namely the relationship between Res and MMP-9 induced by OGD/R in neuron. Methods Primary mouse cerebral cortex neuronal ischemia reperfusion model was created by OGD 4 h /R 20 h. Stock solution (0.1M) of Res was prepared in dimethylsulfoxide (DMSO) and stored at -20℃. For treatment, the Res was diluted in PBS and added to cultures to give the desired final concentrations (2.5, 5 and 10μM). Untreated cultures received the same amount of the carrier solvent (0.1 % DMSO). The duration of treatment is from OGD/R to the end of the experiment. Cell viability was evaluated by TB staining and LDH leakage ratio. Total protein extraction of cells was used for detection of expression of Peroxisome proliferators-activated receptor (PPAR)α,γand MMP-9 protein by Western blot. Total RNA isolated from cells was used for evaluation of MMP-9 mRNA levels by reverse transcription polymerase chain reaction (RT-PCR).Results The results of TB staining showed that OGD 4 h /R 20 h could induced 50% neuronal death, and 0.1% DMSO did not aggravate neuronal damage. Res treatment could reduce cell death under these conditions, and the neuroprotection of Res for neurons was dose-dependent. These results indicated that treatment with 5μM and 10μM of Res showed better therapeutic outcome, and no harmful effects were found. The results of Western blot and RT-PCR showed that normal neurons only expressed very low level of MMP-9 protein. There were low level of PPAR-αand PPAR-γin normal neurons. OGD 4 h/R 20 h could increase transcription and translation of MMP-9 in neurons. At the same time, expressions of PPAR-αand PPAR-γwere also up-regulated by OGD/R injury. Res could inhibit transcription and translation of MMP-9. Expressions of PPAR-αand PPAR-γalso increased further by Res. Bioactivity of Res on MMP-9, PPAR-αandγwas in concentration-dependent manner.Conclusions Res could inhibit transcription and translation of MMP-9 in OGD/R neuron model and activate expressions of PPAR-αand PPAR-γin concentration- dependent manner.3. Mechanisms of protective effects of Res against neuronal injury induced by OGD/R in vitroObjective To confirm mechanisms of protective effects of Res against neuronal injury induced by OGD/R in vitroMethods Primary mouse cerebral cortex neuronal ischemia reperfusion model was created by OGD 4 h /R 20 h. Neurons were grouped according to different reagents. Stock solution of reagents was prepared in DMSO and stored at -20℃. Reagents were added into cultural medium in different dosage and combination, including Res (10μM), activator of PPAR-γtroglitazone (5μM), activator of PPAR-αwy14643 (5μM), antagonist of PPAR-γGW9662 (10μM), and antagonist of PPAR-αMK886 (10μM). Cell viability was evaluated by TB staining and LDH leakage ratio. Total protein extraction of cells was used for detection of expression of PPAR-α, PPAR-γand MMP-9 protein by Western blot. Total RNA isolated from cells was used for evaluation of MMP-9 mRNA levels by RT-PCRResults The results of TB staining showed that OGD 4 h/R 20 h could induced 50% neuronal death, and 0.1% DMSO did not aggravate neuronal damage. Res, troglitazone and wy14643 could decrease neuronal injury caused by OGD/R. Western blot and RT-PCR showed that Res, troglitazone and wy14643 could inhibit up-regulated expression of MMP-9 in OGD/R conditioin. When neurons were co-cultured in GW9662 or MK886 with agonist of PPARs, inhibition effects of troglitazone and wy14643 on MMP-9 and protection effect on neurons were antagonized. Inhibition of MMP-9 and protection of neurons by Res was partially antagonized by MK886, but which was not affected by GW9662. Conclusions Inhibition effects on MMP-9 and protection effect on neurons by Res has relationship with selective activation of PPAR-α, blockage of PPAR-αactivation can partially bring negative impact to physiological function of Res to MMP-9 and neurons. Though both Res and troglitazone can activate expression of PPAR-γ, they have different effect on down-stream target. It may have relation with complex structure of PPARs.更多还原