【作者】 王晓燕；

【导师】 牛侨； 聂继盛；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生学， 2011， 硕士

【副题名】组蛋白去乙酰化酶在其致神经细胞凋亡中的作用

【摘要】 【目的】从体内实验探讨孕期暴露苯并[a]芘对仔鼠神经发育及学习记忆的影响,并通过体内外实验进一步探讨HDAC1、HDAC2及BDNF等在苯并[a]芘致神经细胞凋亡中的作用。【方法】1.动物体内实验研究:1)SD孕鼠随机分为5组,于妊娠第17天每天1次连续3天腹腔注射染毒,分为空白对照组、溶剂对照组、25、50、100 mg/kg B[a]P组,待其自然分娩。2)①PND1、4、7、14、28称量体重,检测发育指标;②Morris水迷宫和旷场实验分别检测仔鼠的学习记忆能力和对新异环境的适应能力;③PND1、4、7、14、28每组处死3只仔鼠取脑组织作病理切片,用硫堇染色及TUNEL实验分别观察海马神经元损失及凋亡情况;④QRT-PCR、免疫组化法和Elisa法检测仔鼠脑组织HDAC1、HDAC2、caspase-3、BDNFmRNA及蛋白的表达。2.体外实验研究:1)大鼠原代神经细胞培养,分为两批:第一批分4个组,以B[a]P同时加入S9对细胞染毒,分为DMSO、低、中、高剂量组,使其终浓度为0、10、20、40μmol/L;第二批分5个组,以20μmol/LB[a]P染毒,分别于染毒0、6、12、24、48h处理细胞。2)①MTT法和流式细胞术分别检测神经细胞活力及凋亡率;②HAT/HDAC activity assay kit检测HAT/HDAC的活性;③QRT-PCR、Western-blot法和Elisa法检测神经细胞HDAC1、HDAC2、caspase-3、BDNFmRNA及蛋白的量效和时效表达。【结果】1.①神经发育指标测试:同一时点染毒组仔鼠体重与对照组比较逐渐降低(P<0.01,P<0.05);与空白对照组、溶剂对照组相比较,中、高剂量组仔鼠张耳时间[(4.1 0.4)、(5.00.4)d]延长(P<0.01)。PND4与空白对照组相比(36.1%),高剂量组平面翻正实验达标率(6.5%)明显降低,PND7与空白对照组、溶剂对照组(80.3%、79.3%)相比,高剂量组平面翻正实验达标率(50.0%)明显降低(P<0.05)。与空白对照组比较,PND12、14各染毒组仔鼠前肢悬挂时间均减少(P<0.01)。与空白对照组相比(94.3%),PND12高剂量组嗅觉定向实验达标率(61.9%)明显降低(P<0.05);②学习记忆能力测试:Morris水迷宫实验结果表明与空白对照组和溶剂对照组比较,各剂量组仔鼠逃避潜伏期明显增加,高剂量组在目标象限停留时间和穿越平台次数减少(P<0.01)。旷场实验结果表明,与空白对照组比较,中、高剂量组仔鼠中央格停留时间明显延长,跨格次数明显减少,高剂量组直立次数明显减少(P<0.05);与溶剂对照组相比,各剂量组跨格次数明显减少(P<0.01,P<0.05);③硫堇染色病理组织结果:空白对照组及溶剂对照组仔鼠海马细胞数量较多,连接紧密,排列规则,神经元尼氏较深;随着染毒剂量的增高,细胞数量逐渐减少,排列逐渐紊乱,连接松解,神经元尼氏体着色较对照组浅,核固缩,核周出现轻度空泡变性;④TUNEL结果:同一时点AI随染毒剂量增加而增高(P<0.01,P<0.05);⑤基因、蛋白检测结果:各组HDAC1、HDAC2、caspase-3mRNA和蛋白的表达水平随染毒剂量的增加逐渐增加, BDNF的表达则下降。与空白对照组相比,高剂量组BDNF mRNA的表达至少减少了50%(P<0.01,P<0.05)。PND28仔鼠血浆中BDNF蛋白水平高剂量组与空白对照组、溶剂对照组相比分别下降了68%、75%(P<0.01,P<0.05)。2.①细胞活力和凋亡率检测结果:不同染毒剂量及时间下,细胞活力显著下降。40μmol/L组神经细胞存活率与DMSO组相比显著减少了50%(P<0.01),48h组与0h组相比也减少了50%(P<0.01).与DMSO组相比,20、40μmol/L组神经细胞的凋亡率明显增加(P<0.05),40μmol/L组凋亡率与10μmol/L组相比也明显增加(P<0.05);与0h组相比,24、48h组神经细胞的凋亡率明显增加(P<0.05);②HAT/HDAC活性测试结果:与DMSO相比, 40μmol/L组HAT活性显著下降了47%(P<0.05),而HDAC活性明显增加了59%(P<0.01);与0h相比,48h组HAT活性显著下降了68%(P<0.01),而HDAC活性增加了142%(P<0.01);③基因检测结果:与DMSO组相比,染毒组HDAC1基因表达有上升趋势,但差异无统计学意义; 40μmol/L组HDAC2、caspase-3mRNA的表达量较对照组明显增加,而BDNF mRNA下降(P<0.01);与0h组相比,48h组HDAC1、HDAC2、caspase-3 mRNA表达明显升高, BDNF表达显著下降(P<0.01,P<0.05);④蛋白检测结果:与DMSO组相比,40μmol/L组HDAC、HDAC2、caspase-3蛋白的表达量明显增加(P<0.05);与0h组相比,24h、48h组caspase-3蛋白,48h组HDAC1、HDAC2蛋白的表达量明显增加(P<0.05)。不同染毒剂量及时间下BDNF蛋白表达水平显著下降(P<0.01)。【结论】孕期暴露B[a]P对仔鼠的神经发育有一定的抑制作用,并可使仔鼠脑组织神经细胞形态结构发生改变,海马神经细胞凋亡,且剂量越高损伤越严重;从体外实验和动物体内实验两方面研究发现,HADC1、HDAC2可能在B[a]P所导致的神经细胞凋亡起重要作用,可能与B[a]P作用后,HDAC1、HDAC2表达上调,组蛋白乙酰化水平减低,导致BDNF表达下降,进而引起神经细胞凋亡有关。更多还原

【Abstract】 [Objective]To study the effect of prenatal exposure to B[a]P on the neurodevelopment and the ability of learning and memory of pup rats,and explore the effect of HDAC1,HDAC2 and BDNF in the B[a]P-induced neurons apoptosis in vivo and in vitro.[Methods]1.Animal experiment :1)Pregnat adult rats were randomly assigned to five groups and given B[a]P by intraperitoneal injection on the day 17 of gestation by daily for three consecutive days at the dosages of 25,50,100 mg/kg B[a]P,olive oil as a solvent group and normal control group respectively,and then expectant naturally.2)①Weighed on days 1,4,7,14 and 28 respectively after pup rats birth, detectd early development indicators of pup rats;②With the Morris water maze and Open-field test detect learning and memory ability and the adaptability to new environment;③Three pup rats of each group were sacrificed after the birth on days 1,4,7,14 and 28 respectively, neurons loss in the CA3 hippocampus and apoptosis of hippocampus neurons were observed by thionin and TUNEL;④The expression levels of HDAC1,HDAC2,caspase-3 and BDNF mRNA and protein were detected by QRT-PCR,immunohistochemistry and Elisa respectively.2. In vitro study:1) The primary cultured neurons of rat were assigned into two lots. The first lot was separated into 4 groups and treated with B[a]P at final concentrations as 0μmol/L (DMSO group),10μmol/L(low-dose group), 20μmol/L(medium-dose group), and 40μmol/L(high-dose group) respectively, and added S9 at the same time, then incubated for 48 hours. The second lot was separated into 5 groups, all treated with 20μmol/L B[a]P, then incubated for 0,6,12,24 and 48h respectively. 2)①The cell viability and apoptosis rates were evaluated with MTT and flow cytometry respectively;②HAT/HDAC activity were detected by HAT/HDAC activity assay kit;③The gene and protein levels of HDAC1,HDAC2,caspase-3 and BDNF were detected by QRT-PCR,western-blot and Elisa respectively.[Results]1.①Neurodevelopment of neonatal rats test: compared to the control and solvent group, offspring weight of B[a]P exposure group reduced gradually(P<0.01,P<0.05);The time of ear opening in middle and high-dose group[(4.1 0.4),(5.0 0.4)d)] posterior to the control and solvent group(P<0.01).Compared to the control group(36.1%) ,the attainment rate of the surface righting reflex test in high-dose on PND4 (6.5%)decreased, and on PND7 (50.0%)decreased significantly compared to the control and solvent group(80.3%,79.3%)(P<0.01).Compared to the control group, time of forelimb hanging test decreased in high-dose on PND12, 14 and decreased significantly on PND14 compared to the solvent group(P<0.01).The attainment rate of olfactory discrimination significantly lower than the control group (94.3%)in high-dose group(61.9%) on PND12(P<0.05);②Learning and memory capability test: Morris water maze test showed that compared to the untreated and solvent group the escape latency of different dose groups were significantly increased, and the time of spatial probe and numbers of traversing flat in high-dose group were shortened significantly(P<0.01).Open-field test showed that center retention time in middle and high-dose group were prolonged compared to control, numbers through lattice reduced, and rearing decreased in high-dose group, there were statistical significance(P<0.05).Compared to the solvent group numbers through lattice in different doses were reduced significantly(P<0.01,P<0.05);③The thionine staining showed: hippocampal neurons of the control group and solvent group were more numerous, connected more closely, lining up in order, neurons nissl staining was more deep, but with the increasing of doses of B[a]P exposed , number of neurons were gradually decreased ,the neurocytes displayed disorderly, the cell junction get loose, neurons nissl staining became lighter than the control, the karyon shrinked,some rings around the neurocyte were observed;④TUNEL results: at the same time point, the AI of hippocampus neurons increased significantly with the increasing dose (P<0.01, P<0.05);⑤Gene and protein results: the expression of HDAC1,HDAC2,caspase-3 mRNA and protein levels were increased as the dose of B[a]P exposed at the same time point,while BDNF decreased(P<0.01, P<0.05).The BDNF protein level in high significantly reduced (50%) as compared to control(P<0.01, P<0.05);PND28 BDNF protein levels in plasma in high dose group was decaeased significantly (68%,75%)compared with the control and the solvent (P<0.01, P<0.05).2.①MTT and Flow cytometry results: neurons viability was significantly decreased at different dose and time group (P<0.01). Cell viability of 40μmol/L group significantly reduced (50%) as compared to DMSO (P<0.01); which at 48h group reduced (50%)compared with 0h(P<0.01).Compared with DMSO group, apoptosis rates of neurons in 20,40μmol/L group were significantly increased(P<0.05), compared with 10μmol/L group, 40μmol/L group also increased (P<0.05);the apoptosis rates at 24,48h after exposure were significantly increased compared with that at 0h (P<0.05);②HAT/HDAC activity results: compared with DMSO group,HAT activity of 40μmol/L group reduced (47%) as compared to DMSO(P<0.05), while HDAC increased(59%)(P<0.01);HAT activity at 48h after exposure was decline (68%)compared with that at 0h,HDAC ascend (142%)(P<0.01);③QRT-PCR results: compared with DMSO group ,HDAC1 mRNA expression increased ,but there were no statistical significance. The caspase-3 and HDAC2 mRNA expression showed significant increments in 40μmol/L group compared with DMSO group(P<0.01), while the expressions of BDNF mRNA of 20,40μmol/L significantly decreased(P<0.01),the caspase-3 mRNA expression in 40μmol/L group increased significantly compared with the 10μmol/L group (P<0.05).The expressions of HDAC1,HDAC2 and caspase-3 mRNA at 48h after exposure increased significantly compared with that at 0h,whereas, BDNF mRNA at 24,48h decreased (P<0.01, P<0.05);④Protein examination results:the HDAC1,HDAC2 and caspase-3 protein levels in 40μmol/L group showed significant increments compared with DMSO group(P<0.05); The expressions of caspase-3 protein at 24h,48h ,HDAC1 and HDAC2 at 48h after exposure increased significantly compared with that at 0h(P<0.05). The gene and protein levels of BDNF decreased significantly as different dose and time (P<0.05).[Conclusion]The prenatal exposure to B[a]P would suppress neonatal rats’neurodevelopment ,and induce the morphology changes of pups’brain nerve cells and hippocampus neural cells apoptosis, the higher dose the more severe injury. From both in vitro study and the animal experiment, it is discovered that HDAC1, HDAC2 may play an important role in B[a]P-induced neuronal apoptosis. After expose to B[a]P, HDAC1 and HDAC2 increased, levels of histone acetylation decreased, which may lead to the expression of BDNF decreased and they may influence neuronal apoptosis.更多还原