【作者】 王昱；

【导师】 俞诗源；

【作者基本信息】 西北师范大学 ， 细胞生物学， 2009， 硕士

【摘要】 目的1)通过给孕小鼠注射海洛因观察并检测发育期小鼠大脑颞叶皮层结构的变化及ADA、SDH、NOS、GSH活性,以探讨海洛因对生长发育期小鼠大脑的毒性及其可能机制。2)采用给仔鼠注射海洛因和麻黄素,探讨海洛因和麻黄素对仔鼠大脑颞叶皮层结构及学习记忆能力影响的机制。方法1)对实验组36例受孕小鼠(第8 d开始)分别连续注射三种不同浓度(1.0 g/L、1.5 g/L和2.0 g/L)的海洛因溶液,对照组12例受孕小鼠注射等量的生理盐水直到小鼠分娩,称量检测各实验组仔鼠在胚胎15d(E15)及生后1d、7d、15d时大脑重量的变化,用生物显微技术观察仔鼠大脑颞叶皮层结构的变化,用比色法检测仔鼠大脑ADA、SDH、NOS、GSH的活性变化。2)海洛因组36只仔鼠采用递增剂量腹腔连续注射海洛因,麻黄素组36只仔鼠采用递增剂量腹腔连续注射麻黄素,对照组36只仔鼠注射等量的生理盐水,通过食饵迷宫观察仔鼠行为学变化,在光学显微镜和透射电镜下观察大脑颞叶皮层细胞结构的变化,用免疫组织化学方法观察了Bax蛋白和角质细胞生长因子(keratinocyte growth factor,KGF)在大脑颞叶皮层的表达,采用比色法检测了大脑颞叶皮层胆碱乙酰基转移酶(ChAT)活性的变化。结果1)孕小鼠注射海洛因影响仔鼠大脑的重量。实验组仔鼠大脑重量明显低于对照组。2)孕小鼠注射海洛因影响仔鼠大脑颞叶皮层的发育。实验组仔鼠大脑颞叶皮层结构不清,神经元发育不良,皮层厚度减少,除在E15和生后1d大脑颞叶皮层第Ⅵ层神经元细胞数量高于对照组外,其它发育期各层神经元细胞数量均小于对照组。3)孕小鼠注射海洛因影响仔鼠大脑ADA、SDH、NOS及GSH的活性。1.0、1.5和2.0g/L三个剂量组在E15时,胎鼠大脑中ADA、SDH及NOS活性高于对照组,差异显著或极显著(P<0.05或P<0.01),GSH活性低于对照组,差异极显著(P<0.01);仔鼠在生后15d的发育过程中,1.0g/L剂量组仔鼠大脑中ADA、SDH、NOS及GSH的活性逐渐恢复到正常水平,1.5g/L剂量组大脑中NOS和SDH的活性逐渐恢复到正常水平,但ADA的活性仍高于对照组,GSH的活性仍低于对照组,2.0g/L剂量组仔鼠大脑中ADA、SDH、NOS的活性在生后15d时仍保持较高的水平,GSH的活性仍低于对照组。双因素方差分析表明剂量因素对结果的影响大于时间因素。4)注射海洛因、麻黄素影响仔鼠的学习记忆能力。除麻黄素组5d时与对照组差异不显著(P > 0.05)外,海洛因和麻黄素组各期仔鼠食饵迷宫试验的总测试次数、错误次数及总测试时间均高于对照组,差异显著或极显著(P < 0.05或P < 0.01);海洛因组仔鼠食饵迷宫的上述三项指标均高于麻黄素组,差异显著或极显著(P < 0.05或P < 0.01)。随着海洛因和麻黄素剂量的递增,仔鼠食饵迷宫试验的总测试次数和错误次数呈上升趋势。5)注射海洛因、麻黄素影响仔鼠大脑颞叶皮层ChAT的活性。注射海洛因、麻黄素5d、10d、15d、20d,仔鼠大脑颞叶皮层ChAT的活性低于对照组,差异显著或极显著(P < 0.05或P < 0.01)。注射海洛因、麻黄素5d到20d,海洛因和麻黄素对大脑颞叶皮层ChAT活性的抑制率都呈上升趋势,且海洛因对大脑颞叶皮层ChAT活性的抑制率始终高于麻黄素。6)注射海洛因、麻黄素影响仔鼠大脑颞叶皮层的显微结构。注射海洛因、麻黄素5d、10d、15 d、20d,仔鼠大脑颞叶皮层锥体神经元树突、轴突萎缩,胞体缩小。海洛因组和麻黄素组仔鼠大脑颞叶皮层凋亡细胞及坏死细胞的数量均高于对照组,差异显著或极显著(P < 0.05或P < 0.01);海洛因组上述两项指标均高于麻黄素组,差异显著或极显著(P < 0.05或P < 0.01)。随着海洛因和麻黄素剂量的递增,仔鼠大脑颞叶皮层凋亡细胞和坏死细胞的数量呈增多趋势。7)注射海洛因和麻黄素影响仔鼠大脑颞叶皮层Bax蛋白和KGF的表达。注射海洛因、麻黄素5d、10d、15d、20d,仔鼠大脑颞叶皮层Bax蛋白和KGF阳性表达细胞的数量均高于对照组,差异显著或极显著(P < 0.05或P < 0.01);海洛因组上述两项指标均高于麻黄素组,差异显著或极显著(P < 0.05或P < 0.01)。随着海洛因和麻黄素剂量的递增,仔鼠大脑颞叶皮层Bax蛋白和KGF阳性表达细胞的数量呈增多趋势。8)注射海洛因、麻黄素影响仔鼠大脑颞叶皮层的超微结构。随着海洛因和麻黄素剂量递增,神经元核膜失去正常圆形轮廓,胞内细胞器结构不正常或空泡化;毛细血管周围基质溶解破坏,管腔狭窄;突触小泡减少。结论1)孕小鼠注射海洛因后对仔鼠大脑发育的影响具有长时程效应。妊娠中晚期接触海洛因对仔鼠脑神经元的迁移具有毒性作用。在生后15d时实验组仔鼠大脑重量、颞叶皮层各层的厚度和细胞密度仍小于对照组,可能是海洛因及其代谢产物对胚胎期神经元具有直接毒性作用的延伸结果。2)孕小鼠注射海洛因引起仔鼠大脑ADA、SDH、NOS活性升高和GSH活性下降。仔鼠大脑组织结构的改变可能与大脑ADA、SDH、NOS及GSH活性的改变有一定的相关性。3)海洛因和麻黄素使仔鼠大脑颞叶皮层ChAT的活性降低。大脑颞叶皮层可能参与了仔鼠记忆的形成。4)海洛因和麻黄素使大脑颞叶皮层组织结构发生改变,引起Bax蛋白和KGF阳性表达增强,大脑颞叶皮层组织受损程度随着剂量的升高而升高,具有明显的剂量效应关系。5)海洛因和麻黄素对仔鼠学习记忆能力有显著损伤,其可能机制与大脑颞叶皮层组织结构受损及ChAT活性下降有一定的相关性。随着海洛因和麻黄素剂量的递增,仔鼠学习记忆能力的损伤加重。更多还原

【Abstract】 Objective1)To explore the possible mechanism of heroin toxicity on the brain during the developmental mice, The structure of cerebral temporal lobe cortex and activities of adenosine deaminase (ADA),succinate dehydrogenase (SDH),nitric oxide synthase (NOS) and glutathione (GSH) of developmental mice were studied.2)To investigate effects of heroin and ephedrine on the structure of cerebral temporal lobe cortex and capability of learning and memory, filial mice were given injection of heroin and ephedrine, and the structure of cerebral temporal lobe cortex and capability of learning and memory were studied.Methods1)36 pregnant mice from the experimental groups at the eighth day began to be continuously injected with three different concentrations (1.0g/L,1.5g/L and 2.0g/L) of heroin, 12 pregnant mice from the control group were injected with the same amount of saline until the birth of filial mice. Cerebral weight of filial mice was weighted. And it was observed that the changes of the cerebral temporal lobe cortex of filial mice by bio-microscopy,and detected that the activities of ADA,SDH,NOS and GSH by colorimetry at embryo 15 days and postnatal ages of 1 day,7 days,15 days from all experimental groups.2)36 filial mice from heroin were given intraperitoneal injection of heroin by gradually increase of doses, 36 filial mice from ephedrine group were injected with ephedrine by gradually increase of doses, and 36 filial mice from the control group were injected with the same amount of saline. The filial mice behavior changes were observed by bait maze. In the same time, the changes of the cell structure of cerebral temporal lobe cortex were observed by light microscope and transmission electron microscope. Expression of Bax protein and keratinocyte growth factor(KGF) was measured by immunohistochemical method, and the ChAT activity was detected by colorimetry.Results 1)Heroin had some adverse effects on cerebral weight of filial mice after injecting heroin in pregnant mice. The cerebral weight of experimental group was significantly lighter than that of control group.2)The development of filial cerebral was affected by heroin. The structure of filial cerebral temporal lobe of experiment group was not clear. The thickness of filial cerebral decreased and the number of neurons reduced except those in the cerebral temporal lobe cortexⅥat embryo15 days and postnatal 1 day.3)Heroin had effects on activities of ADA, SDH, NOS and GSH. Compared with the control group,ADA,SDH and NOS activities in brain increased while GSH activity decreased significantly at embryo 15 days after administration of heroin (P <0.01 or P <0.05),but the recovery of these activities at postnatal 15 days varied with different doses. All of these activities subsequently recovered to the normal level slowly in heroin administration (1.0 g/L);Activities of NOS and SDH also recovered to the normal level while others remained significantly affected at the dose of 1.5g/L;ADA,SDH and NOS activities were still higher and GSH activity were lower than that of the control in 2.0g/L group. Results of Two-Way ANOVA indicated that dose contributed more to heroin-induced brain damage than treatment time.4) Heroin and ephedrine had some effects on learning and memory of filial mice. Total test times, error frequency and total test time of bait maze in the heroin group and ephedrine group were significantly higher than those in the control group (P < 0.05 or P < 0.01) except those at 5 days in the ephedrine group (P > 0.05), and the above-mentioned three indexes of the heroin group were significantly higher than those of the ephedrine group (P < 0.05 or P < 0.01). Total test times and error frequency of bait maze increased by the increase in doses of heroin and ephedrine.5)Heroin and ephedrine had effect on the ChAT activity of filial mice’s cerebral temporal lobe cortex. After administration of heroin and ephedrine at 5,10,15,20 days, ChAT activity was lower than that of the control group (P < 0.05 or P < 0.01). Inhibition rate of ChAT activity increased by the increase in doses of heroin and ephedrine. Inhibition rate of ChAT activity in heroin group was higher than that in ephedrine group from 5 days to 20 days.6)Heroin and ephedrine had some effects on microstructure of filial mice’s cerebral temporal lobe cortex. After administration of heroin and ephedrine at 5,10,15,20 days, the dendrites and axon of pyramidal neurons in cerebral temporal lobe cortex were atrophied and the cell bodies became small. The number of apoptotic or necrotic cells was significantly higher than that of the control group (P < 0.05 or P < 0.01), and the above-mentioned two indexes of the heroin group were significantly higher than those of the ephedrine group (P < 0.05 or P < 0.01). The number of apoptotic or necrotic cells increased by the increase in doses of heroin and ephedrine.7)Heroin and ephedrine had effects on expression of Bax protein and KGF in cerebral temporal lobe cortex. After administration of heroin and ephedrine at 5,10,15,20 days, the number of Bax protein- and KGF-immunopositive neurons was significantly higher than that of the control group(P < 0.05 or P < 0.01), and the above-mentioned two indexes of the heroin group were significantly higher than those of the ephedrine group (P < 0.05 or P < 0.01). The number of Bax protein- and KGF-immunopositive neurons increased by the increase in doses of heroin and ephedrine.8)Heroin and ephedrine had some effects on ultrastructure of filial mice’s cerebral temporal lobe cortex. With the doses of heroin and ephedrine increasing,nuclear membrane lost normal round contour, and the structures of organelles showed abnormal or vacuolar. The matrix around capillary dissolved or were destructed, and capillary lumen became narrow. The synaptic vesicles reduced.Conclusion1) After injecting heroin in pregnant mice, the development of filial mice’s brain was affected for long term. Exposure to heroin at medium and late pregnancy stage had toxic effects on the neurons migration in the filial mice’s brain. Cerebral weight of filial mice, thickness of each cerebral temporal lobe cortex and number of neurons were still lower than that of the control group at postnatal 15 days, which might be the extending results of direct toxicity of heroin and its metabolites on embryonic neurons.2)After injecting heroin in pregnant mice, the activities of ADA、SDH、NOS increased and GSH activity decreased. The damage of the histological structure of filial mice’s brains might be correlated with the changes of ADA,SDH,NOS and GSH activities in mice’s brain. 3)After administration of heroin and ephedrine, the activity of ChAT in cerebral temporal lobe cortex decreased. Cerebral temporal lobe cortex might play an important role in the formation of capability of learning and memory in filial mice.4)Heroin and ephedrine had effects on the histological structure of filial mice cerebral temporal lobe cortex. Positive expression of Bax protein and KGF enhanced. With the concentration increasing, the extent of damage of cerebral temporal lobe cortex also increased, which showed an evident logarithm dose-effect relationship.5)Heroin and ephedrine had great effect on learning and memory in filial mice, and this effects would be related with the damage of the histological structure of cerebral temporal lobe cortex and low activity of ChAT. With the concentration increasing, the extent of damage of learning and memory in filial mice also increased.更多还原