【作者】 荣杰；

【导师】 赵宝玉；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 硕士

【摘要】 苦马豆素因具有较强的细胞毒、免疫调节、细胞保护、抗病毒和抑菌作用,能抑制肿瘤转移、扩散,促细胞凋亡,近年备受国内外学者的关注。研究认为,苦马豆素对多种α-甘露糖苷酶具有抑制作用,对哪种α-甘露糖苷酶的抑制起主导作用还不得而知,是否对α-甘露糖苷酶转录、翻译、加工修饰产生影响还不清楚。本研究以含苦马豆素的甘肃棘豆(Oxytropis kansuensis Bunge)为毒源,用SD大鼠复制苦马豆素中毒模型,通过生化指标、抗氧化指标、病理组织学和分子生物学等研究,探讨苦马豆素对SD大鼠亚急性毒性及α-甘露糖苷酶在mRNA水平的影响;通过免疫组织化学方法,研究高尔基体α-甘露糖苷酶Ⅱ在SD大鼠不同组织的分布,以及SD大鼠甘肃棘豆亚急性中毒时高尔基体α-甘露糖苷酶Ⅱ在不同组织中的蛋白表达情况。研究取得以下结果:1. SD大鼠甘肃棘豆亚急性中毒模型复制64只SD大鼠随机分为4组,即对照组、试验Ⅰ组、试验Ⅱ组、试验Ⅲ组。将甘肃棘豆全草粉碎后,按Ⅰ组添加15%(含苦马豆素0.03‰)、Ⅱ组添加30%(含苦马豆素0.06‰)、Ⅲ组添加45%(含苦马豆素0.09‰)的比例制作混合饲料,饲喂至77天试验Ⅲ组出现疯草中毒临床典型症状。2.甘肃棘豆对SD大鼠血液学指标和生化指标的影响试验结果显示,SD大鼠发生甘肃棘豆亚急性中毒后,RBC、MCV、RDW均降低;GLU、CR、BUN、ALB、TG、ALT变化存在剂量、时间效应,与对照组比较差异显著或差异极显著(P<0.05或P<0.01),并随攻毒时间的延长逐渐升高。3.甘肃棘豆对SD大鼠抗氧化指标的影响甘肃棘豆能使血清中SOD活力和MDA含量下降,CAT活力先升高后降低。试验组大鼠大脑、肝脏、肾脏、脾脏组织SOD活力在试验初期都有升高趋势,但高剂量组在试验后期受到抑制。大脑组织中CAT含量随攻毒剂量和时间先降低后升高,低剂量甘肃棘豆能使肝脏CAT活力升高。试验组肝脏组织MDA含量在第35天与对照组差异极显著,试验后期低剂量组肾脏组织中MDA含量明显升高。4.甘肃棘豆对SD大鼠的病理学影响SD大鼠甘肃棘豆中毒能引起脑组织中部分神经细胞周围出现较大空隙,且HE染色变淡。小脑中浦肯野细胞、脑干网状结构、肾脏、肝脏、脾脏出现以空泡变性为特征的病理变化,肾脏远曲小管病理变化要严重于近曲小管,脾脏边缘区出现大量巨噬细胞。5.甘肃棘豆对SD大鼠α-甘露糖苷酶mRMA表达的影响甘肃棘豆能引起大脑和肾脏MAN2A1 mRNA表达水平的下降,但与对照组比较对大脑影响差异不显著(P>0.05);低剂量甘肃棘豆对肝脏中MAN2A1 mRNA表达具有抑制作用,差异极显著(P<0.01);脾脏结果与肾脏相反,但差异不显著(P>0.05)。大脑、肝脏、肾脏、脾脏的MAN2BA mRNA表达均下降,大脑和脾脏表达差异不显著(P>0.05),肝脏表达差异极显著(P<0.01),肾脏高剂量组差异显著(P<0.05)。6.甘肃棘豆对SD大鼠MAN2A1分布与表达的影响MAN2A1在脑组织中除大脑分子层、海马辐射层,海马始层,齿状回分子层不表达外,其它区域均有分布,表达量与神经细胞和颗粒细胞的数量正相关,这两种细胞的MAN2A1表达量明显高于其他类型的细胞。肾脏集合小管和远曲小管直部MAN2A1表达相对较强,肝脏和脾脏组织种细胞类型较少,分布差异不明显。试验组大脑、小脑、肾脏中MAN2A1表达量随攻毒剂量增加而降低,浦肯野细胞层部分细胞MAN2A1表达消失,肝脏组织在低剂量甘肃棘豆作用下MAN2A1表达就会受到明显抑制。更多还原

【Abstract】 It has been documented that SW has such functions as cytotoxicity, immunoregulation, cytoprotection, antiviral and antibacterial activities etc, which enables it to inhibite the metastasis and the spread of the tumor cells while to stimulate the apoptosis, which makes many researchers focus on it. It has been described that SW is an efficient and specific inhibitor of different kinds ofα-mannosidases, but which is inhibited most is not known. It is also not clear that whether SW has effects on the transcription, translation, processing and modification ofα-mannosidase. In this research, SW poisoning model was reconstructed in SD rats using Oxytropis kansuensis Bunge as the SW resourse, through the monitor of biochemical and antioxidant indexes and the histopathological and molecular biological researches, the subacute toxicity of SW towards SD rats and its effects on MAN2A1 and MAN2B1 at mRNA level were investigated. Furthermore the distribution and expression of MAN2A1 in different tissues of SD rats during the subacute O. kansuensis Bunge intoxication were investigated through immunohistochemical assay. The results are as below:1. Reconstruction of subacute O. kansuensis Bunge poisoning model in SD rats64 SD rats were divided into 4 groups (the control group and experimental groupⅠ,Ⅱ,Ⅲ), the dried plant of the Oxytropis kansuensis Bunge was comminuted, and different amounts of the grass powder (15%, 30% and 45% respectively, and the corresponding SW content was 0.03‰, 0.06‰, 0.09‰) were mixed with the feeds for the three experimental groups. After 77 days, The experimental groupⅢshowed typical clinical symptoms of loco disease.2. Effects of O. kansuensis Bunge on the hematologic and biochemical indicators in SD ratsThe results indicated that, after the poisoning, the values of RBC, MCV, RDW decreased. A time-effect as well as a dose-effect relationship was observed in GLU, CR, BUN, ALB, TG and ALT, and there was significant difference or extremely significant difference compared with the control group (P<0.05 or P<0.01), moreover the values increased with the experimental time extention.3. Effects of O. kansuensis Bunge on antioxidant indexes in SD ratsIt was observed that the levels of serum SOD and MDA reduced in SD rats after intaking O. kansuensis Bunge, while the CAT activity increased at first and then decreased. The SOD activity in the all four tissues of Experiment Groups rose in the initial stage, which decreased in the later stage in high-dose group. The CAT activity decreased at first and then increased with dose of intaking O. kansuensis Bunge rose and time extension in cerebrum, and livers’increased in Low-dose group. The difference of livers’MDA content was extremely significant between the Experiment Groups and the Control Group at the 35th day, and the kidneys’increased significantly in the later stage in Low-dose group.4. The pathological effects of O. kansuensis Bunge on SD ratsDuring the intoxication, there were comparatively large interspaces round the nerve cells in the Thalamus, Hypothalamus and Midbrain etc, which was dyed lighter compared with the normal ones by H-E staining. Purkinje cells, brainstem reticular formation, kidneys, liver and spleen appeared pathological changes with characterized vacuolar degeneration. the pathological changes in proximal convoluted tubule were more serious than thoses in distal convoluted tubule, moreover there were large amount of macrophagocytes in the splenic marginal zone.5. Effects of O. kansuensis Bunge on the expression ofα-mannosidase mRMA in SD ratsO. kansuensis Bunge inhibited the expression of cerebral and renal MAN2A1 mRNA, but the difference was not obvious in cerebrum (P>0.05). Low-dose O. kansuensis Bunge inhibited the expression of hepatic MAN2A1 mRNA while the high-dose one promoted it and the difference was extremely significant (P<0.01), which was opposite with that of spleen’s, but the difference was not obvious in spleen (P>0.05). The expression of MAN2B1 mRNA decreased in the all four tissues, and the differences were not remarkable in cerebrum and spleen (P>0.05), whereras it was extremely significant in liver(P<0.01), and there was obvious difference in the kidneys in high-dose group (P<0.05).6. Effects of O. kansuensis Bunge on the distribution and expression of MAN2A1 in SD ratsIt was found that MAN2A1 expressed in all the areas but the cerebral molecular layer, Rad, Or and Mol in brain. The expression level of MAN2A1 positively correlated with the amount of the nerve cells and the granulocytes, in which MAN2A1 expressed more than other cells. The expression level of MAN2A1 was relatively higher in collecting tubule and distal convoluted tubule in kidneys, while there were no remarkable differences in hepatic and the splenic tissue. Moreover the expression level of MAN2A1 decreased with the increase of the poisonous dosage in cerebrum, cerebellum and kidneys, and the expression in some purkinje cells disappeared, which was inhibited significantly in liver in the Low-dose group.更多还原