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雷洛昔芬对镉染毒去卵巢大鼠肾脏及肾小管上皮细胞的影响

发布时间:2018-03-17 11:06

  本文选题: 切入点:雌激素受体 出处:《桂林医学院》2014年硕士论文 论文类型:学位论文


【摘要】:目的:观察雷洛昔芬对镉染毒大鼠肾脏组织及肾小管上皮细胞中ERa、£1^和MT-la基因表达的影响,探讨该药在镉中毒防治中的保护作用及机制。方法:①动物部分:选取健康雌性SD大鼠32只,随机分为假手术组,去卵巢组,染镉组与雷洛昔芬组。应用放射免疫法测定四组大鼠血清雌激素;Elisa法测定02-微球蛋白;石墨炉原子吸收法测定血镉、尿镉;PCR及免疫组化检测肾脏组织中ER a、ERP和MT-lamRNA及蛋白水平。②细胞部分:选取新生SD大鼠的肾脏组织进行细胞培养,采用Percoll法分离纯化肾小管上皮细胞后原代培养。选取第二代细胞进行实验。实验分为空白组,氯化镉组与雷洛昔芬组。4h后采用MTT法检测细胞活力,,24h后应用PCR及免疫组化检测ERd、ERP及MT-lamRNA及蛋白水平。结果:①动物部分:染镉组与假手术组、去卵巢组相比,雌激素水平明显降低(K0.05),肾脏湿重、肾脏系数、尿02-微球蛋白、血镉及尿镉水平均升高(K0.05),肾脏病理检查发现肾小管出现退行性病变,肾间质出现大面积渗血,RT-PCR及免疫组化结果显示ER0及MT-la蛋白表达水平升高(代0.05)。与染镉组相比,雷洛昔芬组雌激素水平明显升高(K0.05),尿e2-微球蛋白、血镉及尿镉水平均下降(K0.05),肾小管病变减轻,偶见退行性病变,ERP及MT-la蛋白表达水平下降(代0.05),且两者呈正相关(r=0.387,fKO.05)。而ER a mRNA及蛋白表达在染镉组与雷洛昔芬组间变化不显著(乃0.05)。②细胞部分:与空白组相比,氯化镉组大量细胞的形态发生改变,呈圆型,细胞大量死亡,存活率仅空白组的55.3%, PCR及免疫组化结果显示ERP及MT-la mRNA及蛋白表达水平升高(/0.05)。氯化镉组相比,雷洛昔芬组仅少量细胞形态发生改变,细胞存活率上升到88.6%,同时ERP,MT-lamRNA及蛋白表达水平下降(K0.05)。而ERa在三组间表达变化不明显,无统计学意义(AO.05)。结论:镉对肾脏有显著的类雌激素作用,可能通过与ERP结合后刺激MT-la表达量增加,形成Cd-MT化合物损伤肾脏。而雷洛昔芬对镉的类雌激素活性有拮抗作用,可降低镉对肾脏的损伤。这可能与雷洛昔芬与镉竞争性结合肾脏组织ERP后而下调MT-la的表达,使得CM-MT合成减少,从而降低肾脏对镉的敏感性有关。
[Abstract]:Aim: to observe the effects of raloxifene on the expression of ERA, 1 ^ and MT-la genes in renal tissues and tubular epithelial cells of rats exposed to cadmium. To explore the protective effect and mechanism of the drug in the prevention and treatment of cadmium poisoning methods: 32 healthy female SD rats were randomly divided into sham-operation group and ovariectomized group. Cadmium group and raloxifene group. Serum estradiol Elisa assay and graphite furnace atomic absorption spectrometry were used to determine serum cadmium in four groups of rats. The levels of ER AERP, MT-lamRNA and protein in renal tissues were detected by PCR and immunohistochemistry. The renal tissues of newborn SD rats were selected for cell culture. The primary culture of renal tubular epithelial cells was carried out by Percoll method. The second generation cells were selected for experiment. The experiment was divided into blank group. In cadmium chloride group and raloxifene group, the levels of PCR, MT-lamRNA and protein were detected by PCR and immunohistochemistry after 24 hours by MTT assay. Results: the mice in CD group were compared with sham operation group and ovariectomized group, and the levels of ERP, MT-lamRNA and protein in CD 2 + group were compared with those in sham operation group and ovariectomized group. The levels of estrogen, wet weight of kidney, coefficient of kidney, urine 02-microglobulin, blood cadmium and urine cadmium levels were all increased. The renal pathological examination showed that the renal tubules had degenerative lesions. The results of RT-PCR and immunohistochemistry showed that the expression of ER0 and MT-la protein was increased in the renal interstitial tissue. Compared with the cadmium group, the estrogen level in the raloxifene group was significantly higher than that in the cadmium exposed group, and the urine e2-microglobulin was higher in the raloxifene group than in the cadmium exposed group. The levels of blood cadmium and urine cadmium were decreased, and the renal tubule lesion was alleviated. It was occasionally observed that the expression of ERP and MT-la protein in degenerative lesions decreased (0. 05%), and there was a positive correlation between them. However, the expression of ER a mRNA and protein did not change significantly between cadmium exposed group and raloxifene group (0. 05%) (0. 05%), compared with the control group, the expression of ER a mRNA and protein was not significant (0. 05%), and there was no significant difference between CD group and raloxifene group. The morphology of a large number of cells in the cadmium chloride group was round and the cells died in large numbers. The survival rate was only 55.3in the blank group. The PCR and immunohistochemical results showed that the expression levels of ERP and MT-la mRNA and protein were increased by / / 0.05 in the cadmium chloride group, compared with the cadmium chloride group. In raloxifene group, only a few cell morphology changes occurred, the cell survival rate increased to 88.6 and the expression level of ERPnMT-lamRNA and protein decreased. However, the expression of ERa did not change significantly among the three groups. Conclusion: cadmium has significant estrogenic effect on kidney, which may result in the formation of Cd-MT compound to damage kidney by stimulating the increase of MT-la expression after binding to ERP, while raloxifene has antagonistic effect on the estrogenic activity of cadmium. This may be related to the down-regulation of MT-la expression in renal ERP by the competitive binding of raloxifene and cadmium, and the decrease of CM-MT synthesis, which may reduce the sensitivity of kidney to cadmium.
【学位授予单位】:桂林医学院
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R965


本文编号:1624460

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