發(fā)布時(shí)間：2018-03-17 11:06

  本文選題：鋪　切入點(diǎn)：雌激素受體　出處：《桂林醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：觀察雷洛昔芬對鎘染毒大鼠腎臟組織及腎小管上皮細(xì)胞中ERa、￡1^和MT-la基因表達(dá)的影響，探討該藥在鎘中毒防治中的保護(hù)作用及機(jī)制。方法:①動(dòng)物部分：選取健康雌性SD大鼠32只，隨機(jī)分為假手術(shù)組，去卵巢組，染鎘組與雷洛昔芬組。應(yīng)用放射免疫法測定四組大鼠血清雌激素；Elisa法測定02-微球蛋白；石墨爐原子吸收法測定血鎘、尿鎘；PCR及免疫組化檢測腎臟組織中ER a、ERP和MT-lamRNA及蛋白水平。②細(xì)胞部分：選取新生SD大鼠的腎臟組織進(jìn)行細(xì)胞培養(yǎng)，采用Percoll法分離純化腎小管上皮細(xì)胞后原代培養(yǎng)。選取第二代細(xì)胞進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)分為空白組,氯化鎘組與雷洛昔芬組。4h后采用MTT法檢測細(xì)胞活力，，24h后應(yīng)用PCR及免疫組化檢測ERd、ERP及MT-lamRNA及蛋白水平。結(jié)果：①動(dòng)物部分：染鎘組與假手術(shù)組、去卵巢組相比，雌激素水平明顯降低（K0.05)，腎臟濕重、腎臟系數(shù)、尿02-微球蛋白、血鎘及尿鎘水平均升高(K0.05)，腎臟病理檢查發(fā)現(xiàn)腎小管出現(xiàn)退行性病變，腎間質(zhì)出現(xiàn)大面積滲血，RT-PCR及免疫組化結(jié)果顯示ER0及MT-la蛋白表達(dá)水平升高（代0.05)。與染鎘組相比，雷洛昔芬組雌激素水平明顯升高（K0.05),尿e2-微球蛋白、血鎘及尿鎘水平均下降（K0.05)，腎小管病變減輕，偶見退行性病變，ERP及MT-la蛋白表達(dá)水平下降(代0.05)，且兩者呈正相關(guān)(r=0.387，fKO.05)。而ER a mRNA及蛋白表達(dá)在染鎘組與雷洛昔芬組間變化不顯著（乃0.05)。②細(xì)胞部分：與空白組相比，氯化鎘組大量細(xì)胞的形態(tài)發(fā)生改變，呈圓型，細(xì)胞大量死亡，存活率僅空白組的55.3%, PCR及免疫組化結(jié)果顯示ERP及MT-la mRNA及蛋白表達(dá)水平升高(/0.05)。氯化鎘組相比，雷洛昔芬組僅少量細(xì)胞形態(tài)發(fā)生改變，細(xì)胞存活率上升到88.6%，同時(shí)ERP,MT-lamRNA及蛋白表達(dá)水平下降（K0.05)。而ERa在三組間表達(dá)變化不明顯，無統(tǒng)計(jì)學(xué)意義（AO.05)。結(jié)論：鎘對腎臟有顯著的類雌激素作用，可能通過與ERP結(jié)合后刺激MT-la表達(dá)量增加，形成Cd-MT化合物損傷腎臟。而雷洛昔芬對鎘的類雌激素活性有拮抗作用，可降低鎘對腎臟的損傷。這可能與雷洛昔芬與鎘競爭性結(jié)合腎臟組織ERP后而下調(diào)MT-la的表達(dá)，使得CM-MT合成減少，從而降低腎臟對鎘的敏感性有關(guān)。
[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.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965


本文編號(hào)：1624460