本文選題：鎘 + 神經(jīng)元　； 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的探討維生素E(Vitamin E,VE)對鎘致小鼠大腦皮質(zhì)神經(jīng)元與星形膠質(zhì)細胞損傷的保護作用。方法一、體外實驗(1)體外培養(yǎng)原代大腦皮質(zhì)神經(jīng)元和星形膠質(zhì)細胞,并選擇神經(jīng)元特異性蛋白(MAP2)、星形膠質(zhì)細胞特異性蛋白(GFAP)和小膠質(zhì)細胞特異性蛋白(CD11b)進行細胞純度鑒定;(2)以不同濃度鎘溶液染毒,于倒置相差顯微鏡下分別觀察神經(jīng)元和星形膠質(zhì)細胞在不同時間點形態(tài)學(xué)變化,加入維生素E干預(yù)后,觀察維生素E對染鎘皮質(zhì)神經(jīng)元和星形膠質(zhì)細胞的形態(tài)學(xué),分光光度計檢測SOD活性、MDA含量,Western blot檢測Bcl-2/Bax、caspase-3蛋白表達。二、體內(nèi)實驗(1)健康昆明種雄性小鼠60只隨機分為3組,每組20只。慢性鎘中毒組(Cd組):氯化鎘溶液2mg/kg經(jīng)腹腔皮下注射,每周兩次;慢性鎘中毒加維生素E組(Cd+VE組):注射氯化鎘染毒,同時每天進行維生素E溶液10 mg/kg灌胃;對照組:注射生理鹽水。(2)建模13周后,Y形迷宮檢測各組小鼠學(xué)習(xí)記憶能力,檢測大腦皮質(zhì)組織勻漿SOD活性和MDA含量,尼氏染色觀察額葉皮質(zhì)區(qū)神經(jīng)元尼氏體變化,透射電鏡觀察大腦額葉皮質(zhì)神經(jīng)元超微結(jié)構(gòu)變化,免疫組化檢測大腦額葉皮質(zhì)區(qū)GFAP和caspase-3陽性表達細胞,Western blot檢測大腦皮質(zhì)組織caspase-3的蛋白表達。結(jié)果(1)體外培養(yǎng)的原代大腦皮質(zhì)神經(jīng)元和星形膠質(zhì)細胞,經(jīng)免疫熒光鑒定純度均可達到90%以上;(2)體外實驗中,與對照組比較,隨著染鎘濃度的增加和染鎘時間的延長,神經(jīng)元與星形膠質(zhì)細胞形態(tài)學(xué)損傷逐漸加重;加入維生素E干預(yù)后,與單純的染鎘組比較,神經(jīng)元與星形膠質(zhì)細胞形態(tài)學(xué)損傷減輕,細胞的SOD活性升高、MDA含量下降(P0.05),Bcl-/Bax比值升高、caspase-3蛋白表達下降(P0.05);VE干預(yù)組與對照組比較,各項指標(biāo)仍有差異(P0.05);(3)在體研究中,Cd組小鼠學(xué)習(xí)記憶能力明顯低于對照組和Cd+VE組(P0.05);Cd組小鼠大腦皮質(zhì)組織中SOD活性均低于對照組和Cd+VE組、MDA含量均高于對照組和Cd+VE組(P0.05);與對照組和Cd+VE組相比,Cd組大腦皮質(zhì)額葉神經(jīng)元尼氏體減少,神經(jīng)元超微結(jié)構(gòu)損傷嚴重,GFAP與caspase-3陽性細胞增多,大腦皮質(zhì)組織caspase-3蛋白表達顯著增高(P0.05);與對照組比較,Cd+VE組大腦皮質(zhì)神經(jīng)元尼氏體輕度減少,神經(jīng)元超微結(jié)構(gòu)輕度受損,caspase-3與GFAP陽性細胞高于對照組,皮質(zhì)組織caspase-3蛋白表達同樣高于對照組(P0.05)。結(jié)論維生素E對鎘致小鼠大腦皮質(zhì)神經(jīng)元和星形膠質(zhì)細胞的損傷具有一定的保護作用,且維生素E的保護作用與其抗氧化、減少細胞凋亡有關(guān)。
[Abstract]:Objective to investigate the protective effect of vitamin E (Vitamin E) on the injury of cerebral cortex neurons and astrocytes induced by cadmium in mice. Methods 1. Primary cortical neurons and astrocytes were cultured in vitro. Neuron specific protein (MAP2), astrocyte specific protein (GFAP) and microglia specific protein (CD11b) were selected for cell purity identification. The morphologic changes of neurons and astrocytes at different time points were observed under inverted phase contrast microscope. After vitamin E was added, the morphologic changes of neurons and astrocytes in cadmium-exposed cortex were observed. The content of SOD and MDA was measured by spectrophotometer and the expression of Bcl-2 / Bax-caspase-3 protein was detected by Western blot. In vivo experiment (1) 60 healthy Kunming male mice were randomly divided into 3 groups with 20 mice in each group. Chronic cadmium poisoning group (CD group): cadmium chloride solution 2mg/kg was subcutaneously injected twice a week, chronic cadmium intoxication plus vitamin E group (CD VE group) was injected with cadmium chloride, and vitamin E solution was given orally for 10 mg/kg daily. Control group: normal saline was injected. (2) after 13 weeks of modeling, the learning and memory ability, the activity of SOD and the content of MDA in the homogenate of cerebral cortex were measured by Y-shaped maze, and the changes of Nissl body of neurons in frontal cortex were observed by Nissl staining. The ultrastructural changes of neurons in frontal cortex were observed by transmission electron microscope, and the expression of caspase-3 protein in cerebral cortex was detected by immunohistochemistry, and the positive cells of GFAP and caspase-3 were detected by Western blot. Results (1) the purity of primary cortical neurons and astrocytes cultured in vitro was over 90%, (2) compared with the control group, the concentration of cadmium increased and the time of exposure to cadmium prolonged. The morphological damage of neurons and astrocytes was gradually aggravated, and the morphological damage of neurons and astrocytes was alleviated after the addition of vitamin E. The increase of SOD activity and the decrease of MDA content (P0.05) increased the expression of caspase-3 protein in Bcl-P / Bax ratio (P0.05) compared with the control group. There was still significant difference in the indexes (P0.05). The learning and memory ability of mice in the CD group was significantly lower than that in the control group and CD VE group (P0.05). The activity of); (in the cerebral cortex of the mice in the CD VE group was lower than that in the control group and CD VE group, and the content of); (in the CD VE group was higher than that in the control group and CD VE group. Compared with the control group and the CD VE group, the Nissl bodies of the frontal cortex neurons in the CD and C groups were decreased, while those in the VE group were significantly lower than those in the control group and CD VE group. The expression of caspase-3 protein in cerebral cortex was significantly higher than that in the control group (P0.05), and the neuron Nissl body in the CD VE group was slightly decreased compared with the control group. The expression of caspase-3 protein in cortex was also higher than that in control group (P0.05). Conclusion Vitamin E has a protective effect on the injury of cerebral cortex neurons and astrocytes induced by cadmium, and the protective effect of vitamin E is related to its antioxidation and the decrease of apoptosis.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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