發(fā)布時(shí)間：2018-02-03 02:34

  本文關(guān)鍵詞： 鑭 神經(jīng)元 活性氧 線粒體 bcl-2 bax 細(xì)胞凋亡　出處：《中國(guó)醫(yī)科大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 前言 稀土(rare earth, RE)包括17種元素,其中鑭至銪為輕稀土元素,釓至釔為重稀土元素。我國(guó)是稀土資源最多的國(guó)家,近年來(lái)隨著稀土在農(nóng)業(yè)、畜牧業(yè)、工業(yè)、國(guó)防和高技術(shù)產(chǎn)業(yè)及現(xiàn)代生物醫(yī)學(xué)上應(yīng)用的日益擴(kuò)展,稀土元素正在廣泛進(jìn)入環(huán)境,并通過(guò)食物鏈進(jìn)入人體。因此,稀土對(duì)生態(tài)環(huán)境和人體健康的影響及其生物學(xué)效應(yīng),已成為不容忽視的衛(wèi)生學(xué)問(wèn)題。 已有報(bào)道表明RE具有神經(jīng)毒性。稀土礦區(qū)流行病學(xué)調(diào)查顯示,長(zhǎng)期攝入RE使兒童智商和記憶力下降,從而危及礦區(qū)兒童的基本素質(zhì),可能造成深遠(yuǎn)影響。動(dòng)物實(shí)驗(yàn)也證明RE影響學(xué)習(xí)記憶,可能涉及降低抗氧化能力；干擾腦內(nèi)微量元素的代謝；影響酶活力和神經(jīng)遞質(zhì)的釋放等。 鑭作為輕稀土元素的代表,化學(xué)性質(zhì)比較活潑,動(dòng)物實(shí)驗(yàn)表明鑭具有神經(jīng)毒性,但其作用機(jī)制尚不清楚。本研究以大鼠皮層神經(jīng)元為研究對(duì)象,觀察不同劑量鑭處理組神經(jīng)元的線粒體活性,細(xì)胞內(nèi)活性氧含量,線粒體膜電位和bcl-2、baxmRNA表達(dá)的改變以及細(xì)胞凋亡的變化情況,來(lái)探討氯化鑭對(duì)神經(jīng)元的凋亡及其線粒體功能的影響,為闡明稀土元素的神經(jīng)毒性機(jī)制提供新的實(shí)驗(yàn)證據(jù)。 實(shí)驗(yàn)分組 LaCl3用無(wú)血清DMEM配制,終濃度分別為0.01,0.1,1.0,2.0mmol/L,對(duì)照組應(yīng)用無(wú)血清DMEM。 研究方法 1、神經(jīng)元免疫細(xì)胞化學(xué)法純度鑒定 2、MTT法測(cè)定神經(jīng)元線粒體活性 3、DCFH-DA熒光探針?lè)y(cè)定細(xì)胞內(nèi)ROS含量 4、Rhodamine123熒光探針?lè)y(cè)定神經(jīng)元線粒體膜電位 5、RT-PCR法測(cè)定神經(jīng)元bcl-2, bax mRNA表達(dá) 6、流式細(xì)胞儀法測(cè)定細(xì)胞凋亡率 7、倒置顯微鏡觀察神經(jīng)元形態(tài)學(xué)改變 結(jié)果 1、神經(jīng)元的鑒定 顯微鏡下可見(jiàn)絕大部分細(xì)胞胞漿被染成棕黃色,每張玻片選擇10個(gè)視野,計(jì)數(shù)陽(yáng)性細(xì)胞比例,免疫陽(yáng)性細(xì)胞可達(dá)90%以上,因此可以將此原代培養(yǎng)細(xì)胞作為神經(jīng)元的體外模型。 2、神經(jīng)元線粒體活性的改變 鑭暴露后細(xì)胞線粒體活性下降,各劑量組與對(duì)照組相比差異均顯著(p0.01),且呈現(xiàn)一定劑量—反應(yīng)關(guān)系。 3、細(xì)胞內(nèi)活性氧含量的改變 鑭暴露后細(xì)胞內(nèi)ROS水平呈升高趨勢(shì),各劑量組細(xì)胞內(nèi)ROS水平均高于對(duì)照組且統(tǒng)計(jì)學(xué)差異顯著(p0.05)。 4、線粒體膜電位變化 鑭暴露組細(xì)胞線粒體膜電位均降低,并隨鑭暴露濃度的增加而降低顯著,與對(duì)照組比較有明顯差異(p0.05)。 5、bcl-2、bax mRNA表達(dá)改變 鑭暴露組神經(jīng)元bax mRNA表達(dá)增多,同時(shí)bcl-2 mRNA表達(dá)減少,0.1和1.0mmol/L組與對(duì)照組比較均存在顯著差異(p0.01)。 6、細(xì)胞凋亡流式檢測(cè) 氯化鑭暴露組細(xì)胞凋亡率呈升高趨勢(shì),且0.1和1.0mmol/L劑量組與對(duì)照組差異顯著(p0.01)；細(xì)胞壞死率也有增加,但差異不明顯,且低于凋亡率。 結(jié)論 1、氯化鑭能夠使神經(jīng)元內(nèi)ROS水平升高,神經(jīng)元線粒體活性下降,線粒體跨膜電位降低,損傷線粒體功能。 2、氯化鑭可使凋亡調(diào)控基因bax mRNA表達(dá)增多,而bcl-2 mRNA表達(dá)減少,并可誘導(dǎo)大鼠皮層神經(jīng)元凋亡。
[Abstract]:Preface
Rare earth (rare earth, RE) includes 17 elements, including lanthanum to EU for light rare earth elements, yttrium gadolinium to heavy rare earth elements. As our country is the most rare earth resources in recent years, with the rare earth elements in agriculture, animal husbandry, industry, expanding application of national defense and high technology industry and modern medicine. Rare earth elements are widely into the environment, and enter the body through the food chain. Therefore, effect of Rare Earths on the ecological environment and human health and biological effects, has become a health problem can not be ignored.
It has been reported that RE have neurotoxicity. Epidemiological survey of mining area of rare earth, long-term intake of RE children IQ and memory decline, thereby endangering children's basic quality of mining area, may have an impact. Animal experiments also show that RE on learning and memory, may involve lower antioxidant capacity; trace element interference in brain metabolism; enzyme activity and influence the release of neurotransmitters.
La as a representative of light rare earth elements, the chemical nature is lively, the animal experiment showed that lanthanum has neurotoxicity, but its mechanism is still unclear. In this study, rat cortical neurons as the research object, the effects of different doses of La group neuron mitochondrial activity, active oxygen content in cells, mitochondrial membrane potential and Bcl-2 changes the change of baxmRNA expression and cell apoptosis, to investigate the effect of lanthanum chloride on neuronal apoptosis and mitochondrial function, to provide new experimental evidence for clarifying the mechanism of neurotoxicity of rare earth elements.
Experimental grouping
LaCl3 was prepared with serum-free DMEM, the final concentration was 0.01,0.1,1.0,2.0mmol/L, and the control group was serum-free DMEM.
research method
1, the purity identification of neuron immunocytochemical method
2, MTT assay for the determination of neuronal mitochondrial activity
Determination of ROS content in cells by 3, DCFH-DA fluorescence probe
4, Rhodamine123 fluorescence probe method for the determination of mitochondrial membrane potential in neurons
5, RT-PCR method was used to determine the expression of Bcl-2 and Bax mRNA in neurons
6, determination of apoptosis rate by flow cytometry
7, the morphological changes of neurons were observed by inverted microscope
Result
1, identification of neurons
Under microscope, most of the cytoplasm was dyed brown. Each slide selected 10 fields of view, counting the proportion of positive cells, and immunoreactive cells could reach more than 90%. Therefore, this primary culture cell can be used as an in vitro model of neurons.
2, changes in the activity of mitochondria in neurons
After lanthanum exposure, the cell mitochondrial activity decreased, and the difference in each dose group was significantly higher than that of the control group (P0.01), and there was a certain dose response relationship.
3, changes in the content of active oxygen in cells
After lanthanum exposure, the intracellular ROS level increased, and the intracellular ROS level in each dose group was higher than that in the control group and the statistical difference was significant (P0.05).
4, the change of mitochondrial membrane potential
The cell mitochondrial membrane potential in the Lanthanum Exposure group decreased and decreased significantly with the increase of lanthanum exposure concentration, which was significantly different from the control group (P0.05).
Changes in expression of 5, Bcl-2, Bax mRNA
The expression of Bax mRNA in the lanthanum exposed group increased and the expression of Bcl-2 mRNA decreased. There was a significant difference between the 0.1 and 1.0mmol/L groups (P0.01).
6, flow cytometry of cell apoptosis
The apoptosis rate of lanthanum chloride exposure group showed an increasing trend, and there was a significant difference between the 0.1 and 1.0mmol/L dose group and the control group (P0.01), and the cell necrosis rate also increased, but the difference was not obvious, and it was lower than the apoptosis rate.
conclusion
1, lanthanum chloride can increase the level of ROS in neurons, decrease the activity of mitochondria in neurons, decrease the mitochondrial transmembrane potential and damage the function of mitochondria.
2, lanthanum chloride can increase the expression of apoptosis regulating gene Bax mRNA, and decrease the expression of Bcl-2 mRNA, and induce apoptosis of rat cortical neurons.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R363

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