【摘要】：鉛是一種化學(xué)性質(zhì)穩(wěn)定的重金屬毒物,神經(jīng)系統(tǒng)是鉛毒作用的重要靶器官。鉛暴露可降低細(xì)胞中超氧化物歧化酶、過氧化氫酶等抗氧化酶的活性及谷胱甘肽等抗氧化物質(zhì)的含量,引起細(xì)胞氧化損傷,因此鉛的神經(jīng)毒性與氧化應(yīng)激及其所致氧化損傷密切相關(guān)。JWA是活躍的環(huán)境應(yīng)答基因,廣泛參與調(diào)控多種環(huán)境因素誘導(dǎo)的氧化應(yīng)激,保護(hù)細(xì)胞免受氧化損傷。目前尚未見JWA在鉛毒性中作用的相關(guān)報(bào)道,本研究以體外培養(yǎng)的新生大鼠皮質(zhì)神經(jīng)細(xì)胞為研究對(duì)象,研究JWA在鉛神經(jīng)毒性中的表達(dá)變化及可能機(jī)制。目的:觀察鉛對(duì)大鼠大腦皮質(zhì)神經(jīng)細(xì)胞中各氧化應(yīng)激指標(biāo)以及JWA基因表達(dá)的影響,探討JWA基因參與神經(jīng)細(xì)胞鉛氧化應(yīng)激作用可能機(jī)制,為進(jìn)一步從分子水平探討鉛的神經(jīng)毒性機(jī)制提供新的線索和依據(jù)。方法:取新生24 h內(nèi)的SD大鼠皮質(zhì)神經(jīng)細(xì)胞進(jìn)行原代培養(yǎng)并用免疫細(xì)胞化學(xué)方法鑒定;將體外生長(zhǎng)良好的皮質(zhì)神經(jīng)細(xì)胞,分別用終濃度為0μmol/L、31μmol/L、54μmol/L、92μmol/L、150μmol/L醋酸鉛神經(jīng)細(xì)胞維持培養(yǎng)液處理一定時(shí)間,MTT法檢測(cè)細(xì)胞存活情況,并檢測(cè)細(xì)胞超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、過氧化氫酶(CAT)活力和谷胱甘肽(GSH)含量,QRT-PCR技術(shù)檢測(cè)皮質(zhì)神經(jīng)細(xì)胞JWA mRNA的表達(dá)量。結(jié)果:(1)形態(tài)學(xué)觀察和免疫組織化學(xué)鑒定發(fā)現(xiàn),體外原代培養(yǎng)的皮質(zhì)神經(jīng)細(xì)胞在培養(yǎng)第7 d左右生長(zhǎng)狀態(tài)良好,神經(jīng)細(xì)胞所占比例較高,可用于之后鉛損傷模型的建立。(2)隨著醋酸鉛濃度的逐漸升高,培養(yǎng)的大鼠皮質(zhì)神經(jīng)細(xì)胞數(shù)量逐漸減少。染鉛24 h和48 h,92μmol/L和150μmol/L醋酸鉛組同其他劑量組間差異有統(tǒng)計(jì)學(xué)意義(P0.05),92μmol/L醋酸鉛作用下平均OD值分別為0.218±0.008和0.181±0.006,而150μmol/L醋酸鉛作用下平均OD值分別為0.199±0.008和0.166±0.009。篩選出醋酸鉛的適宜作用濃度為92μmol/L,適宜作用時(shí)間為24 h。(3)不同濃度醋酸鉛分別作用皮質(zhì)神經(jīng)元6 h、12 h、24h、48 h,SOD活力、CAT活力和GSH含量較對(duì)照組均有明顯下降(P0.05),而MDA含量較對(duì)照組均有明顯升高(P0.05),皮質(zhì)神經(jīng)細(xì)胞內(nèi)SOD活力、CAT活力和GSH含量與染鉛劑量呈負(fù)相關(guān)關(guān)系(P0.01),MDA含量與染鉛劑量呈正相關(guān)關(guān)系(P0.01)。(4)隨著鉛含量的升高,各鉛處理組細(xì)胞JWA mRNA的表達(dá)水平呈逐漸下降趨勢(shì)(P0.05);鉛處理的同時(shí)加入適當(dāng)?shù)某趸锲缁?JWA mRNA的表達(dá)量差異無統(tǒng)計(jì)學(xué)意義(P0.05),而鉛處理同時(shí)加入適量的過氧化氫酶,JWA mRNA的表達(dá)量升高,且差異有統(tǒng)計(jì)學(xué)意義(P0.05);JWA mRNA表達(dá)量與SOD活力、CAT活力和GSH含量呈正相關(guān)關(guān)系,與MDA含量呈負(fù)相關(guān)關(guān)系(P0.01)。結(jié)論:鉛暴露可導(dǎo)致原代培養(yǎng)的大鼠皮質(zhì)神經(jīng)細(xì)胞發(fā)生氧化應(yīng)激損傷并影響基因JWA的表達(dá)。
[Abstract]:Lead is a kind of heavy metal poison with stable chemical properties. The nervous system is an important target organ of lead toxicity. Lead exposure can reduce the activities of superoxide dismutase, catalase and other antioxidant enzymes, and the content of antioxidants such as glutathione, thus causing oxidative damage to cells. Therefore, the neurotoxicity of lead is closely related to oxidative stress and oxidative damage. JWA is an active environmental response gene, which is widely involved in regulating oxidative stress induced by many environmental factors and protecting cells from oxidative damage. There is no report on the role of JWA in lead toxicity. In this study, the expression of JWA in lead neurotoxicity and its possible mechanism were studied in cultured neonatal rat cortical nerve cells. Aim: to observe the effects of lead on the oxidative stress indexes and the expression of JWA gene in rat cerebral cortical neurons, and to explore the possible mechanism of JWA gene involved in the oxidative stress of nerve cells. It provides a new clue and basis for further exploring the neurotoxic mechanism of lead at molecular level. Methods: the cortical neurons of SD rats within 24 hours were cultured and identified by immunocytochemistry. The cells were treated with 0 渭 mol/L,31 渭 mol/L,54 渭 mol/L,92 渭 mol/L,150 渭 mol/L lead acetate culture medium for a certain time, the survival of the cells was detected by MTT method, the (SOD) activity of superoxide dismutase (SOD) and the content of MDA (MDA) were measured. The activity of catalase (CAT) and the content of glutathione (GSH) were measured. The expression of JWA mRNA in cortical neurons was detected by QRT-PCR. Results: (1) morphological observation and immunohistochemical analysis showed that the primary cultured cortical neurons grew well on the 7th day of culture, and the proportion of neurons was high. It can be used to establish the lead injury model. (2) with the increasing of lead acetate concentration, the number of cultured cortical nerve cells decreased gradually. There was significant difference between lead exposure group and other dose groups in lead exposure for 24 h and 48 h (P0.05). The average OD values of lead acetate group exposed to lead acetate at 92 渭 mol/L were 0.218 鹵0.008 and 0.181 鹵0.006, respectively, and the average OD values of lead acetate group exposed to 150 渭 mol/L lead acetate were 0.199 鹵0.008 and 0.166 鹵0.009, respectively. The optimum concentration of lead acetate was 92 渭 mol/L, for 24 h. (3) the SOD activity, CAT activity and GSH content of cortical neurons treated with lead acetate for 6 h, 12 h, 24 h and 48 h were significantly lower than those of the control group (P0.05), while the content of MDA was significantly lower than that of the control group (P0.05). SOD activity, CAT activity and GSH content in cortical nerve cells were negatively correlated with lead exposure dose (P0.01). (4). The expression of JWA mRNA in the cells of each lead treatment group showed a decreasing trend (P0.05), while the expression of superoxide dismutase, JWA mRNA was not significantly different (P0.05), while the lead treatment with appropriate amount of hydrogen peroxide added at the same time (P0.05). The expression of enzyme, JWA mRNA was increased, The difference was statistically significant (P0.05) the expression of); JWA mRNA was positively correlated with SOD activity, CAT activity and GSH content, and negatively correlated with MDA content (P0.01). Conclusion: lead exposure can induce oxidative stress injury and affect gene JWA expression in primary cultured rat cortical neurons.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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