本文選題：miRNA + 鉛�。� 參考：《第四軍醫(yī)大學(xué)》2013年碩士論文

【摘要】：【背景】 鉛是危害人體健康的一種常見環(huán)境重金屬毒物，分布廣泛，主要靶器官是中樞神經(jīng)系統(tǒng)，嚴(yán)重影響兒童的生長發(fā)育和智力水平，還會造成大腦損傷、精神發(fā)育遲滯、發(fā)育緩慢、行為異常和暴力傾向等，重度鉛暴露甚至可以引起死亡[1-3]。鉛中毒的發(fā)生、發(fā)展非常隱匿，常為低劑量長期慢性暴露所致，，目前尚無特別有效的治療方法。鉛在人體代謝緩慢，半衰期長，慢性蓄積毒性效應(yīng)可一直延續(xù)到成年甚至老年，引起神經(jīng)退行性疾病。因此，揭示鉛的神經(jīng)毒性機(jī)制尤其是對學(xué)習(xí)記憶影響的分子生物學(xué)機(jī)制，提出切實(shí)有效的診療、防治措施刻不容緩。 miRNA通過在轉(zhuǎn)錄后水平負(fù)性調(diào)節(jié)靶mRNA的表達(dá)。miRNA在大腦中的表達(dá)豐度很高，在神經(jīng)發(fā)育、突觸可塑性以及學(xué)習(xí)記憶方面起重要作用。miRNA具有在樹突周圍瞬時(shí)、區(qū)域表達(dá)的特點(diǎn)，對外界刺激具有活動依賴的反應(yīng)模式，且一個miRNA可以同時(shí)調(diào)節(jié)多個靶mRNA，一個mRNA也可以同時(shí)接受多個miRNA的調(diào)節(jié)，使得大腦特異性miRNA成為神經(jīng)可塑性和記憶形成微調(diào)基因表達(dá)的可靠候選。因此我們預(yù)測miRNA在鉛誘導(dǎo)的神經(jīng)毒性和學(xué)習(xí)記憶損傷中可能發(fā)揮了重要的作用。 【目的】 在建立鉛暴露誘導(dǎo)大鼠學(xué)習(xí)記憶能力和海馬神經(jīng)元損傷模型的基礎(chǔ)上，探討miRNA表達(dá)譜的變化；驗(yàn)證miRNA是否參與了鉛誘導(dǎo)的神經(jīng)毒性效應(yīng)；揭示miRNA在鉛誘導(dǎo)的神經(jīng)毒性中的作用和機(jī)制；為鉛中毒的預(yù)防和治療提供實(shí)驗(yàn)依據(jù)。 【方法】 1、通過飲水染鉛、血鉛監(jiān)測的方法建立鉛暴露動物模型；通過體外培養(yǎng)PC12細(xì)胞系染鉛的方法建立細(xì)胞模型。 2、通過水迷宮實(shí)驗(yàn)（Morris Water Maze）監(jiān)測隱蔽平臺潛伏期、去平臺目標(biāo)象限停留時(shí)間等指標(biāo)評價(jià)大鼠空間學(xué)習(xí)記憶能力；運(yùn)用TUNEL、Western blot和免疫組織化學(xué)染色方法檢測鉛暴露大鼠海馬組織損傷狀況。 3、運(yùn)用miRNA芯片（microarry analysis）檢測miRNA表達(dá)譜的改變并通過qRT-PCR技術(shù)加以驗(yàn)證。 4、運(yùn)用生物信息學(xué)方法預(yù)測miRNA靶基因并用Western blot方法驗(yàn)證預(yù)測結(jié)果。 5、運(yùn)用miRNA抑制劑（miRNA inhibitor）建立“Loss of function”模型，初步探究miRNA參與鉛誘導(dǎo)的神經(jīng)毒性的作用機(jī)制。 【結(jié)果】 1、鉛可以引起SD大鼠空間學(xué)習(xí)記憶能力降低 鉛暴露組（包括100ppm、200ppm和300ppm組）大鼠在8周飲水染鉛暴露過程中和對照組大鼠相比，飲水量和體重變化均無統(tǒng)計(jì)學(xué)差異，而血鉛水平隨暴露時(shí)間延長及暴露劑量增加顯著升高。Morris水迷宮實(shí)驗(yàn)結(jié)果顯示染鉛大鼠空間學(xué)習(xí)記憶能力明顯受損，Western blot結(jié)果顯示海馬組織學(xué)習(xí)記憶相關(guān)的谷氨酸受體、NMDA受體等表達(dá)水平降低。 2、鉛可以誘導(dǎo)神經(jīng)元凋亡 300ppm飲水鉛暴露8w大鼠模型和10μmol/L鉛暴露48h PC12細(xì)胞模型TUNEL檢測均顯示鉛可以分別誘導(dǎo)海馬神經(jīng)元和PC12細(xì)胞凋亡增加，提示鉛可能是通過誘導(dǎo)神經(jīng)元凋亡引起學(xué)習(xí)記憶能力改變。 3、鉛誘導(dǎo)海馬組織miRNA表達(dá)譜發(fā)生改變 miRNA芯片結(jié)果顯示斷乳SD大鼠飲水染鉛300ppm8w后海馬組織與對照組相比，7個miRNA表達(dá)水平發(fā)生顯著變化，其中miR-204、miR-211、miR-448、miR-449a、miR-34c、miR-34b表達(dá)水平升高1.5倍以上；miR-494表達(dá)降低為原來的1/6。qRT-PCR驗(yàn)證結(jié)果與芯片結(jié)果一致。篩選其中表達(dá)差異較大的三種miRNA（miR-204、miR-211和miR-448），分別對2w、4w、6w和8w的海馬組織進(jìn)行qRT-PCR驗(yàn)證，結(jié)果表明三種miRNA表達(dá)水平均升高。 4、生物信息學(xué)技術(shù)分析結(jié)果顯示差異表達(dá)顯著的miRNA靶基因與凋亡、LTP/LTD、鈣信號通路等相關(guān)，且三個miRNA具有共同的靶基因即Bcl-2和Itpr1 應(yīng)用生物信息學(xué)技術(shù)（DIANA、TargetScan和miRNA三個網(wǎng)站）分析預(yù)測miRNA的靶基因與凋亡、軸突延伸、鈣信號通路、LTP/LTD、MAPK信號通路、Notch信號通路、緊密連接和神經(jīng)退行性疾病相關(guān)。其中miR-204、miR-211和miR-448擁有共同靶基因，即與凋亡相關(guān)的Bcl-2和與鈣通道相關(guān)的Itpr1，Western blot和qRT-PCR方法驗(yàn)證的結(jié)果（動物和PC12細(xì)胞）與預(yù)測結(jié)果一致。 5、干擾miR-204、miR-211和miR-448表達(dá)，能在一定程度上保護(hù)細(xì)胞免受鉛暴露損傷的影響 應(yīng)用miRNAinhibitor建立“Loss of function”模型，使用MTT、TUNEL等方法驗(yàn)證干擾miRNA后鉛對PC12細(xì)胞活力和凋亡的影響，結(jié)果發(fā)現(xiàn)干擾miRNA表達(dá)后能在一定程度上保護(hù)PC12細(xì)胞免受鉛暴露損傷的影響，其中干涉miR-448表達(dá)后可使細(xì)胞活力提高約20%。 【結(jié)論】 1、鉛暴露可能通過引起海馬神經(jīng)元凋亡影響學(xué)習(xí)記憶功能。 2、鉛暴露可以引起海馬miRNA表達(dá)譜發(fā)生改變，這種改變可能在鉛神經(jīng)毒性作用中發(fā)揮著重要作用。 3、差異表達(dá)顯著的miR-204、miR-211和miR-448可以通過負(fù)性調(diào)節(jié)其共同靶基因與凋亡相關(guān)的Bcl-2和與鈣離子通道相關(guān)的Itpr1影響鉛的神經(jīng)毒性作用。 4、干擾miR-204、miR-211和miR-448表達(dá)，能在一定程度上保護(hù)細(xì)胞免受鉛暴露的損傷。
[Abstract]:[background]
Lead is a common environmental heavy metal poison which is harmful to human health and is widely distributed. The main target organ is the central nervous system, which seriously affects the growth and intelligence level of children. It can also cause brain damage, mental retardation, slow development, abnormal behavior and violence, and severe lead exposure can even cause death in [1-3]. lead. The occurrence of poison is very hidden and often caused by low dose chronic chronic exposure. There is no special effective treatment at present. Lead is slow in metabolism, long half-life and chronic accumulative toxic effect can continue to adult and even old age, causing neurodegenerative diseases. It is urgent to recall the molecular biological mechanism of influence and put forward effective diagnosis and treatment.
The expression of miRNA through the post transcriptional level negatively regulates the expression of target mRNA in the brain, the expression of.MiRNA is high in the brain, and plays an important role in neural development, synaptic plasticity and learning and memory..miRNA has the characteristics of transient, regional expression around the dendrites, a reactive pattern of active dependence on external stimuli, and a miRNA can simultaneously be used. To regulate multiple target mRNA, a mRNA can also accept multiple miRNA regulation, making the brain specific miRNA a reliable candidate for neural plasticity and memory formation. Therefore, we predict that miRNA may play an important role in lead induced neurotoxicity and learning memory damage.
[Objective]
On the basis of establishing the learning and memory ability of rats induced by lead exposure and the damage model of hippocampal neurons, the changes of miRNA expression profiles were explored, and whether miRNA was involved in the neurotoxicity induced by lead was verified, and the role and mechanism of miRNA in lead induced neurotoxicity were revealed, and the experimental basis for the prevention and treatment of lead poisoning was provided.
[method]
1, a lead exposure animal model was established by monitoring lead in drinking water and blood lead monitoring. A cell model was established by culturing PC12 cells in vitro.
2, using the water maze test (Morris Water Maze) to monitor the latent period of the hidden platform, and to evaluate the spatial learning and memory ability of the rats by the target quadrant time of the target, and to detect the damage of the hippocampus in the lead exposed rats by TUNEL, Western blot and immunohistochemistry.
3, miRNA chip (microarry analysis) was used to detect the change of miRNA expression profile and verified by qRT-PCR technology.
4, bioinformatics method was used to predict miRNA target genes and Western blot method was used to verify the prediction results.
5, use miRNA inhibitor (miRNA inhibitor) to establish "Loss of function" model, and explore the mechanism of miRNA involved in lead induced neurotoxicity.
[results]
1, lead can reduce the spatial learning and memory ability of SD rats.
The lead exposure group (including 100ppm, 200ppm and 300ppm) had no significant difference in drinking water amount and weight change during the 8 weeks of exposure to lead exposure in drinking water and the control group, while the blood lead level increased significantly with the exposure time and the increase of exposure dose. The results of the.Morris water maze test showed that the spatial learning and memory ability of the lead exposed rats was clear. Western blot results showed that the expression level of learning and memory related glutamate receptors and NMDA receptors decreased in hippocampus.
2, lead can induce neuron apoptosis
300ppm 8W rat model of drinking lead exposure and TUNEL detection of 48h PC12 cell model of 10 mol/L lead showed that lead could induce the increase of apoptosis in hippocampal neurons and PC12 cells respectively, suggesting that lead may cause the change of learning and memory by inducing neuronal apoptosis.
3, lead induced changes in miRNA expression profiles in hippocampal tissue
The results of miRNA chip showed that after drinking lead 300ppm8w in weaning SD rats, the expression level of 7 miRNA was significantly changed compared with the control group, in which the expression level of miR-204, miR-211, miR-448, miR-449a, miR-34c, miR-34b increased more than 1.5 times; miR-494 expression decreased to the original 1/6.qRT-PCR verification results consistent with the chip results. Three different kinds of miRNA (miR-204, miR-211 and miR-448) were selected to perform qRT-PCR verification on the hippocampus of 2W, 4W, 6W and 8W, and the results showed that the expression level of the three miRNA increased.
4, bioinformatics analysis showed that the significant differentially expressed miRNA target genes were related to apoptosis, LTP/LTD, calcium signaling pathway, and the three miRNA had common target genes, Bcl-2 and Itpr1.
Using bioinformatics (DIANA, TargetScan and miRNA three websites) to analyze and predict the target genes of miRNA and apoptosis, axon extension, calcium signaling pathway, LTP/LTD, MAPK signaling pathway, Notch signaling pathway, close connection and neurodegenerative diseases. MiR-204, miR-211 and miR-448 have common target genes, namely Bcl-2 on apoptosis. The results of validation of Itpr1, Western blot and qRT-PCR methods related to calcium channels (animal and PC12 cells) were consistent with the predicted results.
5, interference with miR-204, miR-211 and miR-448 expression can protect cells from exposure to lead in a certain extent.
The "Loss of function" model was established by using miRNAinhibitor and the effects of MTT and TUNEL were used to verify the effect of lead on the vitality and apoptosis of PC12 cells after interference of miRNA. The results showed that the interference of miRNA expression could protect PC12 cells from the damage of lead exposure to a certain extent, and the activity of cell viability could be increased after the interference of miR-448 expression.
[Conclusion]
1, lead exposure may affect learning and memory function by inducing apoptosis of hippocampal neurons.
2, lead exposure can cause changes in hippocampal miRNA expression profile, which may play an important role in lead neurotoxicity.
3, the significant differentially expressed miR-204, miR-211 and miR-448 can affect the neurotoxicity of lead by negatively regulating its common target gene with apoptosis related Bcl-2 and the Itpr1 associated with calcium ion channels.
4, interference with miR-204, miR-211 and miR-448 expression can protect cells from lead exposure to a certain extent.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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2 鄭偉;周虹;;葡萄糖調(diào)節(jié)蛋白58的結(jié)構(gòu)和功能[J];醫(yī)學(xué)分子生物學(xué)雜志;2007年02期

3 周桂鳳;劉棟;蔣云生;;鉛結(jié)合蛋白研究進(jìn)展[J];國外醫(yī)學(xué)(衛(wèi)生學(xué)分冊);2006年02期

4 劉現(xiàn)芳;馬海燕;曲寶明;金心;李紅;;孕期不同階段鉛暴露對仔鼠腦組織S100B蛋白表達(dá)影響[J];青島大學(xué)醫(yī)學(xué)院學(xué)報(bào);2013年05期

5 顏崇淮;徐健;李e

本文編號：1833801