本文關(guān)鍵詞：小膠質(zhì)細(xì)胞活化產(chǎn)物對原代培養(yǎng)大腦皮質(zhì)神經(jīng)元凋亡的影響　出處：《河北醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的：彌漫性軸索損傷（Diffuse axonal injury，DAI）是一種原發(fā)性彌漫性腦損傷，是最重要的腦創(chuàng)傷類型之一，，是引起死亡、嚴(yán)重致殘及植物生存的主要原因。β-淀粉樣前體蛋白（β-amyloid precursor protein，β-APP）的免疫組織化學(xué)染色是診斷DAI的金標(biāo)準(zhǔn)。正常情況下微量的β-APP沿著軸索通過快速順行軸漿運(yùn)輸至突觸。β淀粉樣蛋白（β-amyloid，Aβ）是β-APP通過蛋白水解作用后產(chǎn)生的一段淀粉樣肽。當(dāng)軸索發(fā)生損傷后，軸漿運(yùn)輸中斷，從而導(dǎo)致β-APP的積聚并在軸突和胞體中大量表達(dá)，大量的Aβ由于β-APP酶解作用的上調(diào)而聚集在神經(jīng)元的核周體，聚集的Aβ具有毒性作用，并可以激活小膠質(zhì)細(xì)胞。 大量研究已證實(shí)，DAI后的神經(jīng)損害不僅發(fā)生在損傷瞬間，且在其后的數(shù)分鐘到數(shù)天內(nèi)還可出現(xiàn)神經(jīng)元延遲性死亡。目前關(guān)于理解DAI發(fā)病過程相關(guān)的病理級聯(lián)反應(yīng)已經(jīng)有了明顯的進(jìn)展，然而其形成機(jī)制尚不完全明確，是法醫(yī)學(xué)和神經(jīng)科學(xué)亟待解決的難題。在DAI的發(fā)病過程中，不僅表現(xiàn)為外力直接作用引起的神經(jīng)元受損和死亡，同時也存在神經(jīng)元凋亡，細(xì)胞凋亡不僅參與了DAI，而且可能在DAI的發(fā)生發(fā)展過程中扮演著重要的角色。Caspase-3屬半胱天冬蛋白酶家族的一員，又被稱作死亡蛋白酶，caspase-3的活化是損傷誘導(dǎo)凋亡的最后共同通路。實(shí)驗(yàn)研究中，多以caspase-3作為研究指標(biāo)來評價細(xì)胞的凋亡。 前期研究表明，在體狀態(tài)下DAI大鼠腦皮質(zhì)和海馬神經(jīng)元軸突中有β-APP的積聚，在損傷的軸索周圍觀察到小膠質(zhì)細(xì)胞的聚集和活化，活化的小膠質(zhì)細(xì)胞可以產(chǎn)生大量的炎癥因子，引發(fā)神經(jīng)元的“二次損傷”，導(dǎo)致神經(jīng)元凋亡。然而，離體狀態(tài)下，Aβ激活的小膠質(zhì)細(xì)胞釋放的TNFα和IL-1β是否可以引發(fā)大腦皮質(zhì)神經(jīng)元凋亡，進(jìn)而參與神經(jīng)元的“二次損傷”及其具體機(jī)制，尚未見詳細(xì)報道。 因此，本實(shí)驗(yàn)采用Aβ1-40作用于原代培養(yǎng)的小膠質(zhì)細(xì)胞，應(yīng)用ELISA和Real Time PCR的方法，通過檢測TNFα和IL-1β含量及其mRNA的表達(dá)觀察小膠質(zhì)細(xì)胞活化情況；用激活的小膠質(zhì)細(xì)胞條件培養(yǎng)基培養(yǎng)大腦皮質(zhì)神經(jīng)元，應(yīng)用Real Time PCR和Hoechst33258染色的方法分別檢測神經(jīng)元caspase-3mRNA的表達(dá)和神經(jīng)元的凋亡率，以探討DAI后小膠質(zhì)細(xì)胞的活化引起神經(jīng)元損傷的可能機(jī)制，為進(jìn)一步闡明DAI病理生理學(xué)機(jī)制奠定基礎(chǔ)，為法醫(yī)學(xué)實(shí)際檢案工作中對DAI損傷的客觀評價提供理論依據(jù)。 方法： 1原代培養(yǎng)新生24h內(nèi)SD乳鼠大腦皮質(zhì)小膠質(zhì)細(xì)胞，用Aβ1-40處理小膠質(zhì)細(xì)胞，隨機(jī)分為4組：對照組、Aβ處理組、Aβ+MC組和MC組。對照組不加處理因素，Aβ處理組加入1μMAβ1-40作用于小膠質(zhì)細(xì)胞6h，Aβ+MC組在加入Aβ1-40前30min加入0.2μM米諾環(huán)素（Minocycline，MC），MC組只加入0.2μM MC。ELISA法檢測細(xì)胞培養(yǎng)上清中TNFα和IL-1β的含量，Real Time PCR檢測細(xì)胞中TNFα和IL-1β mRNA的表達(dá)。 2原代培養(yǎng)新生24h內(nèi)SD乳鼠大腦皮質(zhì)神經(jīng)元，隨機(jī)分為4組：對照組、1/2Aβ-MCM組、1/4Aβ-MCM組、1/8Aβ-MCM組。對照組神經(jīng)元正常培養(yǎng)，1/2Aβ-MCM組、1/4Aβ-MCM組、1/8Aβ-MCM組分別在神經(jīng)元培養(yǎng)基中加入1/2、1/4、1/8Aβ激活的小膠質(zhì)細(xì)胞條件培養(yǎng)基（Aβactivated microglia conditioned media，Aβ-MCM），每組包括5個不同時相：1h、3h、6h、12h、24h。Real Time PCR檢測各組細(xì)胞中caspase-3mRNA的表達(dá)。選取1/2Aβ-MCM組，采用Hoechst33258染色，觀察神經(jīng)元的凋亡率。 用SPSS13.0軟件進(jìn)行統(tǒng)計學(xué)分析，數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（Mean±SD）表示。各組均數(shù)的比較行單因素方差分析（ANOVA），以最小顯著差法（least significant difference，LSD）作兩兩比較，以P＜0.05為有顯著性差異。 結(jié)果： 1Aβ1-40對小膠質(zhì)細(xì)胞的作用及米諾環(huán)素對其作用的影響 應(yīng)用1μM Aβ1-40作用于小膠質(zhì)細(xì)胞6h后，細(xì)胞培養(yǎng)上清中TNFα和IL-1β的含量明顯升高，與對照組相比有顯著性差異（P＜0.01）；加入0.2μM米諾環(huán)素和Aβ1-40共同作用于小膠質(zhì)細(xì)胞后，明顯抑制了TNFα和IL-1β的升高，與Aβ處理組相比明顯下降。 應(yīng)用1μM Aβ1-40作用于小膠質(zhì)細(xì)胞6h后，小膠質(zhì)細(xì)胞中TNFα、IL-1βmRNA的表達(dá)明顯升高，與對照組相比有顯著性差異（P＜0.01）；加入0.2μM米諾環(huán)素和Aβ1-40共同作用于小膠質(zhì)細(xì)胞后，明顯抑制了TNFα、IL-1β mRNA的表達(dá)，與Aβ處理組相比明顯下降。 2Aβ-MCM對神經(jīng)元caspase-3mRNA表達(dá)的影響 在神經(jīng)元培養(yǎng)基中加入1/2量的Aβ-MCM后，各時間點(diǎn)神經(jīng)元caspase-3mRNA的表達(dá)均升高，與對照組相比均有顯著性差異（P＜0.05），其中以6h時升高最明顯（P＜0.01）。在神經(jīng)元培養(yǎng)基中加入1/4量和1/8量的Aβ-MCM，各時間點(diǎn)神經(jīng)元caspase-3mRNA的表達(dá)與對照組相比均無顯著性差異。 3Aβ-MCM對神經(jīng)元凋亡率的影響 應(yīng)用Hoechst33258染色觀察神經(jīng)元的凋亡率，結(jié)果顯示，1/2Aβ-MCM處理后各時間點(diǎn)均可見熒光強(qiáng)度較高的凋亡神經(jīng)元細(xì)胞核，并伴有染色質(zhì)的濃縮、聚集。隨機(jī)選取5個視野，300個細(xì)胞計數(shù)并進(jìn)行統(tǒng)計學(xué)分析，結(jié)果顯示，1/2Aβ-MCM作用6h時，神經(jīng)元凋亡率最高。 結(jié)論： 1成功建立了Aβ1-40激活的小膠質(zhì)細(xì)胞條件培養(yǎng)基促大腦皮質(zhì)神經(jīng)元損傷模型。 2Aβ1-40激活的小膠質(zhì)細(xì)胞產(chǎn)生TNFα和IL-1β，導(dǎo)致大腦皮質(zhì)神經(jīng)元caspase-3mRNA表達(dá)上調(diào)和神經(jīng)元凋亡率增加。
[Abstract]:Objective: diffuse axonal injury (Diffuse axonal, injury, DAI) is a kind of primary diffuse brain injury is one of the most important types of traumatic brain injury, is the main reason to cause death, serious disability and plant survival. Beta amyloid precursor protein (beta -amyloid precursor protein, P -APP). Immunohistochemical staining is the gold standard for the diagnosis of DAI. Under normal circumstances, the trace of beta -APP along the axon by fast anterograde axoplasmic transport to synapses. Amyloid beta (beta -amyloid and A beta) is a beta amyloid peptide -APP by proteolysis after production. When the cable is damaged, shaft slurry transport disruption, which leads to the accumulation of -APP and beta expression in axons and cell bodies, a large number of A beta and aggregation in the neuronal perikaryon by beta -APP enzyme increases, aggregation of A beta has toxic effect, and can activate microglia.
A large number of studies have confirmed that neural damage after DAI injury occurs not only in the moment, and after a few minutes to several days can also appear delayed neuron death. The pathological cascade is about understanding the pathogenesis of DAI related has made significant progress, but its formation mechanism is not entirely clear, is a difficult problem in forensic medicine science and neuroscience needs to be solved. In the pathogenesis of DAI, not only for the injury and death caused by external force directly but also the existence of neurons, neuronal apoptosis, apoptosis not only in DAI, and may be a member of.Caspase-3 plays an important role in the development of DAI is a caspase family that is called death protease, caspase-3 activation is the final common pathway of apoptosis induced by injury. In the experiment with caspase-3 as the research index to evaluate cell Apoptosis.
Previous studies have shown that in vivo cerebral cortex and hippocampal neuronal axons in DAI rats with beta -APP accumulation, around the injury axonal observed in microglia activation and aggregation, activated microglia can produce numerous inflammatory factors, causing neurons two "damage", leading to neuronal apoptosis however, the isolated condition, whether A beta activated microglia release of TNF alpha and IL-1 beta could induce apoptosis in cortical neurons, and to participate in the "two neuron injury" and its specific mechanism has not yet been reported in detail.
Therefore, this experiment adopts the A beta 1-40 in primary cultured microglia, ELISA and Real Time PCR method, by detecting the expression of TNF alpha and IL-1 beta content and mRNA observation of the activation of microglia; medium cortical neurons with conditions of activation of microglia cultured respectively, detection methods Real Time and PCR Hoechst33258 staining neurons caspase-3mRNA expression and neuron apoptosis rate, to investigate the DAI after the activation of microglia may cause neuronal damage mechanism, which lays the foundation for the study of mechanism to further elucidate the pathophysiology of DAI, and provide a theoretical basis for objective evaluation of DAI damage in actual prosecution for forensic case work.
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