本文選題：TCDD + 小膠質(zhì)細(xì)胞　； 參考：《南通大學(xué)》2014年碩士論文

【摘要】：目的觀察TCDD誘導(dǎo)小膠質(zhì)細(xì)胞炎性激活及繼而引發(fā)神經(jīng)元凋亡的作用機(jī)制。方法1.本實(shí)驗(yàn)擬采用HAPI小膠質(zhì)細(xì)胞觀察TCDD誘導(dǎo)小膠質(zhì)細(xì)胞炎癥激活。(1)首先采用半定量逆轉(zhuǎn)錄-多聚酶鏈反應(yīng)(reverse transcription polymerase chain reaction,RT-PCR)檢測(cè)10 nmol/ml(nM)TCDD誘導(dǎo)的HAPI小膠質(zhì)細(xì)胞細(xì)胞因子TNF-α?xí)r間效應(yīng)關(guān)系(0.5、1、3、4、6、12 h)和IL-1βm RNA表達(dá)水平以及利用酶聯(lián)免疫吸附試驗(yàn)(enzyme linked immunosorbent assay,ELISA)觀察兩者分泌水平改變。(2)利用半定量RT-PCR觀察TCDD誘導(dǎo)非基因通路兩種主要激酶cPLA2和COX-2 mRNA表達(dá)水平變化。(3)利用蛋白免疫印跡(Western blot)檢測(cè)TCDD引起的HAPI細(xì)胞的總蛋白中IκBα、p-IκBα和p-p65的蛋白水平變化,為進(jìn)一步確證NF-κB通路的激活,核漿分離后分別檢測(cè)TCDD誘導(dǎo)HAPI細(xì)胞后其核內(nèi)和胞漿內(nèi)p65的變化,并用細(xì)胞免疫熒光檢測(cè)在正常和TCDD刺激下p65入核情況。2、為了研究小膠質(zhì)細(xì)胞活化后對(duì)神經(jīng)元的影響,本實(shí)驗(yàn)主要研究被公認(rèn)的小膠質(zhì)細(xì)胞分泌的一氧化氮(NO)對(duì)原代神經(jīng)元影響機(jī)制。(1)利用半定量rt-pcr和westernblot分別檢測(cè)inos的mrna水平和蛋白質(zhì)的表達(dá)水平變化,利用griess法檢測(cè)tcdd刺激hapi細(xì)胞后引起no分泌的劑量效應(yīng)(0.01、0.1、1、10、50、100nm)和時(shí)間效應(yīng)(0.5、1、3、4、6、12h)關(guān)系。westernblot檢測(cè)引起no釋放的主要信號(hào)通路mapks各蛋白(p38、jnk、erk)的變化情況。(2)利用tcdd誘導(dǎo)hapi細(xì)胞激活后的條件培養(yǎng)基(cm)培養(yǎng)大鼠原代神經(jīng)元細(xì)胞,運(yùn)用細(xì)胞計(jì)數(shù)試劑盒(cellcounterkit-8,cck-8)和乳酸脫氫酶釋放(ldhrelease)試劑盒檢測(cè)不同時(shí)間點(diǎn)(0.5、1、3、4、6、12h)cm對(duì)原代神經(jīng)元細(xì)胞活力和細(xì)胞毒性作用影響。運(yùn)用末端脫氧核苷酰基轉(zhuǎn)移酶介導(dǎo)的dutp切口末端標(biāo)記(terminaldeoxynucleotidyltransferase-mediatedbiotinylated-dutpnick-endlabeling,tunel)染色檢測(cè)炎性活化的hapi細(xì)胞cm對(duì)原代神經(jīng)元凋亡的作用。(3)加入mapks信號(hào)通路抑制劑預(yù)處理hapi細(xì)胞,運(yùn)用westernblot和griess方法分別檢測(cè)對(duì)tcdd誘導(dǎo)的mapks信號(hào)通路蛋白表達(dá)和no釋放的改變,以及利用rt-pcr和westernblot檢測(cè)對(duì)誘導(dǎo)no合成inos的變化。同時(shí)運(yùn)用cck-8和ldh試劑盒檢測(cè)分別檢測(cè)抑制劑預(yù)處理后的hapi細(xì)胞cm對(duì)原代神經(jīng)元細(xì)胞活力和細(xì)胞毒性作用改變。最后,用tunel染色檢測(cè)抑制劑預(yù)處理后的hapi細(xì)胞cm對(duì)原代神經(jīng)元凋亡作用的影響。結(jié)果1、(1)rt-pcr和elisa結(jié)果顯示,與對(duì)照組(dmso)相比,10nmtcdd能夠誘導(dǎo)hapi細(xì)胞以時(shí)間依賴方式轉(zhuǎn)錄和分泌tnf-α,在3h與對(duì)照組相比顯著增加(p0.05),并在4h達(dá)到高峰。同時(shí),il-1β轉(zhuǎn)錄和分泌也增加。(2)rt-pcr結(jié)果顯示,10nmtcdd能夠誘導(dǎo)hapi細(xì)胞非基因通路激酶cpla2和cox-2的轉(zhuǎn)錄水平增加,并分別在0.5h和1h明顯升高(p0.05)。(3)westernblot結(jié)果顯示,10nmtcdd可以誘導(dǎo)hapi細(xì)胞nf-κb信號(hào)通路激活,包括iκb磷酸化和泛素化降解,p65磷酸化,同時(shí)核漿分離結(jié)果顯示,核內(nèi)p65增加,胞漿內(nèi)p65明顯減少。細(xì)胞免疫熒光結(jié)果表明hapi細(xì)胞在tcdd誘導(dǎo)下p65可以由胞漿轉(zhuǎn)移入胞核。2、(1)rt-pcr和westernblot結(jié)果表明,10nmtcdd能夠誘導(dǎo)hapi細(xì)胞inos的轉(zhuǎn)錄水平和蛋白水平以劑量依賴和時(shí)間依賴方式增加。griess方法檢測(cè)結(jié)果顯示tcdd能夠誘導(dǎo)hapi細(xì)胞以劑量依賴和時(shí)間依賴方式釋放NO,并可隨著時(shí)間延長增多。Western blot結(jié)果也顯示,調(diào)控iNOS表達(dá)并引起NO合成增加MAPKs中的p38和JNK表達(dá)水平明顯增加,但ERK沒有發(fā)生明顯變化。(2)CCK-8和LDH釋放試劑盒分析結(jié)果顯示TCDD刺激過的HAPI細(xì)胞的CM能夠引起大鼠原代神經(jīng)元細(xì)胞活力降低和LDH釋放增加。同時(shí)TUNEL染色也證實(shí)了該CM可以引起大鼠原代神經(jīng)元細(xì)胞發(fā)生明顯凋亡。(3)分別加入p38 MAPK和JNK抑制劑SB202190和SP600125預(yù)處理HAPI細(xì)胞后,Western blot和Griess法分別證實(shí)TCDD誘導(dǎo)的p38 MAPK和JNK的磷酸化激活和NO升高被抑制。RT-PCR和Western blot也顯示iNOS的轉(zhuǎn)錄和蛋白表達(dá)的增多也被抑制。CCK-8和LDH釋放試劑盒也顯示CM誘導(dǎo)的大鼠原代神經(jīng)元細(xì)胞活力下降和LDH釋放也被抑制,最后,TUNEL染色發(fā)現(xiàn)CM誘導(dǎo)的原代神經(jīng)元TUNEL陽性細(xì)胞增多也明顯被抑制。結(jié)論1、(1)TCDD能夠引起HAPI小膠質(zhì)細(xì)胞的炎性激活,并且釋放TNF-α、IL-1β等細(xì)胞因子;(2)TCDD可經(jīng)過非基因通路迅速激活cPLA2和COX-2等激酶;(3)TCDD可以誘導(dǎo)HAPI細(xì)胞NF-κB信號(hào)通路激活。2、(1)TCDD誘導(dǎo)HAPI小膠質(zhì)細(xì)胞p38 MAPK和JNK信號(hào)通路誘導(dǎo)iNOS激酶增加并從而誘導(dǎo)NO合成增加;(2)小膠質(zhì)細(xì)胞炎性激活釋放的NO能夠引起原代神經(jīng)元損傷,甚至凋亡;(3)抑制p38 MAPK和JNK信號(hào)通路可以抑制NO釋放,從而抑制小膠質(zhì)細(xì)胞炎性激活引起的原代神經(jīng)元損傷和凋亡。
[Abstract]:Objective To observe the mechanism of TCDD induced inflammatory activation of microglia and to induce neuronal apoptosis. Methods 1. this experiment was designed to observe the activation of microglia induced by TCDD by HAPI microglia. (1) first, the semi quantitative reverse transcription polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) was used to detect 1 0 nmol/ml (nM) TCDD induced HAPI microglia cytokine TNF- alpha time effect relationship (0.5,1,3,4,6,12 h) and the expression level of IL-1 beta m RNA and the use of enzyme linked immunosorbent assay (enzyme linked) to observe the changes in secretory levels. (2) two kinds of non gene pathways were induced by semi quantitative observation. The changes in the expression level of kinase cPLA2 and COX-2 mRNA. (3) the changes in the level of I kappa B alpha, p-I kappa B alpha and p-p65 protein in the total protein of TCDD induced by TCDD (Western blot) were detected by protein immunoblotting (Western blot). In order to study the effect of the activation of microglia on the neurons after the activation of p65 under normal and TCDD stimulation, the effect of nitric oxide (NO) on the primary neurons was investigated by cell immunofluorescence. (1) a semi quantitative RT-PCR and Westernblot were used to detect mRNA of iNOS, respectively. Changes in level and protein expression level, Griess method was used to detect the dose effect (0.01,0.1,1,10,50100nm) and time effect (0.5,1,3,4,6,12h) of NO secretion induced by hapi cells stimulated by hapi cells. The changes of MAPKs proteins (p38, JNK, ERK) were the main signaling pathways of no release by.Westernblot detection. (2) Cell activated conditioned medium (CM) was used to cultivate primary neuron cells in rats. The cell count Kit (cellcounterkit-8, CCK-8) and lactate dehydrogenase release (ldhrelease) kit were used to detect the effects of different time points (0.5,1,3,4,6,12h) cm on the cell viability and cytotoxicity of primary neurons. Use of terminal deoxynucleoside transfer Enzyme mediated dUTP incision terminal labeling (terminaldeoxynucleotidyltransferase-mediatedbiotinylated-dutpnick-endlabeling, TUNEL) staining to detect the apoptosis of inflammatory activated hapi cells cm on primary neurons. (3) hapi fine cells were pretreated with MAPKs signaling inhibitor, and Westernblot and Griess were used to detect TCDD respectively. The changes in the induced MAPKs signaling pathway protein expression and no release, and the changes in the induced NO synthesis of iNOS by RT-PCR and Westernblot. Meanwhile, CCK-8 and LDH kits were used to detect the changes in the viability and cytotoxicity of the hapi cell cm after pre-treatment of the inhibitor, respectively. Finally, the TUNEL staining was used. The effect of hapi cell cm on the apoptosis of primary neurons after pretreatment. Results 1, (1) RT-PCR and ELISA results showed that 10nmtcdd could induce hapi cells to transcribe and secrete tnf- alpha in time dependent manner, compared with the control group (DMSO), and increased significantly in 3H compared with the control group (P0.05), and reached the peak in 4H. Meanwhile, IL-1 beta transcript and (2) RT-PCR results showed that 10nmtcdd could induce the increase in the transcriptional level of non gene pathway kinase cPLA2 and COX-2 in hapi cells, and increased significantly in 0.5h and 1H (P0.05). (3) Westernblot results showed that 10nmtcdd could induce activation of hapi cell nf- kappa signal transduction pathway, including phosphorylation and ubiquitination, and phosphorylation, At the same time, the results of nuclear plasma separation showed that the p65 in the nucleus increased and the intracellular p65 decreased obviously. The results of cell immunofluorescence showed that p65 could be transferred from the cytoplasm to the nucleus.2 under the induction of TCDD, and (1) RT-PCR and Westernblot showed that 10nmtcdd could induce the iNOS transcription level and protein level of hapi cells in a dose dependent and time dependent manner. The results of.Griess method detection showed that TCDD could induce hapi cells to release NO in a dose dependent and time dependent manner, and the result of increasing.Western blot showed that the expression of iNOS was regulated and the p38 and JNK expression level in NO synthesis increased MAPKs, but ERK did not change obviously. (2) The analysis of the release kit showed that the CM of HAPI cells stimulated by TCDD could cause the decrease of primary neuron cell viability and the increase of LDH release in rats. TUNEL staining also confirmed that the CM could induce obvious apoptosis in the primary neuron cells of rats. (3) SB202190 and SP600125 pretreatment of p38 MAPK and JNK inhibitors were added, respectively. After the cell, Western blot and Griess methods confirmed that the activation of p38 MAPK and JNK induced by TCDD and the increase of NO were inhibited by.RT-PCR and Western blot also showed that the transcription of iNOS and the increase of protein expression were also inhibited. Finally, TUNEL staining showed that the number of TUNEL positive cells induced by CM was also obviously inhibited. Conclusion 1, (1) TCDD can cause inflammatory activation of HAPI microglia and release TNF- alpha, IL-1 beta and other cytokines, and (2) TCDD can rapidly activate cPLA2 and COX-2 and other kinases through non gene pathways; (3) TCDD can induce HAPI cells Activation of.2, (1) TCDD induced HAPI microglia p38 MAPK and JNK signaling pathway to induce iNOS kinase to induce the increase of iNOS kinase and induce the increase of NO synthesis; (2) the inflammatory activation and release of microglia can induce primary neuronal damage and even apoptosis; (3) inhibition of p38 MAPK and JNK signal pathways can inhibit the release of microglia and thus inhibit microglia. Inflammatory activation induces primary neuronal damage and apoptosis.

【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R114
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本文編號(hào)：1861113