TCDD通过炎性激活小胶质细胞诱导神经元凋亡的机制研究

发布时间：2018-05-08 10:56

  本文选题：TCDD + 小胶质细胞　； 参考：《南通大学》2014年硕士论文

【摘要】：目的观察TCDD诱导小胶质细胞炎性激活及继而引发神经元凋亡的作用机制。方法1.本实验拟采用HAPI小胶质细胞观察TCDD诱导小胶质细胞炎症激活。(1)首先采用半定量逆转录-多聚酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测10 nmol/ml(nM)TCDD诱导的HAPI小胶质细胞细胞因子TNF-α时间效应关系(0.5、1、3、4、6、12 h)和IL-1βm RNA表达水平以及利用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)观察两者分泌水平改变。(2)利用半定量RT-PCR观察TCDD诱导非基因通路两种主要激酶cPLA2和COX-2 mRNA表达水平变化。(3)利用蛋白免疫印迹(Western blot)检测TCDD引起的HAPI细胞的总蛋白中IκBα、p-IκBα和p-p65的蛋白水平变化,为进一步确证NF-κB通路的激活,核浆分离后分别检测TCDD诱导HAPI细胞后其核内和胞浆内p65的变化,并用细胞免疫荧光检测在正常和TCDD刺激下p65入核情况。2、为了研究小胶质细胞活化后对神经元的影响,本实验主要研究被公认的小胶质细胞分泌的一氧化氮(NO)对原代神经元影响机制。(1)利用半定量rt-pcr和westernblot分别检测inos的mrna水平和蛋白质的表达水平变化,利用griess法检测tcdd刺激hapi细胞后引起no分泌的剂量效应(0.01、0.1、1、10、50、100nm)和时间效应(0.5、1、3、4、6、12h)关系。westernblot检测引起no释放的主要信号通路mapks各蛋白(p38、jnk、erk)的变化情况。(2)利用tcdd诱导hapi细胞激活后的条件培养基(cm)培养大鼠原代神经元细胞,运用细胞计数试剂盒(cellcounterkit-8,cck-8)和乳酸脱氢酶释放(ldhrelease)试剂盒检测不同时间点(0.5、1、3、4、6、12h)cm对原代神经元细胞活力和细胞毒性作用影响。运用末端脱氧核苷酰基转移酶介导的dutp切口末端标记(terminaldeoxynucleotidyltransferase-mediatedbiotinylated-dutpnick-endlabeling,tunel)染色检测炎性活化的hapi细胞cm对原代神经元凋亡的作用。(3)加入mapks信号通路抑制剂预处理hapi细胞,运用westernblot和griess方法分别检测对tcdd诱导的mapks信号通路蛋白表达和no释放的改变,以及利用rt-pcr和westernblot检测对诱导no合成inos的变化。同时运用cck-8和ldh试剂盒检测分别检测抑制剂预处理后的hapi细胞cm对原代神经元细胞活力和细胞毒性作用改变。最后,用tunel染色检测抑制剂预处理后的hapi细胞cm对原代神经元凋亡作用的影响。结果1、(1)rt-pcr和elisa结果显示,与对照组(dmso)相比,10nmtcdd能够诱导hapi细胞以时间依赖方式转录和分泌tnf-α,在3h与对照组相比显著增加(p0.05),并在4h达到高峰。同时,il-1β转录和分泌也增加。(2)rt-pcr结果显示,10nmtcdd能够诱导hapi细胞非基因通路激酶cpla2和cox-2的转录水平增加,并分别在0.5h和1h明显升高(p0.05)。(3)westernblot结果显示,10nmtcdd可以诱导hapi细胞nf-κb信号通路激活,包括iκb磷酸化和泛素化降解,p65磷酸化,同时核浆分离结果显示,核内p65增加,胞浆内p65明显减少。细胞免疫荧光结果表明hapi细胞在tcdd诱导下p65可以由胞浆转移入胞核。2、(1)rt-pcr和westernblot结果表明,10nmtcdd能够诱导hapi细胞inos的转录水平和蛋白水平以剂量依赖和时间依赖方式增加。griess方法检测结果显示tcdd能够诱导hapi细胞以剂量依赖和时间依赖方式释放NO,并可随着时间延长增多。Western blot结果也显示,调控iNOS表达并引起NO合成增加MAPKs中的p38和JNK表达水平明显增加,但ERK没有发生明显变化。(2)CCK-8和LDH释放试剂盒分析结果显示TCDD刺激过的HAPI细胞的CM能够引起大鼠原代神经元细胞活力降低和LDH释放增加。同时TUNEL染色也证实了该CM可以引起大鼠原代神经元细胞发生明显凋亡。(3)分别加入p38 MAPK和JNK抑制剂SB202190和SP600125预处理HAPI细胞后,Western blot和Griess法分别证实TCDD诱导的p38 MAPK和JNK的磷酸化激活和NO升高被抑制。RT-PCR和Western blot也显示iNOS的转录和蛋白表达的增多也被抑制。CCK-8和LDH释放试剂盒也显示CM诱导的大鼠原代神经元细胞活力下降和LDH释放也被抑制,最后,TUNEL染色发现CM诱导的原代神经元TUNEL阳性细胞增多也明显被抑制。结论1、(1)TCDD能够引起HAPI小胶质细胞的炎性激活,并且释放TNF-α、IL-1β等细胞因子;(2)TCDD可经过非基因通路迅速激活cPLA2和COX-2等激酶;(3)TCDD可以诱导HAPI细胞NF-κB信号通路激活。2、(1)TCDD诱导HAPI小胶质细胞p38 MAPK和JNK信号通路诱导iNOS激酶增加并从而诱导NO合成增加;(2)小胶质细胞炎性激活释放的NO能够引起原代神经元损伤,甚至凋亡;(3)抑制p38 MAPK和JNK信号通路可以抑制NO释放,从而抑制小胶质细胞炎性激活引起的原代神经元损伤和凋亡。
[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.

【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R114
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本文编号：1861113