药物对细菌脂多糖诱导的BV2小胶质细胞的抗作用及其机制的研究

发布时间：2018-03-28 04:13

本文选题：BV2　切入点：小胶质细胞　出处：《宁夏医科大学》2014年硕士论文

【摘要】：小胶质细胞是中枢神经系统中常驻的巨噬样细胞，在中枢神经系统的免疫反应中发挥着核心作用。在损伤或感染因素如细菌脂多糖（Lipopolysaccharide，LPS）等的刺激下，小胶质细胞被迅速激活。活化的小胶质细胞可诱导蛋白酶的激活，释放各种促炎性细胞因子和谷氨酸，并生成大量活性氧和活性氮，导致周围其他神经元和神经胶质细胞受损和丢失。小胶质细胞的激活参与了很多神经退行性疾病的发生、发展。因此，目前很多研究都在试图通过抑制小胶质细胞的激活来治疗神经退行性疾病。纳洛酮（naloxone）是一种阿片类受体拮抗剂，而右旋美沙芬是阿片类受体激动剂。它们都被证实具有神经保护作用，但其机制目前尚不十分清楚。我们的研究旨在阐明纳洛酮和右美沙芬是否可以通过抑制LPS（细菌脂多糖）激活的BV2小胶质细胞炎症因子的释放而起到神经保护作用。结果显示纳洛酮和右美沙芬都显著地抑制了BV2小胶质细胞内热休克蛋白60（HSP60）的表达及释放。我们假设被小胶质细胞释放至胞外的HSP60可以与Toll-like receptor-4（TLR-4）结合，激活NF-κB（nuclear factor-kappa b）和caspase-3，从而诱导各种细胞因子的释放而产生炎症反应。于是我们检测了两种药物对LPS诱导的BV2细胞内TLR-4，NF-κB和caspase-3的活性的影响。结果发现它们显著抑制了TLR-4，NF-κB和caspase-3的表达。同时，细胞中的炎症因子NO，iNOS，IL-6，IL-1β，TNF-α的释放都被抑制了。这些结果提示纳洛酮和右美沙芬可抑制小胶质细胞的激活，它们的抗炎作用可能是通过抑制HSP60-TLR-4-NF-κB信号通路而发挥的。研究目的：明确药物（纳洛酮和右美沙芬）对于LPS激活的BV2小胶质细胞的抑制作用，并探讨药物发挥抗炎作用的分子机制，从而明确这些药物在小胶质细胞介导的炎症反应中的作用。研究方法： 1首先对体外培养的BV2小胶质细胞进行不同浓度的药物处理后，通过CCK8实验检测药物对LPS诱导的BV2小胶质细胞活性的影响。 2同时，通过Western blot和免疫荧光染色技术检测LPS诱导的BV2小胶质细胞内HSP60，HSF1，iNOS，caspase-3，NF-κB和TLR-4的表达情况。 3通过ELISA（酶联免疫吸附实验）检测细胞培液上清中的炎症因子IL-6，IL-1β，TNF-α和HSP60的含量；通过iNOS活力的测定和Griess Reagent检测细胞培液上清中iNOS的活力和NO的含量。主要结果： 1CCK8检测结果提示，与对照组相比，单独LPS处理BV2小胶质细胞后，细胞的活性明显降低，将不同浓度的药物与LPS诱导的BV2小胶质细胞共孵育24小时后，随着浓度的增加，，明显地提升了LPS诱导的BV2小胶质细胞的活性。 2通过Western blot和免疫荧光法检测发现，与对照组相比，LPS单独处理BV2小胶质细胞后，细胞内的HSF1，HSP60，iNOS，caspase-3，NF-κB，TLR-4的表达水平明显上调；药物组有效抑制了LPS诱导的这些蛋白的表达，提示药物可能通过抑制HSP60-TLR-4-NF-κB信号通路而发挥抗炎症反应。 3通过ELISA检测发现，与对照组相比，LPS处理BV2小胶质细胞后，细胞上清液中的炎症因子IL-6，IL-1β，TNF-α和HSP60明显增多，而药物组有效抑制了LPS激活的BV2小胶质细胞炎症因子的释放。 4通过Griess Reagent检测发现，与对照组相比，LPS处理BV2小胶质细胞后，细胞上清液中的NO明显增多，iNOS活力也增加了，而药物组有效抑制了LPS激活的BV2小胶质细胞中这些炎性变化。结论： 1药物对于LPS激活的BV2小胶质细胞引起的炎症反应具有抑制作用。 2药物的抗炎作用可能是通过抑制HSP60-TLR-4-NF-κB信号通路来完成的。
[Abstract]:Microglia is a giant cell - like cell resident in the central nervous system and plays a central role in the immune response of the central nervous system . The activated microglial cells can induce the activation of the protease , release various pro - inflammatory cytokines and glutamic acid , and generate a large amount of active oxygen and active nitrogen , resulting in the damage and loss of other neurons and glial cells . Activation of the microglial cells is involved in the development and development of many neurodegenerative diseases . Therefore , many studies are now trying to treat neurodegenerative diseases by inhibiting the activation of microglial cells .

These results suggest that naloxone and dexmesafen can inhibit the expression of TLR - 4 , NF - 魏B and caspase - 3 in BV2 cells induced by LPS . The results suggest that naloxone and dexmesafen can inhibit the activation of TLR - 4 , NF - 魏B and caspase - 3 in BV2 cells . These results suggest that both naloxone and dexmesafen can inhibit the activation of TLR - 4 , NF - 魏B and caspase - 3 . These results suggest that naloxone and dexmesfin can inhibit the activation of microglial cells , and their anti - inflammatory effects may be by inhibiting the signaling pathway of HSP60 - TLR - 4 - NF - 魏B .

Purpose of study :

The inhibitory effect of drugs ( naloxone and dexmesafen ) on the activation of LPS - activated BV2 microglial cells was defined , and the molecular mechanism of anti - inflammatory action of drugs was discussed , and the role of these drugs in the inflammatory reaction mediated by microglial cells was defined .

Study method :

1 . After treatment with different concentrations of BV2 microglial cells cultured in vitro , the effect of drug on the activity of BV2 microglial cells induced by LPS was detected by CC8 .

At the same time , the expression of HSP60 , HSF , iNOS , caspase - 3 , NF - 魏B and TLR - 4 in BV2 microglial cells induced by LPS was detected by Western blot and immunofluorescence staining .

3 . The levels of IL - 6 , IL - 1尾 , TNF - 伪 and HSP60 were detected by ELISA ( enzyme linked immunosorbent assay ) .
The activity of iNOS and the content of NO were detected by the determination of iNOS activity and Griess Reagent .

Main results :

Compared with the control group , the activity of BV2 microglial cells induced by LPS was significantly decreased and the activity of LPS - induced BV2 microglial cells was significantly increased after 24 hours incubation with LPS - induced BV2 microglial cells .

2 . Compared with the control group , the expression level of HSF1 , HSP60 , iNOS , caspase - 3 , NF - 魏B and TLR - 4 in the cells was significantly increased compared with the control group by Western blot and immunofluorescence assay .
The drug group effectively inhibited LPS - induced expression of these proteins , suggesting that the drug might exert an anti - inflammatory response by inhibiting the HSP60 - TLR - 4 - NF - 魏B signal pathway .

3 . Compared with the control group , the inflammatory cytokines IL - 6 , IL - 1尾 , TNF - 伪 and HSP60 in the supernatant of the cells were significantly increased compared with the control group , while the drug group effectively inhibited the release of LPS - activated BV2 microglial cells .

4 By Griess Reagent test , after LPS treatment of BV2 microglial cells , the cells

The NO in supernatant increased and the activity of iNOS increased , while the drug group effectively inhibited these inflammatory changes in BV2 microglial cells activated by LPS .

Conclusion :

1 drug has an inhibitory effect on inflammatory responses induced by LPS - activated BV2 microglial cells .

The anti - inflammatory effect of the drug may be accomplished by inhibiting the HSP60 - TLR - 4 - NF - 魏B signal pathway .

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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