本文選題：BV2　切入點(diǎn)：小膠質(zhì)細(xì)胞　出處：《寧夏醫(yī)科大學(xué)》2014年碩士論文

【摘要】：小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)中常駐的巨噬樣細(xì)胞，在中樞神經(jīng)系統(tǒng)的免疫反應(yīng)中發(fā)揮著核心作用。在損傷或感染因素如細(xì)菌脂多糖（Lipopolysaccharide，LPS）等的刺激下，小膠質(zhì)細(xì)胞被迅速激活�；罨男∧z質(zhì)細(xì)胞可誘導(dǎo)蛋白酶的激活，釋放各種促炎性細(xì)胞因子和谷氨酸，并生成大量活性氧和活性氮，導(dǎo)致周圍其他神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞受損和丟失。小膠質(zhì)細(xì)胞的激活參與了很多神經(jīng)退行性疾病的發(fā)生、發(fā)展。因此，目前很多研究都在試圖通過抑制小膠質(zhì)細(xì)胞的激活來治療神經(jīng)退行性疾病。 納洛酮（naloxone）是一種阿片類受體拮抗劑，而右旋美沙芬是阿片類受體激動劑。它們都被證實具有神經(jīng)保護(hù)作用，但其機(jī)制目前尚不十分清楚。我們的研究旨在闡明納洛酮和右美沙芬是否可以通過抑制LPS（細(xì)菌脂多糖）激活的BV2小膠質(zhì)細(xì)胞炎癥因子的釋放而起到神經(jīng)保護(hù)作用。結(jié)果顯示納洛酮和右美沙芬都顯著地抑制了BV2小膠質(zhì)細(xì)胞內(nèi)熱休克蛋白60（HSP60）的表達(dá)及釋放。我們假設(shè)被小膠質(zhì)細(xì)胞釋放至胞外的HSP60可以與Toll-like receptor-4（TLR-4）結(jié)合，激活NF-κB（nuclear factor-kappa b）和caspase-3，從而誘導(dǎo)各種細(xì)胞因子的釋放而產(chǎn)生炎癥反應(yīng)。于是我們檢測了兩種藥物對LPS誘導(dǎo)的BV2細(xì)胞內(nèi)TLR-4，NF-κB和caspase-3的活性的影響。結(jié)果發(fā)現(xiàn)它們顯著抑制了TLR-4，NF-κB和caspase-3的表達(dá)。同時，細(xì)胞中的炎癥因子NO，iNOS，IL-6，IL-1β，TNF-α的釋放都被抑制了。這些結(jié)果提示納洛酮和右美沙芬可抑制小膠質(zhì)細(xì)胞的激活，它們的抗炎作用可能是通過抑制HSP60-TLR-4-NF-κB信號通路而發(fā)揮的。 研究目的： 明確藥物（納洛酮和右美沙芬）對于LPS激活的BV2小膠質(zhì)細(xì)胞的抑制作用，并探討藥物發(fā)揮抗炎作用的分子機(jī)制，從而明確這些藥物在小膠質(zhì)細(xì)胞介導(dǎo)的炎癥反應(yīng)中的作用。 研究方法： 1首先對體外培養(yǎng)的BV2小膠質(zhì)細(xì)胞進(jìn)行不同濃度的藥物處理后，通過CCK8實驗檢測藥物對LPS誘導(dǎo)的BV2小膠質(zhì)細(xì)胞活性的影響。 2同時，通過Western blot和免疫熒光染色技術(shù)檢測LPS誘導(dǎo)的BV2小膠質(zhì)細(xì)胞內(nèi)HSP60，HSF1，iNOS，caspase-3，NF-κB和TLR-4的表達(dá)情況。 3通過ELISA（酶聯(lián)免疫吸附實驗）檢測細(xì)胞培液上清中的炎癥因子IL-6，IL-1β，TNF-α和HSP60的含量；通過iNOS活力的測定和Griess Reagent檢測細(xì)胞培液上清中iNOS的活力和NO的含量。 主要結(jié)果： 1CCK8檢測結(jié)果提示，與對照組相比，單獨(dú)LPS處理BV2小膠質(zhì)細(xì)胞后，細(xì)胞的活性明顯降低，將不同濃度的藥物與LPS誘導(dǎo)的BV2小膠質(zhì)細(xì)胞共孵育24小時后，隨著濃度的增加，，明顯地提升了LPS誘導(dǎo)的BV2小膠質(zhì)細(xì)胞的活性。 2通過Western blot和免疫熒光法檢測發(fā)現(xiàn)，與對照組相比，LPS單獨(dú)處理BV2小膠質(zhì)細(xì)胞后，細(xì)胞內(nèi)的HSF1，HSP60，iNOS，caspase-3，NF-κB，TLR-4的表達(dá)水平明顯上調(diào)；藥物組有效抑制了LPS誘導(dǎo)的這些蛋白的表達(dá)，提示藥物可能通過抑制HSP60-TLR-4-NF-κB信號通路而發(fā)揮抗炎癥反應(yīng)。 3通過ELISA檢測發(fā)現(xiàn)，與對照組相比，LPS處理BV2小膠質(zhì)細(xì)胞后，細(xì)胞上清液中的炎癥因子IL-6，IL-1β，TNF-α和HSP60明顯增多，而藥物組有效抑制了LPS激活的BV2小膠質(zhì)細(xì)胞炎癥因子的釋放。 4通過Griess Reagent檢測發(fā)現(xiàn)，與對照組相比，LPS處理BV2小膠質(zhì)細(xì)胞后，細(xì)胞 上清液中的NO明顯增多，iNOS活力也增加了，而藥物組有效抑制了LPS激活的BV2小膠質(zhì)細(xì)胞中這些炎性變化。 結(jié)論： 1藥物對于LPS激活的BV2小膠質(zhì)細(xì)胞引起的炎癥反應(yīng)具有抑制作用。 2藥物的抗炎作用可能是通過抑制HSP60-TLR-4-NF-κB信號通路來完成的。
[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 - 偽 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 - 偽 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 .

【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

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