本文關(guān)鍵詞： 羊布魯桿菌 Omp31 小膠質(zhì)細(xì)胞 自噬 炎癥 NF-κB(P65)　出處：《寧夏醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：1.目的本研究:⑴用不同濃度Omp31處理BV-2細(xì)胞6h(文獻(xiàn)報道誘導(dǎo)自噬的時間),一是明確Omp31能否誘導(dǎo)BV-2細(xì)胞自噬,二是確定Omp31誘導(dǎo)自噬的合適濃度。⑵用合適濃度的Omp31處理BV-2細(xì)胞不同時間,目的是驗(yàn)證BV-2細(xì)胞發(fā)生自噬的時間是否和文獻(xiàn)報道時間吻合,以便進(jìn)一步研究自噬在BV-2細(xì)胞炎癥中的機(jī)制。⑶用不同濃度的Rapa和3-MA處理BV-2細(xì)胞24h,以確定Rapa誘導(dǎo)自噬的濃度及3-MA抑制自噬的濃度。⑷用Omp31、Rapa及3-MA處理BV-2細(xì)胞,以確定自噬能否通過調(diào)控NF-κB(P65)通路影響TNF-α的表達(dá)。2.方法⑴羊布魯桿菌Omp31誘導(dǎo)BV-2小膠質(zhì)細(xì)胞自噬①(0、0.17、0.5、1.5、4.5、13.5)μg/m L Omp31處理BV-2細(xì)胞6h,用Western blot法檢測自噬相關(guān)蛋白LC3B的表達(dá);RT-q PCR檢測LC3B m RNA的相對表達(dá)水平;透射電鏡觀察BV-2細(xì)胞內(nèi)的自噬體;ELISA法檢測TNF-α、IL-6及IL-10的表達(dá);②0.5μg/m L Omp31處理BV-2細(xì)胞(0、6、12、24)h,用Western blot檢測自噬相關(guān)蛋白LC3B的含量;RT-q PCR檢測LC3B m RNA的相對表達(dá)水平;透射電鏡觀察BV-2細(xì)胞內(nèi)的自噬體;ELISA法檢測TNF-α、IL-6及IL-10的表達(dá)。⑵Omp31誘導(dǎo)的自噬對BV-2細(xì)胞NF-κB(P65)通路的作用①(2.5、5、10)μM Rapa、(2.5、5、10)m M 3-MA預(yù)處理BV-2細(xì)胞24h,用We stern blot檢測自噬標(biāo)志物L(fēng)C3BⅡ蛋白的含量;②Omp31、Rapa及3-MA處理BV-2細(xì)胞6h,用Western blot檢測自噬相關(guān)蛋白B eclin-1、LC3B、P62以及NF-κB(P65)通路相關(guān)蛋白IκBα、p-P65、t-P65的表達(dá);ELIS A法檢測TNF-α的表達(dá);RT-q PCR檢測(Beclin-1、LC3B、TNF-α)m RNA的相對表達(dá);透射電鏡觀察細(xì)胞內(nèi)自噬體;免疫熒光技術(shù)檢測p-P65蛋白入核。3.結(jié)果⑴不同濃度Omp31處理BV-2細(xì)胞6h,發(fā)現(xiàn)0.5μg/m LOmp31能使LC3B蛋白及m RNA的表達(dá)量增加,與其余各組相比差異有統(tǒng)計學(xué)意義(P0.05),且能使LC3BⅡ蛋白含量增加(P0.05),同時在透射電鏡下可見較多自噬體;⑵各濃度組TNF-α、IL-6表達(dá)量均較對照組增加(P0.05),0.5μg/m L實(shí)驗(yàn)組TNF-α、IL-6表達(dá)量較其他實(shí)驗(yàn)組低(P0.01)。各實(shí)驗(yàn)組IL-10表達(dá)量較對照組均降低(P0.05),且不具有濃度依賴性;⑶0.5μg/m LOmp31處理BV-2細(xì)胞不同時間,結(jié)果顯示,各實(shí)驗(yàn)組LC3B蛋白的表達(dá)及LC3BⅡ蛋白含量較對照組增加(P0.05),6h組LC3BⅡ蛋白含量及LC3Bm RNA表達(dá)量較其余各組增加(P0.01),并且在透射電鏡下可見較多自噬體;⑷各時間組TNF-α、IL-6表達(dá)量均較對照組增加(P0.01),且具有時間依賴性,而IL-10表達(dá)量均較對照組減少(P0.01),與時間無關(guān);⑸5μM Rapa預(yù)處理BV-2細(xì)胞后LC3BⅡ蛋白含量較各組均增加(P0.01),2.5m M3-MA預(yù)處理BV-2細(xì)胞能降低LC3BⅡ蛋白含量,與其余各組相比差異有統(tǒng)計學(xué)意義(P0.01);⑹Omp31、Rapa和3-MA處理BV-2細(xì)胞,結(jié)果顯示:Omp31和Rapa均能促進(jìn)L C3B及Beclin-1蛋白表達(dá),亦能促進(jìn)二者m RNA的表達(dá),且均能增加LC3BⅡ蛋白含量,以上結(jié)果與對照組結(jié)果相比差異均有統(tǒng)計學(xué)意義(P0.05)。3-MA對LC3B蛋白和m RN A的表達(dá)以及Beclin-1蛋白的表達(dá)無影響,但較對照組能降低LC3BⅡ蛋白含量,并且抑制Beclin-1 m RNA的表達(dá)(P0.05)。Omp31和Rapa都能降低P62蛋白含量,而3-M A則導(dǎo)致P62蛋白含量增加,與對照組相比均有統(tǒng)計學(xué)差異(P0.05)。在透射電鏡下可見Omp31和Rapa均能誘導(dǎo)自噬體形成,而3-MA對自噬體形成有抑制作用。⑺Omp31、Rapa及3-MA均能使IκBα和t-P65蛋白含量降低,與對照組相比差異有統(tǒng)計學(xué)意義(P0.01)。Omp31能增加p-P65蛋白水平,而Rapa和3-MA都能使p-P65蛋白水平降低,與對照組相比均有統(tǒng)計學(xué)差異(P0.05)。用免疫熒光技術(shù)檢測p-P65蛋白入核,可知Omp31能增加胞核內(nèi)p-P65蛋白的含量,而Rapa及3-MA使胞核內(nèi)p-P65蛋白含量減少;⑻Omp31與對照組相比能增加TNF-α表達(dá)量(P0.01),而Rapa及3-MA則使TN F-α表達(dá)量降低(P0.01)。與對照組相比,Omp31、Rapa和3-MA均能使TNF-αm RNA表達(dá)量降低(P0.01)。4.結(jié)論⑴羊布魯桿菌Omp31能誘導(dǎo)BV-2細(xì)胞自噬,并促進(jìn)TNF-α、IL-6表達(dá),抑制IL-10表達(dá);⑵自噬能負(fù)性調(diào)控NF-κB(P65)信號通路,進(jìn)而抑制TNF-α的表達(dá)。
[Abstract]:The purpose of this study is as follows: 1. BV-2 cells were treated with different concentrations of Omp31 6h (reported to induce autophagy time) is clear, a Omp31 can induce autophagy in BV-2 cells, the two is to determine the appropriate concentration of Omp31 induced autophagy. 2 with appropriate concentration of Omp31 treated BV-2 cells at different time, to verify whether the autophagy of BV-2 cells time and time are reported in the literature, in order to further study the mechanism of autophagy in BV-2 cells inflammation. 3 with different concentrations of Rapa and 3-MA in BV-2 cells treated with 24h, to determine the concentration of 3-MA and Rapa induced autophagy inhibition of autophagy. The concentration of Omp31, Rapa and 3-MA treated BV-2 cells, to determine whether the regulation of autophagy the expression of NF- B (P65) pathway on expression of.2. TNF- alpha 1 methods of Brucella melitensis Omp31 induced autophagy in BV-2 microglia 1 (0,0.17,0.5,1.5,4.5,13.5) g/m L Omp31 6h in BV-2 cells treated with Western blot method. To detect the expression of autophagy related protein LC3B; LC3B m RNA RT-q to detect the relative expression level of PCR; TEM observation of autophagosomes in BV-2 cells; detection of TNF- alpha ELISA method, the expression of IL-6 and IL-10; the 0.5 g/m L Omp31 BV-2 (0,6,12,24) H cells, determination of autophagy related protein LC3B by Western blot check the relative expression of LC3B m RNA; RT-q PCR; transmission electron microscope was used to observe autophagy in BV-2 cells; detection of TNF- alpha ELISA method, the expression of IL-6 and IL-10. The Omp31 induced autophagy of BV-2 cells NF- kappa B (P65) function pathway (2.5,5,10) M (Rapa, 2.5,5,10) pretreatment of BV-2 cells with 24h m M 3-MA, marking content of LC3B II protein by We stern blot of Omp31 Rapa, detection of autophagy; 3-MA and BV-2 cells treated with Western blot 6h, detection of autophagy related protein B eclin-1, LC3B, P62 and NF- K B (P65) pathway related protein I kappa B alpha, p-P65. The expression of t-P65; ELIS A娉曟嫻婽NF-偽鐨勮〃杈，

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