本文選題：肥大細胞 + 豬布魯菌��； 參考：《河北大學(xué)》2014年碩士論文

【摘要】：目的： 為研究肥大細胞對布魯菌的模式識別與免疫應(yīng)答效應(yīng)，進一步闡明布魯菌病的發(fā)病機制，為布魯菌病的治療和疫苗研發(fā)探索新途徑與新方法。 方法： 1．分別用布魯菌S2菌株、WboA-S2菌株和大腸桿菌以MOI50:1感染肥大細胞，分別在感染后1h、12h和24h時收集上清，ELISA方法測上清中IL-6、TNF-α、LT、IL-1β、組胺、類胰蛋白酶、IL-12、IFN-γ的含量，以未處理的肥大細胞為對照。 2．RT-PCR法檢測正常肥大細胞TLR2、4、8與9mRNA的表達。 3．分別用布魯菌S2菌株、WboA-S2菌株和大腸桿菌以MOI50:1感染肥大細胞，在感染后1h、12h和24h時收集細胞，RT-PCR法測細胞TLR4、8與9mRNA的表達。 4．用RNAi技術(shù)沉默肥大細胞TLR4，并設(shè)未沉默對照組，用RT-PCR法和流式細胞術(shù)檢測其沉默效果。 5.分別用布魯菌S2菌株、WboA-S2菌株和大腸桿菌以MOI50:1感染沉默組與未沉默組肥大細胞，分別在感染后1h、12h和24h時收集上清，ELISA方法測上清中IL-6、TNF-α、LT與組胺的含量。 結(jié)果： 1．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞，1h、12h、24h感染組IL-6含量明顯高于正常組（P0.05），且隨著感染時間的延長，IL-6含量呈增加趨勢。S2菌株感染組IL-6含量與WboA-S2菌株感染組IL-6含量比較無統(tǒng)計學(xué)意義（P㧐0.05）。 2．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞，1h感染組TNF-α含量與正常組相比無統(tǒng)計學(xué)意義（P㧐0.05），12h、24h各感染組TNF-α含量均高于正常組（P0.05）。S2菌株感染組TNF-α含量與WboA-S2菌株感染組TNF-α含量比較無統(tǒng)計學(xué)意義（P㧐0.05）。 3．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞，1h、12h、24h感染組LT含量高于正常組（P0.05）。而且同一感染組在不同感染時間LT含量沒有明顯變化（P㧐0.05）。S2菌株感染組LT含量與WboA-S2菌株感染組LT含量比較無統(tǒng)計學(xué)意義（P㧐0.05）。 4．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞，1h、12h、24h感染組組胺含量高于正常組（P0.05）。S2菌株、大腸桿菌感染肥大細胞后組胺含量呈顯先增加，再減少，然后又增加的趨勢。WboA-S2菌株感染肥大細胞后1h、12h、24h組胺含量沒有明顯變化。24h WboA-S2菌株感染組組胺含量明顯高于S2菌株感染組胺含量（P0.05）。 5．在布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞的各時間點上清中均未檢測到IL-1β、類胰蛋白酶、IL-12和IFN-γ。 6．肥大細胞表達高水平的TLR4和TLR9mRNA，TLR8mRNA的表達豐度較低。值得注意的是肥大細胞P815不表達TLR2。 7．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞后，在12h、24h時，TLR4mRNA的表達發(fā)生了變化。S2菌株感染肥大細胞后，TLR4mRNA的表達隨感染時間的延長逐漸增加。WboA-S2菌株感染肥大細胞后12h，TLR4mRNA的表達減少，24h時增多。大腸桿菌感染肥大細胞后12h，TLR4mRNA的表達顯著增多，24h則減少。 8．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞后，在12h、24h時，TLR9mRNA的表達發(fā)生了變化。S2菌株感染肥大細胞后，TLR9mRNA的表達12h時增加，24h時減少。WboA-S2菌株感染肥大細胞后，TLR9mRNA的表達12h時無明顯變化，24h表達量增加。大腸桿菌感染肥大細胞后，TLR9mRNA的表達12h時顯著減少，24h時顯著增加。 9．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞后，在12h、24h時TLR8mRNA的表達沒有明顯變化。 10．確定并優(yōu)化了TLR4RNAi沉默條件：24孔板每孔加1μl轉(zhuǎn)染試劑、6pmol TLR4siRNA、1×105個肥大細胞，此條件下TLR4mRNA和蛋白表達抑制效果最好，轉(zhuǎn)染效率能達到50%。 11．布魯菌S2菌株、WboA-S2菌株和大腸桿菌感染肥大細胞，TLR4mRNA沉默組IL-6含量明顯高于相應(yīng)未沉默組（P0.05），TNF-α含量和LT含量與相應(yīng)未沉默組相比均無統(tǒng)計學(xué)意義（P㧐0.05）。 12．肥大細胞TLR4mRNA沉默組的組胺含量與相應(yīng)未沉默組相比有統(tǒng)計學(xué)意義（P0.05）。S2菌株、大腸桿菌感染肥大細胞后，RNA沉默組組胺含量在1h時低于相應(yīng)未沉默組，12h時高于相應(yīng)未沉默組，24h時又低于相應(yīng)未沉默組。WboA-S2菌株感染肥大細胞后，RNA沉默組組胺含量在1h時低于相應(yīng)未沉默組，12h時和24h時均高于相應(yīng)未沉默組。結(jié)論： 1．布魯菌S2菌株和WboA-S2菌株均可刺激肥大肥大細胞釋放IL-6、TNF-α、LT和組胺，，但不釋放類胰蛋白酶、IL-1β、IL-12和IFN-γ； 2．WboA基因的表達產(chǎn)物可能是刺激肥大細胞釋放組胺的主要模式配體； 3．肥大細胞表達高水平的TLR4和TLR9mRNA，但TLR8mRNA的表達豐度較低，不表達TLR2； 4． TLR4和TLR9參與肥大細胞對布魯菌的識別，TLR8在識別布魯菌中沒有明顯 作用； 5．在布魯菌刺激肥大細胞分泌IL-6和組胺的過程中，TLR4是肥大細胞的主要模式識別受體，但由于布魯菌WboA-S2菌株與S2菌株刺激肥大細胞產(chǎn)生IL-6的作用相似，故推測TLR4除可識別布魯菌的LPS外，尚可識別其他菌體成分，進而激活肥大細胞分泌IL-6。但布魯菌激活肥大細胞分泌TNF-α和LT的過程與TLR4介導(dǎo)的模式識別無關(guān)。
[Abstract]:Objective:
In order to study the pattern recognition and immune response effect of mast cells to Brucella, further elucidate the pathogenesis of brucellosis and explore new ways and new methods for the treatment and vaccine research and development of brucellosis.
Method:
1. strains of Brucella S2, WboA-S2 and Escherichia coli were infected with mast cells with MOI50:1, respectively, at 1H, 12h and 24h after infection. The ELISA method was used to measure IL-6, TNF- alpha, LT, IL-1 beta, histamine, tryptase, IL-12, and gamma, with the untreated mast cells as the control.
2.RT-PCR method was used to detect the expression of TLR2,4,8 and 9mRNA in normal mast cells.
3. strains of Brucella S2, WboA-S2 and Escherichia coli were infected with mast cells by MOI50:1, and cells were collected at 1H, 12h and 24h after infection. The expression of TLR4,8 and 9mRNA was measured by RT-PCR method.
4. mast cell TLR4 was silenced by RNAi technique, and the control group was not silenced. The silencing effect was detected by RT-PCR and flow cytometry.
5. with Brucella S2 strain, WboA-S2 strain and Escherichia coli mast cells with MOI50:1 infection silent group and non silencing group, the supernatant was collected at 1H, 12h and 24h after infection, and ELISA method was used to measure the content of IL-6, TNF- alpha, LT and histamine in the supernatant.
Result:
1. Brucella S2, WboA-S2 and Escherichia coli infected mast cells, 1H, 12h, 24h infection group IL-6 content was significantly higher than normal group (P0.05), and with the prolongation of the infection time, IL-6 content increased the.S2 strain infection group IL-6 content and WboA-S2 strain infected group IL-6 content was not statistically significant (0.05).
2. Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cells, 1H infection group TNF- alpha content was not statistically significant compared with the normal group (P? 0.05), 12h, 24h infection group TNF- alpha content was higher than the normal group (P0.05).S2 strain infected group TNF- alpha content and WboA-S2 strain infection group no statistical significance (0.05).
3. Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cell, 1H, 12h, 24h infection group LT content was higher than normal group (P0.05). Moreover, the same infection group had no significant change in LT content at different infection time (P? 0.05).S2 strain infected group, LT content compared with the WboA-S2 strain infection group content was not statistically significant (0.05).
4. Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cell, 1H, 12h, 24h infection group histamine content is higher than normal group (P0.05).S2 strain, Escherichia coli infected mast cells increased first, then decreased, and then the trend.WboA-S2 strain infected mast cells after 1h, 12h, 24h histamine content did not significantly change. The histamine content of infected group.24h WboA-S2 was significantly higher than that of S2 strain (P0.05).
5. no IL-1 beta, tryptase, IL-12 and IFN- gamma were detected in the supernatants of Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cells at each time point.
6. mast cells express high levels of TLR4 and TLR9mRNA, and the abundance of TLR8mRNA is relatively low. It is worth noting that mast cells P815 does not express TLR2..
7. Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cells, at 12h, 24h, the expression of TLR4mRNA changed after.S2 strain infected mast cells, TLR4mRNA expression increased gradually with the prolongation of infection time, gradually increasing.WboA-S2 strain infected mast cells, TLR4mRNA expression decreased, 24h increased. Escherichia coli infection fertilizer After 12h, TLR4mRNA expression increased significantly, while 24h decreased.
8. Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cells, at 12h, 24h, the expression of TLR9mRNA changed after.S2 strain infected mast cells, TLR9mRNA expression increased, and 24h reduced.WboA-S2 strains infected mast cells when 24h, TLR9mRNA table did not change obviously when 12h, the expression increased. Escherichia coli After infection with mast cells, the expression of TLR9mRNA decreased significantly while 12h increased significantly at 24h.
9. there was no significant change in TLR8mRNA expression at 12h and 24h after Brucella S2 strain, WboA-S2 strain and Escherichia coli infected mast cells.
10. the conditions of TLR4RNAi silencing were determined and optimized: the transfection reagents, 6pmol TLR4siRNA, 1 x 105 mast cells per pore of 24 orifice plates, 6pmol TLR4siRNA and 1 x 105 mast cells were the best, and the transfection efficiency could reach 50%..
11. Brucella S2, WboA-S2 and Escherichia coli infected mast cells, and the content of IL-6 in TLR4mRNA silencing group was significantly higher than that in the corresponding non silent group (P0.05). The content of TNF- alpha and the content of LT were not statistically significant compared with that of the corresponding non silencing group (P? 0.05).
12. the histamine content in the mast cell TLR4mRNA silencing group was statistically significant compared with that of the corresponding non silencing group (P0.05).S2 strain. After Escherichia coli infected mast cells, the content of histamine in the RNA silencing group was lower than that in the corresponding non silent group at 1H, while 12h was higher than that in the corresponding non silencing group, while 24h was lower than that of the corresponding non silent group of.WboA-S2 strain infected with mast cells when 24h. The histamine content in the RNA silencing group was lower than that in the corresponding silent group at 1H, and higher than that in the corresponding silent group at 12h and 24h.
1. Brucella S2 strain and WboA-S2 strain could stimulate mast cells to release IL-6, TNF- alpha, LT and histamine, but did not release tryptase, IL-1 beta, IL-12 and IFN- gamma.
The expression product of 2.WboA gene may be the main ligand to stimulate histamine release from mast cells.
3. mast cells express high levels of TLR4 and TLR9mRNA, but the abundance of TLR8mRNA is low, and TLR2 is not expressed.
4.TLR4 and TLR9 were involved in the recognition of Brucella by mast cells, and TLR8 was not obvious in identifying Brucella.
Effect;
5. in the process of Brucella stimulating mast cells to secrete IL-6 and histamine, TLR4 is the main pattern recognition receptor of mast cells. But because the WboA-S2 strain of Brucella is similar to the effect of S2 strain on mast cells producing IL-6, it is presumed that TLR4 can recognize other bacterial components and activate mast cells in addition to identifying the LPS of Brucella. Secreting IL-6., but the process of Brucella activating mast cells to secrete TNF- alpha and LT has nothing to do with TLR4 mediated pattern recognition.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R516.7

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