肥大细胞对布鲁菌模式识别与应答效应的体外研究

发布时间：2018-07-10 02:42

本文选题：肥大细胞 + 猪布鲁菌　；参考：《河北大学》2014年硕士论文

【摘要】：目的：为研究肥大细胞对布鲁菌的模式识别与免疫应答效应，进一步阐明布鲁菌病的发病机制，为布鲁菌病的治疗和疫苗研发探索新途径与新方法。方法： 1．分别用布鲁菌S2菌株、WboA-S2菌株和大肠杆菌以MOI50:1感染肥大细胞，分别在感染后1h、12h和24h时收集上清，ELISA方法测上清中IL-6、TNF-α、LT、IL-1β、组胺、类胰蛋白酶、IL-12、IFN-γ的含量，以未处理的肥大细胞为对照。 2．RT-PCR法检测正常肥大细胞TLR2、4、8与9mRNA的表达。 3．分别用布鲁菌S2菌株、WboA-S2菌株和大肠杆菌以MOI50:1感染肥大细胞，在感染后1h、12h和24h时收集细胞，RT-PCR法测细胞TLR4、8与9mRNA的表达。 4．用RNAi技术沉默肥大细胞TLR4，并设未沉默对照组，用RT-PCR法和流式细胞术检测其沉默效果。 5.分别用布鲁菌S2菌株、WboA-S2菌株和大肠杆菌以MOI50:1感染沉默组与未沉默组肥大细胞，分别在感染后1h、12h和24h时收集上清，ELISA方法测上清中IL-6、TNF-α、LT与组胺的含量。结果： 1．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞，1h、12h、24h感染组IL-6含量明显高于正常组（P0.05），且随着感染时间的延长，IL-6含量呈增加趋势。S2菌株感染组IL-6含量与WboA-S2菌株感染组IL-6含量比较无统计学意义（P㧐0.05）。 2．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞，1h感染组TNF-α含量与正常组相比无统计学意义（P㧐0.05），12h、24h各感染组TNF-α含量均高于正常组（P0.05）。S2菌株感染组TNF-α含量与WboA-S2菌株感染组TNF-α含量比较无统计学意义（P㧐0.05）。 3．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞，1h、12h、24h感染组LT含量高于正常组（P0.05）。而且同一感染组在不同感染时间LT含量没有明显变化（P㧐0.05）。S2菌株感染组LT含量与WboA-S2菌株感染组LT含量比较无统计学意义（P㧐0.05）。 4．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞，1h、12h、24h感染组组胺含量高于正常组（P0.05）。S2菌株、大肠杆菌感染肥大细胞后组胺含量呈显先增加，再减少，然后又增加的趋势。WboA-S2菌株感染肥大细胞后1h、12h、24h组胺含量没有明显变化。24h WboA-S2菌株感染组组胺含量明显高于S2菌株感染组胺含量（P0.05）。 5．在布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞的各时间点上清中均未检测到IL-1β、类胰蛋白酶、IL-12和IFN-γ。 6．肥大细胞表达高水平的TLR4和TLR9mRNA，TLR8mRNA的表达丰度较低。值得注意的是肥大细胞P815不表达TLR2。 7．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞后，在12h、24h时，TLR4mRNA的表达发生了变化。S2菌株感染肥大细胞后，TLR4mRNA的表达随感染时间的延长逐渐增加。WboA-S2菌株感染肥大细胞后12h，TLR4mRNA的表达减少，24h时增多。大肠杆菌感染肥大细胞后12h，TLR4mRNA的表达显著增多，24h则减少。 8．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞后，在12h、24h时，TLR9mRNA的表达发生了变化。S2菌株感染肥大细胞后，TLR9mRNA的表达12h时增加，24h时减少。WboA-S2菌株感染肥大细胞后，TLR9mRNA的表达12h时无明显变化，24h表达量增加。大肠杆菌感染肥大细胞后，TLR9mRNA的表达12h时显著减少，24h时显著增加。 9．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞后，在12h、24h时TLR8mRNA的表达没有明显变化。 10．确定并优化了TLR4RNAi沉默条件：24孔板每孔加1μl转染试剂、6pmol TLR4siRNA、1×105个肥大细胞，此条件下TLR4mRNA和蛋白表达抑制效果最好，转染效率能达到50%。 11．布鲁菌S2菌株、WboA-S2菌株和大肠杆菌感染肥大细胞，TLR4mRNA沉默组IL-6含量明显高于相应未沉默组（P0.05），TNF-α含量和LT含量与相应未沉默组相比均无统计学意义（P㧐0.05）。 12．肥大细胞TLR4mRNA沉默组的组胺含量与相应未沉默组相比有统计学意义（P0.05）。S2菌株、大肠杆菌感染肥大细胞后，RNA沉默组组胺含量在1h时低于相应未沉默组，12h时高于相应未沉默组，24h时又低于相应未沉默组。WboA-S2菌株感染肥大细胞后，RNA沉默组组胺含量在1h时低于相应未沉默组，12h时和24h时均高于相应未沉默组。结论： 1．布鲁菌S2菌株和WboA-S2菌株均可刺激肥大肥大细胞释放IL-6、TNF-α、LT和组胺，，但不释放类胰蛋白酶、IL-1β、IL-12和IFN-γ； 2．WboA基因的表达产物可能是刺激肥大细胞释放组胺的主要模式配体； 3．肥大细胞表达高水平的TLR4和TLR9mRNA，但TLR8mRNA的表达丰度较低，不表达TLR2； 4． TLR4和TLR9参与肥大细胞对布鲁菌的识别，TLR8在识别布鲁菌中没有明显作用； 5．在布鲁菌刺激肥大细胞分泌IL-6和组胺的过程中，TLR4是肥大细胞的主要模式识别受体，但由于布鲁菌WboA-S2菌株与S2菌株刺激肥大细胞产生IL-6的作用相似，故推测TLR4除可识别布鲁菌的LPS外，尚可识别其他菌体成分，进而激活肥大细胞分泌IL-6。但布鲁菌激活肥大细胞分泌TNF-α和LT的过程与TLR4介导的模式识别无关。
[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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.7

【参考文献】