PRRSV感染对Marc-145细胞IKKγ基因表达水平影响研究
发布时间：2019-05-23 02:06
【摘要】：目的：通过分析PRRSV感染MARC-145细胞疾病模型中IKKγmRNA和蛋白表达量的变化,揭示NF-κB通路的调节因子与PRRSV之间的关系,为进一步研究通路与病毒感染之间的关系奠定基础。方法：通过间接免疫荧光(IFA)法评价建立的PRRSV感染Marc-145细胞模型；通过MTT法检测LY294002抑制率；实验分对照组、抑制剂(LY294002)组、病毒(PRRSV)组、抑制剂(LY294002)+病毒(PRRSV)组,其中对照组为只加培养基,抑制剂组为只加10μMY294002的溶液,病毒组为只加100TCID50的PRRS V组,抑制剂+病毒组为加入lO^M LY294002溶液和100TCIDso的病毒稀释液。通过qPCR和Western blot检测不同处理组的IKKγ表达量的变化,探究IKKγ参与抗PRRSV的分子免疫机制。结果：①成功建立了PRRSV感染Marc-145细胞模型。②MTT比色法检测P13K抑制剂LY294002对Marc-145细胞的抑制率,与正常细胞组比较均有抑制,且抑制程度随浓度升高而增强。48h内,抑制率随时间的延长呈递增趋势;当抑制时间超过48h后,抑制下降。③荧光定量PCR结果显示：与对照相比,PRRSV组IKKγmRNA水平上调,差异显著(P0.05),LY294002组mRNA水平下调,差异极显著(P0.01),LY293002+PRRSV组]mRNA水平下调,差异极显著(P0.01)。④Western blot结果显示：与对照组相比,感染PRRSV后IKKγ蛋白水平水平上调,加入抑制剂LY294002后IKKγ蛋白表达下调,I.Y294002+PRRSV组,IKKγ蛋白表达下调。结论：①上游信号通路影响下游信号通路的信号转导。②LY294002抑制剂影响了PRRSV复制转录过程。③IKKγ在感染PRRSV后的分子免疫协调机制中发挥作用。
[Abstract]:Aim: to analyze the changes of IKK 纬 mRNA and protein expression in MARC-145 cell disease model infected with PRRSV, and to reveal the relationship between the regulatory factors of NF- 魏 B pathway and PRRSV, so as to lay a foundation for further study on the relationship between NF- kB pathway and viral infection. Methods: the model of Marc-145 cells infected with PRRSV was evaluated by indirect immunofluorescence (IFA) assay, and the inhibition rate of LY294002 was detected by MTT assay. The experiment was divided into control group, inhibitor (LY294002) group, virus (PRRSV) group and inhibitor (LY294002) virus (PRRSV) group. The control group was supplemented with medium only, the inhibitor group was only supplemented with 10 渭 MY294002 solution, and the virus group was PRRSV group with 100TCID50 only. The inhibitor virus group was the virus diluent with Lo ^ M LY294002 solution and 100TCIDso. The expression of IKK 纬 in different treatment groups was detected by qPCR and Western blot, and the molecular immune mechanism of IKK 纬 involved in anti-PRRSV was explored. Results: 1 the Marc-145 cell model of PRRSV infection was successfully established. 2 the inhibition rate of P13K inhibitor LY294002 on Marc-145 cells was detected by MTT colorimetric assay, which was significantly higher than that of the normal cell group, and the degree of inhibition increased within 48 hours. The inhibition rate increased with the prolongation of time. When the inhibition time was more than 48 hours, the inhibition decreased. 3 the results of fluorescence quantitative PCR showed that the level of IKK 纬 mRNA in PRRSV group was significantly higher than that in control group (P 0.05), and the level of mRNA in LY294002 group was significantly lower than that in control group (P 0.01). The level of mRNA in LY293002 PRRSV group was significantly lower than that in control group (P 0.01). The results of 4Western blot showed that compared with the control group, the level of IKK 纬 protein was up-regulated after infection with PRRSV, and the expression of IKK 纬 protein was down-regulated after LY294002 was added to I.Y294002 PRRSV group, and the expression of IKK-纬 protein was down-regulated in I.Y294002 PRRSV group. The expression of IKK 纬 protein was down-regulated. Conclusion: 1 upstream signal pathway affects the signal transduction of downstream signal pathway. 2Lyn294002 inhibitor affects the process of PRRSV replication and transcription. 3IKK 纬 plays a role in the molecular immune coordination mechanism after PRRSV infection.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2483499
[Abstract]:Aim: to analyze the changes of IKK 纬 mRNA and protein expression in MARC-145 cell disease model infected with PRRSV, and to reveal the relationship between the regulatory factors of NF- 魏 B pathway and PRRSV, so as to lay a foundation for further study on the relationship between NF- kB pathway and viral infection. Methods: the model of Marc-145 cells infected with PRRSV was evaluated by indirect immunofluorescence (IFA) assay, and the inhibition rate of LY294002 was detected by MTT assay. The experiment was divided into control group, inhibitor (LY294002) group, virus (PRRSV) group and inhibitor (LY294002) virus (PRRSV) group. The control group was supplemented with medium only, the inhibitor group was only supplemented with 10 渭 MY294002 solution, and the virus group was PRRSV group with 100TCID50 only. The inhibitor virus group was the virus diluent with Lo ^ M LY294002 solution and 100TCIDso. The expression of IKK 纬 in different treatment groups was detected by qPCR and Western blot, and the molecular immune mechanism of IKK 纬 involved in anti-PRRSV was explored. Results: 1 the Marc-145 cell model of PRRSV infection was successfully established. 2 the inhibition rate of P13K inhibitor LY294002 on Marc-145 cells was detected by MTT colorimetric assay, which was significantly higher than that of the normal cell group, and the degree of inhibition increased within 48 hours. The inhibition rate increased with the prolongation of time. When the inhibition time was more than 48 hours, the inhibition decreased. 3 the results of fluorescence quantitative PCR showed that the level of IKK 纬 mRNA in PRRSV group was significantly higher than that in control group (P 0.05), and the level of mRNA in LY294002 group was significantly lower than that in control group (P 0.01). The level of mRNA in LY293002 PRRSV group was significantly lower than that in control group (P 0.01). The results of 4Western blot showed that compared with the control group, the level of IKK 纬 protein was up-regulated after infection with PRRSV, and the expression of IKK 纬 protein was down-regulated after LY294002 was added to I.Y294002 PRRSV group, and the expression of IKK-纬 protein was down-regulated in I.Y294002 PRRSV group. The expression of IKK 纬 protein was down-regulated. Conclusion: 1 upstream signal pathway affects the signal transduction of downstream signal pathway. 2Lyn294002 inhibitor affects the process of PRRSV replication and transcription. 3IKK 纬 plays a role in the molecular immune coordination mechanism after PRRSV infection.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【参考文献】
相关期刊论文 前3条
1 刘开泰;韩秀敏;张阳;;IκB激酶结构和功能研究进展[J];大连医科大学学报;2007年01期
2 Irene M Rodríguez-Gómez;Jaime Gómez-Laguna;Librado Carrasco;;Impact of PRRSV on activation and viability of antigen presenting cells[J];World Journal of Virology;2013年04期
3 胡永亮;宋伦;;IKK激酶的NF-κB非相关性功能研究进展[J];生物技术通讯;2015年02期
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