LPS诱导L929细胞β防御素-2和schlafen-2基因表达及其信号转导

发布时间：2018-01-23 21:49

  本文关键词： LPS 小鼠β防御素-2 小鼠schlafen 小鼠成纤维细胞 信号转导　出处：《兰州大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：通过研究LPS对于小鼠成纤维细胞(L929)β-防御素(defb)和schlafen (slfn)基因表达的影响,以及P38MAPK和NF-κB信号转导通路对defb2和slfn2基因表达的调控,探讨成纤维细胞防御病原微生物作用的机制,为进一步研究机体抗感染机理提供理论基础和实验依据。 方法：以低、中、高剂量组LPS(50ng/ml,200ng/ml,500ng/ml)分别处理L929细胞2h,4h,6h,以PBS处理L929细胞相同时间段作为对照组,提取细胞总RNA,用RT-PCR和荧光定量PCR的方法检测defb1,2和slfn1,2,3基因表达；应用Western Blotting的方法检测defb2蛋白的表达。BMS345541和PD169316两种信号转导通路的化学抑制剂预处理细胞,再用LPS刺激细胞后观察defb2和slfn2基因、蛋白表达的变化,探讨defb2和slfn2基因表达的信号转导通路。 结果： 1.LPS诱导小鼠成纤维细胞β-防御素-2(defb2)的表达,其在中剂量组、2h时基因、蛋白表达最强；p-防御素-1(defb1)则未被诱导表达。 2. schlafen2 (slfn2)在中剂量组、2h时其相对mRNA表达最多；在不同浓度和不同时间下,与对照组相比,slfnl,3的相对mRNA表达均无明显差异。 3.IL-6相对mRNA表达在2h组基因表达最强,4h和6h组相对mRNA表达逐渐下降；IL-1相对mRNA表达在2h、4h、6h组与对照组相比均无明显差异。 4.信号转导效应分子TLR4、MYD88、NF-κB的相对]nRNA表达在2h、4h、6h组与对照组相比均有统计学意义。 5.与LPS组相比,BMS345541和PD169316组defb2相对mRNA与蛋白的表达均明显降低,slfn2相对mRNA表达也明显降低。 结论：LPS诱导小鼠成纤维细胞β-防御素-2基因的表达,上调schlafen-2和白细胞介素-6基因的表达,对p-防御素-1、schlafen-1,-3和白细胞介素-1基因的表达无影响；小鼠成纤维细胞defb2和slfn2基因表达都受到P38MAPK及NF-κB信号转导通路调控。提示成纤维细胞可能通过诱导defb2、上调schlafen-2和白细胞介素-6的表达发挥机体抗感染的作用。
[Abstract]:Objective: to investigate the effect of LPS on the expression of 尾 -defb and schlafen slfn in mouse fibroblasts. And the regulation of P38 MAPK and NF- 魏 B signal transduction pathway on the expression of defb2 and slfn2 genes, to explore the mechanism of fibroblast defense against pathogenic microorganisms. It provides theoretical and experimental basis for further study on the mechanism of anti-infection. Methods: L929 cells were treated with L929 cells for 2 h and 4 h for 6 h with low, medium and high doses of LPSN 50ng / ml, 200ng / ml and 500ng / ml, respectively. L929 cells were treated with PBS at the same time as control group. Total RNAs were extracted from L929 cells. Defb1m-2 and slfn1m-2 were detected by RT-PCR and fluorescence quantitative PCR. 3Gene expression; Application of Western. The expression of defb2 protein. BMS345541 and PD169316 signal transduction pathway were detected by Blotting. The changes of defb2 and slfn2 gene and protein expression were observed after stimulation with LPS, and the signal transduction pathway of defb2 and slfn2 gene expression was discussed. Results: 1. LPS-induced expression of 尾 -defin -2tdefb _ 2 (尾 -defb _ 2) in mouse fibroblasts was the strongest at 2 h in the middle dose group. The expression of P-defin-1 was not induced. 2.The relative mRNA expression of schlafen2 / slfn2 was the highest at 2 h in the middle dose group. There was no significant difference in the expression of mRNA between the two groups at different concentrations and at different time. 3. The relative mRNA expression of IL-6 decreased gradually in the groups of 2h and 6h, respectively. There was no significant difference in the expression of IL-1 relative mRNA between the two groups at 2 h and 4 h after 6 h compared with the control group. 4. The expression of nRNA in the signal transduction effector molecule TLR4, MYD8, NF- 魏 B was significantly higher than that in the control group. 5. Compared with LPS group, the expression of defb2 and protein in BMS345541 and PD169316 group were significantly lower than those in LPS group. The expression of slfn2 was also significantly decreased relative to mRNA. Conclusion the expression of 尾-defensin-2 gene in mouse fibroblasts was induced by 1: 1 LPS, and the expression of schlafen-2 and IL-6 genes was up-regulated, and the expression of 尾-defensin-1 was up-regulated. The expression of Schlafen-1- 3 and interleukin-1 gene was not affected. The expression of defb2 and slfn2 genes in mouse fibroblasts were regulated by P38 MAPK and NF- 魏 B signal transduction pathway, suggesting that fibroblasts might induce defb2. Upregulation of schlafen-2 and IL-6 expression may play an anti-infection role.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.12

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