腺病毒E1A蛋白对糖皮质激素抗炎作用的影响及其机制研究
发布时间：2019-06-26 15:41
【摘要】：目的：探讨腺病毒E1A蛋白对细胞炎症反应及皮质激素抗炎作用的影响及其可能的机制，并进一步了解腺病毒潜伏感染对COPD发生、发展的影响，为寻找COPD有效的治疗方法打下基础。 方法1.稳定表达腺病毒E1A蛋白的人肺泡上皮细胞株的构建：将已构建成功的pneo-E1A、pneo质粒转染人肺泡上皮细胞，通过G418抗性筛选和单克隆化操作最终获得稳定表达腺病毒E1A蛋白的抗性细胞克隆；用RT-PCR、western blot及免疫细胞化学等方法对E1A基因表达进行鉴定；2.腺病毒E1A基因对人肺泡上皮细胞炎症因子的影响及其机制：10ng.ml-1TNFα作用于E1A+组细胞和E1A-组细胞，分别于刺激因素作用24h后收集细胞上清，用ELISA试剂盒检测细胞因子IL-8的表达；收集细胞悬液用流式细胞术检测ICAM-1蛋白的表达；3.腺病毒E1A蛋白对不同浓度糖皮质激素抗炎作用的影响及其机制：3.1分别用终浓度10~(-5)mol.L~(-1)、10~(-6)mol.L~(-1)、10~(-7)mol.L~(-1)、10~(-8)mol.L~(-1)的DXM作用于E1A-组细胞及E1A+组细胞，检测细胞HDAC1、HDAC2的表达情况；3.2用人ELISA试剂盒、流式细胞术检测腺病毒E1A蛋白对TNF-α诱导及DXM干预下细胞因子IL-8及ICAM-1表达的影响；3.3用western blot检测腺病毒E1A蛋白对TNF-α诱导及DXM干预下HDAC1、HDAC2、GR、NF-κB的影响，用比色法检测TNF-α作用及DXM干预对HDAC活性的影响。 结果1. pneo质粒及pneo-E1A质粒转染A549细胞后经G418抗性筛选，经RT-PCR检测E1A+组细胞克隆能扩增出238bp和375bp特异性片段，，而E1A-组细胞无特异性条带显示；免疫组化结果显示：E1A+组细胞克隆均在细胞核内出现棕黄色细胞染色，而E1A-组细胞未见阳性染色；western blot结果显示：E1A+细胞克隆出现大小约为45～48、50～52kD E1A特异性的蛋白条带，而E1A-细胞组未检测出蛋白条带，稳定表达E1A蛋白的人肺泡上皮细胞株构建成功；2．在TNF-α作用下，E1A+组细胞和E1A-组细胞IL-8、ICAM-1蛋白表达增加，相对于E1A-组细胞，E1A+组细胞能明显上调TNF-α作用下炎症因子的表达；3.不同浓度的地塞米松均可以明显增加E1A+和E1A-组细胞HDAC1及HDAC2的蛋白表达，10~(-5)mol.l~(-1)地塞米松作用最强。E1A蛋白对HDAC1、HDAC2表达无明显影响。4.糖皮质激素主要通过与GR结合作用于HDAC及NF-κB发挥抗炎作用，用western blot检测TNF-α或DXM干预下E1A+和E1A-组细胞HDAC1、HDAC2、GR和NF-κB的表达情况；用比色法检测各组细胞HDAC的活性。结果显示在E1A+和E1A-组细胞，DXM能拮抗TNF-α抑制HDAC1、HDAC2蛋白表达的作用；DXM均能抑制TNF-α诱导的NF-κB活化作用，腺病毒E1A蛋白对GR入核作用无影响。 结论1.腺病毒E1A蛋白能够放大致炎因素诱导下炎症因子IL-8、ICAM-1的表达；2.E1A蛋白主要通过上调转录因子NF-κB的转录活性促进炎症因子的表达；3.糖皮质激素主要通过作用于GR、HDAC及NF-κB发挥抗炎作用，腺病毒E1A蛋白对其抗炎作用无影响。
[Abstract]:Aim: to explore the effect of adenoviral E1A protein on cellular inflammatory response and corticosteroid anti-inflammatory effect and its possible mechanism, and to further understand the effect of adenoviral latent infection on the occurrence and development of COPD, so as to lay a foundation for finding an effective treatment for COPD. Method 1. Construction of human alveolar epithelial cell line stably expressing adenoviral E1A protein: the constructed pneo-E1A,pneo plasmid was transferred into human alveolar epithelial cells, and the resistant cell clone stably expressing adenoviral E1A protein was obtained by G418 resistance screening and single cloning, and the expression of E1A gene was identified by RT-PCR,western blot and immunocytochemistry. The effect of adenoviral E1A gene on inflammatory factors in human alveolar epithelial cells and its mechanism: 10ng.ml-1TNF 伪 acted on E1A group cells and E1A-group cells, respectively, after 24 hours of stimulation, the cells were collected for 24 hours, the expression of cytokine IL-8 was detected by ELISA kit, the expression of ICAM-1 protein was detected by flow cytometry, and the expression of ICAM-1 protein was detected by flow cytometry. The effect of adenoviral E1A protein on the anti-inflammatory effect of different concentrations of glucocorticoid and its mechanism: 3.1 the expression of HDAC1,HDAC2 was detected by the final concentration of 10 ~ (- 5) mol.L~ (- 1), 10 ~ (- 6) mol.L~ (- 1), 10 ~ (- 7) mol.L~ (- 1), 10 ~ (- 8) mol.L~ (- 1) DXM in E1A group and E1A group, respectively. 3.2. the effects of adenoviral E1A protein on the expression of cytokines IL-8 and ICAM-1 induced by TNF- 伪 and the intervention of DXM were detected by flow cytometry with human ELISA kit, the effects of adenoviral E1A protein on TNF- 伪 induction and DXM intervention were detected by western blot, and the effects of TNF- 伪 and DXM intervention on HDAC activity were detected by colorimetric assay. Results 1. Pneo plasmid and pneo-E1A plasmid were selected by G418 resistance screening. 238bp and 375bp specific fragments could be amplified by RT-PCR in E1A group, but no specific bands were found in E1A-group. The results of immunohistochemistry showed that all the cell clones in E1A group showed brownish yellow cell staining in the nucleus, but no positive staining was found in E1A-group. Western blot results showed that the size of E1A cell clone was about 45 脳 48 and 50 卤52 KD E1A specific protein band, but no protein band was detected in E1A-cell group, and the human alveolar epithelial cell line stably expressing E1A protein was constructed successfully. 2. The expression of IL-8,ICAM-1 protein in E1A group and E1A-group was increased under the action of TNF- 伪. Compared with E1A-group, E1A group could significantly up-regulate the expression of inflammatory factors under the action of TNF- 伪. Different concentrations of dexamethasone could significantly increase the protein expression of HDAC1 and HDAC2 in E1A and E1A-groups. 10 ~ (- 5) mol.l~ (- 1) dexamethasone had the strongest effect on the expression of HDAC1,HDAC2. E1A protein had no significant effect on the expression of HDAC1,HDAC2. Glucocorticoids mainly play an anti-inflammatory effect on HDAC and NF- kappa B by binding to GR. The expression of HDAC1,HDAC2,GR and NF- 魏 B in E1A and E1A-groups under the intervention of TNF- 伪 or DXM was detected by western blot, and the activity of HDAC in each group was detected by colorimetric assay. The results showed that DXM could antagonize the inhibitory effect of TNF- 伪 on the expression of HDAC1,HDAC2 protein in E1A and E1A-cells, while DXM could inhibit the activation of NF- 魏 B induced by TNF- 伪, but the adenoviral E1A protein had no effect on the entry of GR into the nucleus. Conclusion 1. Adenoviral E1A protein could amplify the expression of inflammatory factor IL-8,ICAM-1 induced by inflammatory factors, and 2.E1A protein promoted the expression of inflammatory factor mainly by up-regulating the transcriptional activity of transcription factor NF- kappa B. Glucocorticoids play an anti-inflammatory effect on GR,HDAC and NF- kappa B, but adenoviral E1A protein has no effect on its anti-inflammatory effect.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9
本文编号：2506295
[Abstract]:Aim: to explore the effect of adenoviral E1A protein on cellular inflammatory response and corticosteroid anti-inflammatory effect and its possible mechanism, and to further understand the effect of adenoviral latent infection on the occurrence and development of COPD, so as to lay a foundation for finding an effective treatment for COPD. Method 1. Construction of human alveolar epithelial cell line stably expressing adenoviral E1A protein: the constructed pneo-E1A,pneo plasmid was transferred into human alveolar epithelial cells, and the resistant cell clone stably expressing adenoviral E1A protein was obtained by G418 resistance screening and single cloning, and the expression of E1A gene was identified by RT-PCR,western blot and immunocytochemistry. The effect of adenoviral E1A gene on inflammatory factors in human alveolar epithelial cells and its mechanism: 10ng.ml-1TNF 伪 acted on E1A group cells and E1A-group cells, respectively, after 24 hours of stimulation, the cells were collected for 24 hours, the expression of cytokine IL-8 was detected by ELISA kit, the expression of ICAM-1 protein was detected by flow cytometry, and the expression of ICAM-1 protein was detected by flow cytometry. The effect of adenoviral E1A protein on the anti-inflammatory effect of different concentrations of glucocorticoid and its mechanism: 3.1 the expression of HDAC1,HDAC2 was detected by the final concentration of 10 ~ (- 5) mol.L~ (- 1), 10 ~ (- 6) mol.L~ (- 1), 10 ~ (- 7) mol.L~ (- 1), 10 ~ (- 8) mol.L~ (- 1) DXM in E1A group and E1A group, respectively. 3.2. the effects of adenoviral E1A protein on the expression of cytokines IL-8 and ICAM-1 induced by TNF- 伪 and the intervention of DXM were detected by flow cytometry with human ELISA kit, the effects of adenoviral E1A protein on TNF- 伪 induction and DXM intervention were detected by western blot, and the effects of TNF- 伪 and DXM intervention on HDAC activity were detected by colorimetric assay. Results 1. Pneo plasmid and pneo-E1A plasmid were selected by G418 resistance screening. 238bp and 375bp specific fragments could be amplified by RT-PCR in E1A group, but no specific bands were found in E1A-group. The results of immunohistochemistry showed that all the cell clones in E1A group showed brownish yellow cell staining in the nucleus, but no positive staining was found in E1A-group. Western blot results showed that the size of E1A cell clone was about 45 脳 48 and 50 卤52 KD E1A specific protein band, but no protein band was detected in E1A-cell group, and the human alveolar epithelial cell line stably expressing E1A protein was constructed successfully. 2. The expression of IL-8,ICAM-1 protein in E1A group and E1A-group was increased under the action of TNF- 伪. Compared with E1A-group, E1A group could significantly up-regulate the expression of inflammatory factors under the action of TNF- 伪. Different concentrations of dexamethasone could significantly increase the protein expression of HDAC1 and HDAC2 in E1A and E1A-groups. 10 ~ (- 5) mol.l~ (- 1) dexamethasone had the strongest effect on the expression of HDAC1,HDAC2. E1A protein had no significant effect on the expression of HDAC1,HDAC2. Glucocorticoids mainly play an anti-inflammatory effect on HDAC and NF- kappa B by binding to GR. The expression of HDAC1,HDAC2,GR and NF- 魏 B in E1A and E1A-groups under the intervention of TNF- 伪 or DXM was detected by western blot, and the activity of HDAC in each group was detected by colorimetric assay. The results showed that DXM could antagonize the inhibitory effect of TNF- 伪 on the expression of HDAC1,HDAC2 protein in E1A and E1A-cells, while DXM could inhibit the activation of NF- 魏 B induced by TNF- 伪, but the adenoviral E1A protein had no effect on the entry of GR into the nucleus. Conclusion 1. Adenoviral E1A protein could amplify the expression of inflammatory factor IL-8,ICAM-1 induced by inflammatory factors, and 2.E1A protein promoted the expression of inflammatory factor mainly by up-regulating the transcriptional activity of transcription factor NF- kappa B. Glucocorticoids play an anti-inflammatory effect on GR,HDAC and NF- kappa B, but adenoviral E1A protein has no effect on its anti-inflammatory effect.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9
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