【摘要】：目的：探討腺病毒E1A蛋白對(duì)細(xì)胞炎癥反應(yīng)及皮質(zhì)激素抗炎作用的影響及其可能的機(jī)制，并進(jìn)一步了解腺病毒潛伏感染對(duì)COPD發(fā)生、發(fā)展的影響，為尋找COPD有效的治療方法打下基礎(chǔ)。 方法1.穩(wěn)定表達(dá)腺病毒E1A蛋白的人肺泡上皮細(xì)胞株的構(gòu)建：將已構(gòu)建成功的pneo-E1A、pneo質(zhì)粒轉(zhuǎn)染人肺泡上皮細(xì)胞，通過G418抗性篩選和單克隆化操作最終獲得穩(wěn)定表達(dá)腺病毒E1A蛋白的抗性細(xì)胞克隆；用RT-PCR、western blot及免疫細(xì)胞化學(xué)等方法對(duì)E1A基因表達(dá)進(jìn)行鑒定；2.腺病毒E1A基因?qū)θ朔闻萆掀ぜ?xì)胞炎癥因子的影響及其機(jī)制：10ng.ml-1TNFα作用于E1A+組細(xì)胞和E1A-組細(xì)胞，分別于刺激因素作用24h后收集細(xì)胞上清，用ELISA試劑盒檢測(cè)細(xì)胞因子IL-8的表達(dá)；收集細(xì)胞懸液用流式細(xì)胞術(shù)檢測(cè)ICAM-1蛋白的表達(dá)；3.腺病毒E1A蛋白對(duì)不同濃度糖皮質(zhì)激素抗炎作用的影響及其機(jī)制：3.1分別用終濃度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-組細(xì)胞及E1A+組細(xì)胞，檢測(cè)細(xì)胞HDAC1、HDAC2的表達(dá)情況；3.2用人ELISA試劑盒、流式細(xì)胞術(shù)檢測(cè)腺病毒E1A蛋白對(duì)TNF-α誘導(dǎo)及DXM干預(yù)下細(xì)胞因子IL-8及ICAM-1表達(dá)的影響；3.3用western blot檢測(cè)腺病毒E1A蛋白對(duì)TNF-α誘導(dǎo)及DXM干預(yù)下HDAC1、HDAC2、GR、NF-κB的影響，用比色法檢測(cè)TNF-α作用及DXM干預(yù)對(duì)HDAC活性的影響。 結(jié)果1. pneo質(zhì)粒及pneo-E1A質(zhì)粒轉(zhuǎn)染A549細(xì)胞后經(jīng)G418抗性篩選，經(jīng)RT-PCR檢測(cè)E1A+組細(xì)胞克隆能擴(kuò)增出238bp和375bp特異性片段，，而E1A-組細(xì)胞無特異性條帶顯示；免疫組化結(jié)果顯示：E1A+組細(xì)胞克隆均在細(xì)胞核內(nèi)出現(xiàn)棕黃色細(xì)胞染色，而E1A-組細(xì)胞未見陽性染色；western blot結(jié)果顯示：E1A+細(xì)胞克隆出現(xiàn)大小約為45～48、50～52kD E1A特異性的蛋白條帶，而E1A-細(xì)胞組未檢測(cè)出蛋白條帶，穩(wěn)定表達(dá)E1A蛋白的人肺泡上皮細(xì)胞株構(gòu)建成功；2．在TNF-α作用下，E1A+組細(xì)胞和E1A-組細(xì)胞IL-8、ICAM-1蛋白表達(dá)增加，相對(duì)于E1A-組細(xì)胞，E1A+組細(xì)胞能明顯上調(diào)TNF-α作用下炎癥因子的表達(dá)；3.不同濃度的地塞米松均可以明顯增加E1A+和E1A-組細(xì)胞HDAC1及HDAC2的蛋白表達(dá)，10~(-5)mol.l~(-1)地塞米松作用最強(qiáng)。E1A蛋白對(duì)HDAC1、HDAC2表達(dá)無明顯影響。4.糖皮質(zhì)激素主要通過與GR結(jié)合作用于HDAC及NF-κB發(fā)揮抗炎作用，用western blot檢測(cè)TNF-α或DXM干預(yù)下E1A+和E1A-組細(xì)胞HDAC1、HDAC2、GR和NF-κB的表達(dá)情況；用比色法檢測(cè)各組細(xì)胞HDAC的活性。結(jié)果顯示在E1A+和E1A-組細(xì)胞，DXM能拮抗TNF-α抑制HDAC1、HDAC2蛋白表達(dá)的作用；DXM均能抑制TNF-α誘導(dǎo)的NF-κB活化作用，腺病毒E1A蛋白對(duì)GR入核作用無影響。 結(jié)論1.腺病毒E1A蛋白能夠放大致炎因素誘導(dǎo)下炎癥因子IL-8、ICAM-1的表達(dá)；2.E1A蛋白主要通過上調(diào)轉(zhuǎn)錄因子NF-κB的轉(zhuǎn)錄活性促進(jìn)炎癥因子的表達(dá)；3.糖皮質(zhì)激素主要通過作用于GR、HDAC及NF-κB發(fā)揮抗炎作用，腺病毒E1A蛋白對(duì)其抗炎作用無影響。
[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 偽 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- 偽 and the intervention of DXM were detected by flow cytometry with human ELISA kit, the effects of adenoviral E1A protein on TNF- 偽 induction and DXM intervention were detected by western blot, and the effects of TNF- 偽 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 鹵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- 偽. Compared with E1A-group, E1A group could significantly up-regulate the expression of inflammatory factors under the action of TNF- 偽. 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- 偽 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- 偽 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- 偽, 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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R563.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 方怡,冉丕鑫;腺病毒感染與慢性阻塞性肺疾病發(fā)病機(jī)制研究現(xiàn)狀[J];國外醫(yī)學(xué).呼吸系統(tǒng)分冊(cè);2005年06期

2 陳娟;張錦;鄭西衛(wèi);郭園園;付欣;李冰;冉丕鑫;;腺病毒E1A蛋白對(duì)大鼠肺泡上皮細(xì)胞及人肺腺癌細(xì)胞凋亡的影響[J];細(xì)胞與分子免疫學(xué)雜志;2010年05期

3 方怡;李冰;陳娟;劉啟才;冉丕鑫;;腺病毒潛伏感染對(duì)大鼠肺泡上皮細(xì)胞氧化/抗氧化平衡的影響[J];中國病理生理雜志;2008年06期

4 周玉民,冉丕鑫;慢性阻塞性肺疾病的流行病學(xué)[J];中國呼吸與危重監(jiān)護(hù)雜志;2004年02期

5 ;慢性阻塞性肺疾病診治指南(2007年修訂版)[J];中華結(jié)核和呼吸雜志;2007年01期

6 陳娟;李冰;羅健東;張丹丹;冉丕鑫;;腺病毒早表達(dá)蛋白1A對(duì)大鼠細(xì)胞間黏附分子1的影響[J];中華結(jié)核和呼吸雜志;2007年08期

7 劉升明,王小平,王大禮,周玉民,呂嘉春,鄭勁平,鐘南山,冉丕鑫;廣東部分地區(qū)慢性阻塞性肺疾病發(fā)病狀況調(diào)查[J];中華醫(yī)學(xué)雜志;2005年11期

本文編號(hào)：2506295