本文選題：支氣管哮喘 + NF-kB��； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:組蛋白去乙�；�(HDAC)是一類廣泛存在于真核細(xì)胞中的蛋白酶,通過使組蛋白去乙�；鴮�(duì)基因的表達(dá)調(diào)控起重要作用。研究發(fā)現(xiàn)哮喘患者中,HDAC的活性是降低的,隨著乙�；潭鹊纳�,炎癥細(xì)胞、炎癥介質(zhì)亦會(huì)增加;而近來發(fā)現(xiàn)維生素D3不僅參與礦物質(zhì)代謝,在免疫方面也起了作用。研究發(fā)現(xiàn)1,25(OH)_2D_3的缺乏可能會(huì)影響哮喘的發(fā)病機(jī)制,然而它在調(diào)節(jié)特定分子的轉(zhuǎn)錄機(jī)制方面尚不清楚。目前,地塞米松和1,25(OH)_2D_3在哮喘中對(duì)氣道HDAC表達(dá)的影響尚未見報(bào)道。本實(shí)驗(yàn)通過復(fù)制OVA哮喘小鼠模型,并使用地塞米松(Dex)和1,25(OH)_2D_3進(jìn)行干預(yù),來觀察其對(duì)哮喘小鼠氣道HDAC2表達(dá)和活性的影響,以及它與地塞米松(Dex)在抑制氣道炎癥細(xì)胞因子分泌方面是否有協(xié)同作用。方法:50只約20-45g的雌性Balb/c小鼠,隨機(jī)編為1-50號(hào):對(duì)照組(N組)為1-10號(hào)、OVA哮喘組(M組)為11-20號(hào)、維生素D組(V組)為21-30號(hào)、Dex組(D組)為31-40號(hào)、維生素D+Dex組(DV組)為41-50號(hào)。各組小鼠分別在第3天、第10天的同一時(shí)間接受腹腔注射致敏,第22天開始至26天連續(xù)5天進(jìn)行滴鼻激發(fā)。其中維生素D治療組小鼠于每次激發(fā)前30min予以1,25(OH)_2D_3(0.25ug/kg)腹腔注射治療;D組小鼠在每次激發(fā)前30min,予以地塞米松磷酸鈉(1mg/kg)腹腔注射治療;dv組于每次激發(fā)前30min予以1,25(oh)2d3(0.25ug/kg)、地塞米松磷酸鈉(1mg/kg)腹腔注射治療,對(duì)照組采用等容量的pbs腹腔注射及激發(fā),末次激發(fā)24小時(shí)后處死小鼠;取病變肺組織行he染色,支氣管肺泡灌洗液(balf)作細(xì)胞分類計(jì)數(shù),造模成功后采用眶靜脈取血用酶聯(lián)免疫法(elisa)檢測(cè)血清中il-4水平;用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(rt-pcr)法檢測(cè)肺組織中nf-kbp65表達(dá)情況;用蛋白質(zhì)印跡法(westernblot)檢測(cè)hdac2含量。結(jié)果:1、n組小鼠好動(dòng),精神食欲佳,he染色肺組織層結(jié)構(gòu)清晰,杯狀細(xì)胞散在且少,氣道、血管及周圍肺組織極少見到炎性細(xì)胞浸潤;ova組小鼠可見不同程度精神、食欲下降,毛發(fā)臟亂,激發(fā)后可見到明顯呼吸困難、噴嚏、嘴唇爪紫紺,有的可見二便失禁,he染色可見肺組織結(jié)構(gòu)破壞,有大量的炎性細(xì)胞,氣道內(nèi)可見脫落的壞死細(xì)胞,氣道粘膜增厚,有的可見管腔塌陷;地塞米松治療組和1,25(oh)2d3治療組可見肺組織各層結(jié)構(gòu)清晰,浸潤的炎癥細(xì)胞減少,平滑肌變薄。2、m組與n相比,balf中嗜酸性粒細(xì)胞百分比升高(p0.05);小鼠血清il-4含量升高(p0.05);而維生素d和地塞米松干預(yù)組動(dòng)物balf中白細(xì)胞總數(shù)和嗜酸性粒細(xì)胞數(shù)量明顯低于哮喘組(均p0.05),小鼠血清il-4含量明顯降低(p0.05);dv組分別與d組、v組相比,balf中嗜酸粒數(shù)量減低(p0.05),小鼠血清il-4量也降低(p0.05)。3、nf-kbp65基因表達(dá)結(jié)果:m組小鼠nf-kbp65量明顯比對(duì)照組升高(均p0.05);同m組相比,d、v、dv組nf-kbp65基因表達(dá)水平降低(均p0.05);與d組、v組相比,dv組nf-kbp65基因表達(dá)水平降低(P0.05)。4、Western blot檢測(cè)顯示:M組小鼠肺組織中HDAC2蛋白水平明顯比對(duì)照組小鼠降低(均P0.05),而維生素D和地塞米松治療后HDAC水平顯著升高,地塞米松組較維生素D組水平更高(均P0.05)。結(jié)論:1、OVA誘導(dǎo)哮喘小鼠肺組織NF-kB p65表達(dá)水平升高,HDAC2蛋白水平降低。2、1,25(OH)_2D_3能抑制哮喘小鼠肺組織中Th2型細(xì)胞因子的分泌,下調(diào)NF-kB p65mRNA水平,并能顯著增加HDAC2蛋白表達(dá)和酶活性。3、1,25(OH)_2D_3和地塞米松在抑制細(xì)胞因子釋放和NF-kB p65的表達(dá),增加HDAC2的表達(dá)和活性方面具有協(xié)同效應(yīng)。4、1,25(OH)_2D_3可能成為一種治療哮喘的新型HDAC2活化劑。
[Abstract]:Objective: histone deacetylase (HDAC) is a kind of protease widely existed in eukaryotic cells. It plays an important role in the regulation of gene expression by deacetylation of histone. It is found that the activity of HDAC is reduced in asthmatic patients. As the degree of acetylation is higher, inflammatory cells and inflammatory mediators are also increased; Vitamin D3 is not only involved in mineral metabolism, but also plays a role in immunity. The study found that the lack of 1,25 (OH) _2D_3 may affect the pathogenesis of asthma. However, it is not clear in regulating the transcriptional mechanism of specific molecules. At present, the effect of dexamethasone and 1,25 (OH) _2D_3 on airway HDAC expression has not yet been reported. This experiment was conducted by replicating the model of OVA asthma mice and using dexamethasone (Dex) and 1,25 (OH) _2D_3 to observe the effect of its effect on airway HDAC2 expression and activity in asthmatic mice and whether it had a synergistic effect with dexamethasone (Dex) in inhibiting the secretion of airway inflammatory cytokines. Method: 50 female Balb/c mice with approximately 20-45g. The random number was No. 1-50: the control group (group N) was No. 1-10, the OVA asthma group (group M) was No. 11-20, the vitamin D group (group V) was No. 21-30, the Dex group (group D) was 31-40, and the vitamin D+Dex group (DV group) was No. 41-50. The mice in each group were treated with intraperitoneal sensitization at the same time of third days and tenth days, and the nose was triggered on twenty-second days to 26 days for 26 days. 1,25 (OH) _2D_3 (0.25ug/kg) was administered by intraperitoneal injection of 1,25 (OH) _2D_3 (0.25ug/kg) before each stimulation in the group of vitamin D treatment group; the mice in group D were injected with dexamethasone sodium phosphate (1mg/kg) before each stimulation, and the DV group was treated by intraperitoneal injection of dexamethasone sodium phosphate before each excitation, and the control of dexamethasone sodium phosphate was administered by intraperitoneal injection. The group was injected with equal volume PBS intraperitoneally and excited, and the mice were killed after 24 hours of last excitation; he staining and bronchoalveolar lavage fluid (BALF) were used to count the pathological lung tissue. The serum IL-4 level was detected by ELISA for the use of the orbital vein after successful modeling. The reverse transcriptase polymerase chain reaction (RT-PCR) method was used to detect the serum IL-4 level. The expression of NF-kBp65 in lung tissue was measured and the content of HDAC2 was detected by Western blot (Westernblot). Results: 1, the mice in group n were very active, with good mental appetite, the structure of lung tissue was clear with HE staining, the goblet cells were scattered and few, and the airway, blood vessels and surrounding lung tissues were rarely seen in inflammatory cell infiltration; the mice of group ova showed different degrees of spirit and loss of appetite. The hair is messy and can be stimulated to see obvious dyspnea, sneezing, lip cyanosis, and some two incontinence. HE staining shows the destruction of the lung tissue, a large number of inflammatory cells, necrotic cells in the airway, the thickening of the airway mucosa, and the collapse of the lumen; the dexamethasone treatment group and the 1,25 (OH) 2D3 treatment group can see the Lung Group The structure of each layer was clear, the infiltration of inflammatory cells decreased and the smooth muscle thinned.2. Compared with N, the percentage of eosinophils in the M group increased (P0.05), and the serum IL-4 content in mice increased (P0.05), while the total number of white blood cells and eosinophils in the vitamin D and dexamethasone intervention group were significantly lower than that in the asthma group (P0.05), and the mice were significantly lower than those in the asthma group (P0.05). The content of serum IL-4 decreased significantly (P0.05), and in group DV, the number of eosinophils in BALF group decreased (P0.05) compared with group D and V group, and the serum IL-4 of mice decreased (P0.05).3, NF-kBp65 gene expression results: the M group mice increased significantly than those in the control group. Compared with group DV, the level of NF-kBp65 gene expression decreased (P0.05).4, and Western blot detection showed that the level of HDAC2 protein in the lung tissue of the M group was significantly lower than that of the control group (all P0.05), while the HDAC level after the treatment of vitamin D and dexamethasone was significantly higher, and the level of dexamethasone group was higher than that of the vitamin D group. Conclusion: 1, induced asthma. The expression level of NF-kB p65 in lung tissue of asthmatic mice was increased. The decrease of HDAC2 protein level by.2,1,25 (OH) _2D_3 could inhibit the secretion of Th2 type cytokines in the lung tissues of asthmatic mice, reduce the p65mRNA level of NF-kB, and significantly increase the expression of HDAC2 protein and enzyme activity.3,1,25 (OH) and dexamethasone in inhibiting the release of cytokines and the expression of NF-kB. Synergistic effect of increasing expression and activity of HDAC2.4,1,25 (OH) _2D_3 may become a new HDAC2 activator for treating asthma.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R562.25

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本文編號(hào)：1869339