组蛋白去乙酰化酶在哮喘小鼠气道的表达及维生素D和地塞米松对其影响的研究

发布时间：2018-05-10 12:37

本文选题：支气管哮喘 + NF-kB　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:组蛋白去乙酰化酶(HDAC)是一类广泛存在于真核细胞中的蛋白酶,通过使组蛋白去乙酰化而对基因的表达调控起重要作用。研究发现哮喘患者中,HDAC的活性是降低的,随着乙酰化程度的升高,炎症细胞、炎症介质亦会增加;而近来发现维生素D3不仅参与矿物质代谢,在免疫方面也起了作用。研究发现1,25(OH)_2D_3的缺乏可能会影响哮喘的发病机制,然而它在调节特定分子的转录机制方面尚不清楚。目前,地塞米松和1,25(OH)_2D_3在哮喘中对气道HDAC表达的影响尚未见报道。本实验通过复制OVA哮喘小鼠模型,并使用地塞米松(Dex)和1,25(OH)_2D_3进行干预,来观察其对哮喘小鼠气道HDAC2表达和活性的影响,以及它与地塞米松(Dex)在抑制气道炎症细胞因子分泌方面是否有协同作用。方法:50只约20-45g的雌性Balb/c小鼠,随机编为1-50号:对照组(N组)为1-10号、OVA哮喘组(M组)为11-20号、维生素D组(V组)为21-30号、Dex组(D组)为31-40号、维生素D+Dex组(DV组)为41-50号。各组小鼠分别在第3天、第10天的同一时间接受腹腔注射致敏,第22天开始至26天连续5天进行滴鼻激发。其中维生素D治疗组小鼠于每次激发前30min予以1,25(OH)_2D_3(0.25ug/kg)腹腔注射治疗;D组小鼠在每次激发前30min,予以地塞米松磷酸钠(1mg/kg)腹腔注射治疗;dv组于每次激发前30min予以1,25(oh)2d3(0.25ug/kg)、地塞米松磷酸钠(1mg/kg)腹腔注射治疗,对照组采用等容量的pbs腹腔注射及激发,末次激发24小时后处死小鼠;取病变肺组织行he染色,支气管肺泡灌洗液(balf)作细胞分类计数,造模成功后采用眶静脉取血用酶联免疫法(elisa)检测血清中il-4水平;用逆转录聚合酶链式反应(rt-pcr)法检测肺组织中nf-kbp65表达情况;用蛋白质印迹法(westernblot)检测hdac2含量。结果:1、n组小鼠好动,精神食欲佳,he染色肺组织层结构清晰,杯状细胞散在且少,气道、血管及周围肺组织极少见到炎性细胞浸润;ova组小鼠可见不同程度精神、食欲下降,毛发脏乱,激发后可见到明显呼吸困难、喷嚏、嘴唇爪紫绀,有的可见二便失禁,he染色可见肺组织结构破坏,有大量的炎性细胞,气道内可见脱落的坏死细胞,气道粘膜增厚,有的可见管腔塌陷;地塞米松治疗组和1,25(oh)2d3治疗组可见肺组织各层结构清晰,浸润的炎症细胞减少,平滑肌变薄。2、m组与n相比,balf中嗜酸性粒细胞百分比升高(p0.05);小鼠血清il-4含量升高(p0.05);而维生素d和地塞米松干预组动物balf中白细胞总数和嗜酸性粒细胞数量明显低于哮喘组(均p0.05),小鼠血清il-4含量明显降低(p0.05);dv组分别与d组、v组相比,balf中嗜酸粒数量减低(p0.05),小鼠血清il-4量也降低(p0.05)。3、nf-kbp65基因表达结果:m组小鼠nf-kbp65量明显比对照组升高(均p0.05);同m组相比,d、v、dv组nf-kbp65基因表达水平降低(均p0.05);与d组、v组相比,dv组nf-kbp65基因表达水平降低(P0.05)。4、Western blot检测显示:M组小鼠肺组织中HDAC2蛋白水平明显比对照组小鼠降低(均P0.05),而维生素D和地塞米松治疗后HDAC水平显著升高,地塞米松组较维生素D组水平更高(均P0.05)。结论:1、OVA诱导哮喘小鼠肺组织NF-kB p65表达水平升高,HDAC2蛋白水平降低。2、1,25(OH)_2D_3能抑制哮喘小鼠肺组织中Th2型细胞因子的分泌,下调NF-kB p65mRNA水平,并能显著增加HDAC2蛋白表达和酶活性。3、1,25(OH)_2D_3和地塞米松在抑制细胞因子释放和NF-kB p65的表达,增加HDAC2的表达和活性方面具有协同效应。4、1,25(OH)_2D_3可能成为一种治疗哮喘的新型HDAC2活化剂。
[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.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

【参考文献】