人抗原R与转化生长因子β1之间的反馈调节对哮喘气道重塑发生发展的作用

发布时间：2018-05-03 23:24

本文选题：哮喘 + 人抗原R　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景目前全球患有哮喘人数超过3亿,其患病率与死亡率猛速增加,寻求有效的治疗手段刻不容缓,而最根本的治疗突破点离不开机制的探索。慢性炎症和气道高反应是哮喘发病的主要机理,近年来关于气道重塑及控制气道重塑的药物研发迎来了高峰。人抗原R(Human antigen R,HuR)是一种RNA结合蛋白,主要通过与目的基因的3'UTR结合发挥功能,包括COX-2、CyclinD、VEGF、p53等。研究HuR与TGF-β1对哮喘气道重塑发生发展的机制是本课题的核心,旨在为哮喘控制方面提供新的方向。在本研究中,Western印记、PCR、免疫荧光等发现在PDGF刺激下,气道平滑肌细胞HuR、TGF-β1、α-SMA和COI-I表达呈时间依赖性。利用RNA干扰技术阻断HuR表达后,TGF-β1、α-SMA和COI-I表达降低。然而,利用RNA干扰技术降低TGF-β1表达,HuR、COI-I表达也明显降低,并且额外增加TGF-β1刺激,HuR和COI-I表达可部分恢复。综合以上分析,我们猜测在气道平滑肌细胞内可能存在HuR/TGF-β1的反馈调节,且此反馈调节对哮喘状态下气道重塑的发生发展有影响。分别用OVA和PBS刺激BALB/c小鼠以构建哮喘小鼠和对照。研究发现,OVA诱导的哮喘小鼠,HE染色发现小鼠肺实质及气道周围大量炎症细胞的聚集,Western印记、RT-PCR及免疫组织化学技术均证实在OVA诱导的哮喘小鼠,肺组织内高表达HuR、TGF-β 1,从而在小鼠体内证实了 HuR、TGF-β 1对气道重塑的作用。目的1.采用Western印记、RT-PCR、免疫组织化学等检测气道平滑肌细胞内HuR、TGF-β1等的表达。2.siRNA干扰HuR,检测ASM形态学及功能学改变。此外,利用特异siRNA干扰TGF-β1表达或者增加外源性TGF-[β1刺激ASM,检测HuR、α-SMA和胶原酶Ⅰ的表达,探讨HuR及TGF-β1对气道重塑的作用。3.探讨哮喘小鼠体内HuR与TGF-β1对气道重塑的作用。方法1.PDGF(20μg/L)分别刺激气道平滑肌细胞0、6、12和24h。分别检测各时间点细胞内相关指标RNA水平及蛋白表达水平。RNA干扰技术分别阻断HuR、TGF-β1表达,Western印迹检测干扰效率,探讨HuR与TGF-β1之间的作用。2.为探讨哮喘小鼠气道重塑发生与HuR/TGF反馈调节的作用,BALB/c小鼠分别用卵蛋白(OVA)和PBS致敏,收集肺泡灌洗液检测炎性细胞。切取小鼠肺组织采用Western印记、RT-PCR和免疫组织化学检测HuR、TGF-β1、α-SMA和胶原酶Ⅰ的表达。结果1.PDGF分别在0、6、12和24h刺激培养的气道平滑肌细胞,与Oh相比,细胞内TGF-β1 RNA水平分别增加了 27%,60%和87%(P0.05),蛋白表达分别增加了 31%,69%和106%(P0.05),α-SMA和胶原酶Ⅰ表达也随时间依递增。表明哮喘状态下TGF-β1、α-SMA和胶原酶Ⅰ呈时间依赖性表达。2.干扰HuR表达,干扰组细胞凋亡率(3.315%)较对照组(1.236%)增加(P0.05),HuR可能参与调控ASM形态学改变。干扰组细胞内及细胞上清中TGF-β1、α-SMA和胶原酶Ⅰ表达较对照组显著降低(P0.05)。进一步给予平滑肌细胞actinomycinD刺激,发现干扰组TGF-β1 mRNA稳定性较对照组明显降低(P0.05)。因此我们推测HuR主要通过增加TGF-β1 mRNA稳定性从而调控TGF-β1表达并进而影响α-SMA和胶原酶Ⅰ的表达。3.siRNA干扰TGF-β1后,CCK8检测发现干扰组细胞增殖明显降低,细胞内TGF-β1表达较对照组也降低了 23.8%,然而增加外源性TGF-β1(4ng/ml)刺激,HuR和COI-I表达恢复,说明TGF-β1参与调控HuR与COI-I的表达。4.OVA组小鼠肺组织内炎性反应较PBS组更显著,OVA组诱导的小鼠BALF内细胞总数 7.56 ± 1.53 × 106/ml,其他细胞如 Macrophages、Eosnophils、Lymphocytes 及 Neutrophils 所占比例依次:28.20 ± 1.95%,35.40 ±2.02%,26.70±2.38%和 7.08±0.74%,PBS 刺激组小鼠 BALF 内细胞总数 3.69±0.98×106/ml,相应细胞所占的比例依次:68.88±2.73%,6.25±0.95%,18.12±1.58%和3.02±0.63%。(P0.05)。Sirius Red Staining 发现 OVA 组小鼠肺间质内弥漫Ⅰ型和Ⅲ型胶原沉积。5.PCR、Western印记联合免疫组织化学共同显示OVA组小鼠肺组织内高表达HuR、TGF-β1等,在小鼠进一步证实了 HuR与TGF-β1对气道重塑的作用。结论1.HuR与TGF-β1共同调控α-SMA和COI-I的表达,从而影响气道重塑。2.HuR、TGF-β1和α-SMA在哮喘小鼠肺组织内高表达,肺间质内存在大量Ⅰ型和Ⅲ型胶原沉积。
[Abstract]:Background the prevalence of asthma in the world is more than 300 million, its morbidity and mortality rate increases rapidly, and it is urgent to seek effective treatment. The most fundamental treatment breakthroughs cannot be separated from the mechanism. Chronic inflammation and airway hyperreaction are the main mechanisms of asthma. In recent years, airway remodeling and airway remodeling are controlled. Human antigen R (Human antigen R (HuR)) is a RNA binding protein, which mainly functions by combining with the 3'UTR of the target gene, including COX-2, CyclinD, VEGF, p53 and so on. The mechanism of the development of the airway remodeling of the asthma and beta 1 is the core of this topic and aims to provide a new direction for the control of asthma. In this study, Western, PCR, and immunofluorescence were found to be time dependent on the expression of HuR, TGF- beta 1, alpha -SMA and COI-I in airway smooth muscle cells under PDGF stimulation. TGF- beta 1, alpha -SMA and COI-I expression decreased after blocking HuR expression by RNA interference. However, the expression of beta 1 was reduced by the interference technique. And additional TGF- beta 1 stimulation, HuR and COI-I expression can be partially restored. We hypothesized that the feedback regulation of HuR/TGF- beta 1 may exist in the airway smooth muscle cells, and this feedback regulation has an effect on the development of airway remodeling in asthma state. BALB/c mice were stimulated by OVA and PBS to construct asthma mice and pairs, respectively. The study found that OVA induced asthma mice, HE staining found a large number of inflammatory cells in the lung parenchyma and around the airway, Western imprint, RT-PCR and immunohistochemical techniques proved OVA induced asthma mice, high expression of HuR, TGF- beta 1 in the lung tissue, which confirmed the effect of HuR and TGF- beta 1 on airway remodeling in mice. Objective 1. to detect the morphological and functional changes of ASM in airway smooth muscle cells by means of Western imprint, RT-PCR, immunohistochemistry and other expressions of HuR, TGF- beta 1, etc., and to detect the morphological and functional changes of ASM. In addition, the expression of HuR, alpha and collagenase I was detected by specific siRNA interference TGF- beta 1 or exogenous TGF-[beta 1 stimulation ASM. The effect of TGF- beta 1 on airway remodeling.3. to explore the effect of HuR and TGF- beta 1 on airway remodeling in asthmatic mice. Methods 1.PDGF (20 mu g/L) respectively stimulated the 0,6,12 and 24h. of airway smooth muscle cells to detect the levels of RNA and protein expression level in each time point respectively, and.RNA interference technique blocked HuR, TGF- beta 1 expressed, respectively. Detection of interference efficiency, the role of.2. between HuR and TGF- beta 1 was used to investigate the effect of.2. on airway remodeling and HuR/TGF feedback in asthmatic mice. BALB/c mice were sensitized with ovalbumin (OVA) and PBS, and pulmonary alveolar lavage fluid was collected to detect inflammatory cells. The lung tissue was cut by Western imprint, RT-PCR and immunohistochemistry were used to detect HuR, TGF. - expression of beta 1, alpha -SMA and collagenase I. Results 1.PDGF stimulated airway smooth muscle cells in 0,6,12 and 24h respectively. Compared with Oh, the level of intracellular TGF- beta 1 RNA increased by 27%, 60% and 87% (P0.05), and protein expression increased by 31%, 69% and 106% (P0.05), and the expression of alpha -SMA and collagenase I was also increasing with time. TGF- beta 1, alpha -SMA and collagenase I expressed.2. interference with HuR expression in a time dependent manner. The apoptosis rate of the interference group increased (3.315%) (P0.05), and HuR might participate in the regulation of the morphological changes of ASM. The expression of TGF- beta 1 in the cell and cell supernatant of the interference group was significantly lower than that in the control group (P0.05). ActinomycinD stimulation of smooth muscle cells found that the stability of TGF- beta 1 mRNA in the interference group was significantly lower than that of the control group (P0.05). Therefore, we speculate that HuR mainly regulates the expression of TGF- beta 1 by increasing the stability of TGF- beta 1 mRNA and then affects the expression of the expression of alpha -SMA and collagenase I after.3.siRNA interference TGF- beta 1. CCK8 detection found the cell proliferation in the interference group. The expression of TGF- beta 1 was decreased by 23.8% compared with the control group, but the expression of exogenous TGF- beta 1 (4ng/ml) was increased and the expression of HuR and COI-I was restored. The expression of TGF- beta 1 involved in the regulation of HuR and COI-I. The inflammatory response in the lung tissue of the.4.OVA group mice was more significant than that in the PBS group. The total number of cells in the OVA group induced by the OVA group was 7.56 + 1.53 * *. The proportion of other cells, such as Macrophages, Eosnophils, Lymphocytes and Neutrophils, were 28.20 + 1.95%, 35.40 + 2.02%, 26.70 + 2.38% and 7.08 + 0.74%. The total number of cells in the PBS stimulation group was 3.69 + 0.98 * 106/ml, and the proportion of the corresponding cells was 68.88 + 2.73%, 6.25 +, and 0.63%. (P0.05).Si. Rius Red Staining found that type I and type III collagen deposition in the lung interstitium of OVA mice was diffuse.5.PCR. Western imprint combined with immunohistochemistry showed the high expression of HuR and TGF- beta 1 in the lung tissue of group OVA mice. The effect of HuR and TGF- beta 1 on airway remodeling was further confirmed in mice. Conclusion 1.HuR and TGF- beta 1 jointly regulate the alpha and airway remodeling. The expression of I, which affects airway remodeling,.2.HuR, TGF- beta 1 and alpha -SMA are highly expressed in lung tissue of asthmatic mice. There are a large number of type I and type III collagen deposits in the lung interstitium.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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