本文選題：哮喘 + 人抗原R��； 參考：《山東大學》2017年碩士論文

【摘要】：研究背景目前全球患有哮喘人數超過3億,其患病率與死亡率猛速增加,尋求有效的治療手段刻不容緩,而最根本的治療突破點離不開機制的探索。慢性炎癥和氣道高反應是哮喘發(fā)病的主要機理,近年來關于氣道重塑及控制氣道重塑的藥物研發(fā)迎來了高峰。人抗原R(Human antigen R,HuR)是一種RNA結合蛋白,主要通過與目的基因的3'UTR結合發(fā)揮功能,包括COX-2、CyclinD、VEGF、p53等。研究HuR與TGF-β1對哮喘氣道重塑發(fā)生發(fā)展的機制是本課題的核心,旨在為哮喘控制方面提供新的方向。在本研究中,Western印記、PCR、免疫熒光等發(fā)現在PDGF刺激下,氣道平滑肌細胞HuR、TGF-β1、α-SMA和COI-I表達呈時間依賴性。利用RNA干擾技術阻斷HuR表達后,TGF-β1、α-SMA和COI-I表達降低。然而,利用RNA干擾技術降低TGF-β1表達,HuR、COI-I表達也明顯降低,并且額外增加TGF-β1刺激,HuR和COI-I表達可部分恢復。綜合以上分析,我們猜測在氣道平滑肌細胞內可能存在HuR/TGF-β1的反饋調節(jié),且此反饋調節(jié)對哮喘狀態(tài)下氣道重塑的發(fā)生發(fā)展有影響。分別用OVA和PBS刺激BALB/c小鼠以構建哮喘小鼠和對照。研究發(fā)現,OVA誘導的哮喘小鼠,HE染色發(fā)現小鼠肺實質及氣道周圍大量炎癥細胞的聚集,Western印記、RT-PCR及免疫組織化學技術均證實在OVA誘導的哮喘小鼠,肺組織內高表達HuR、TGF-β 1,從而在小鼠體內證實了 HuR、TGF-β 1對氣道重塑的作用。目的1.采用Western印記、RT-PCR、免疫組織化學等檢測氣道平滑肌細胞內HuR、TGF-β1等的表達。2.siRNA干擾HuR,檢測ASM形態(tài)學及功能學改變。此外,利用特異siRNA干擾TGF-β1表達或者增加外源性TGF-[β1刺激ASM,檢測HuR、α-SMA和膠原酶Ⅰ的表達,探討HuR及TGF-β1對氣道重塑的作用。3.探討哮喘小鼠體內HuR與TGF-β1對氣道重塑的作用。方法1.PDGF(20μg/L)分別刺激氣道平滑肌細胞0、6、12和24h。分別檢測各時間點細胞內相關指標RNA水平及蛋白表達水平。RNA干擾技術分別阻斷HuR、TGF-β1表達,Western印跡檢測干擾效率,探討HuR與TGF-β1之間的作用。2.為探討哮喘小鼠氣道重塑發(fā)生與HuR/TGF反饋調節(jié)的作用,BALB/c小鼠分別用卵蛋白(OVA)和PBS致敏,收集肺泡灌洗液檢測炎性細胞。切取小鼠肺組織采用Western印記、RT-PCR和免疫組織化學檢測HuR、TGF-β1、α-SMA和膠原酶Ⅰ的表達。結果1.PDGF分別在0、6、12和24h刺激培養(yǎng)的氣道平滑肌細胞,與Oh相比,細胞內TGF-β1 RNA水平分別增加了 27%,60%和87%(P0.05),蛋白表達分別增加了 31%,69%和106%(P0.05),α-SMA和膠原酶Ⅰ表達也隨時間依遞增。表明哮喘狀態(tài)下TGF-β1、α-SMA和膠原酶Ⅰ呈時間依賴性表達。2.干擾HuR表達,干擾組細胞凋亡率(3.315%)較對照組(1.236%)增加(P0.05),HuR可能參與調控ASM形態(tài)學改變。干擾組細胞內及細胞上清中TGF-β1、α-SMA和膠原酶Ⅰ表達較對照組顯著降低(P0.05)。進一步給予平滑肌細胞actinomycinD刺激,發(fā)現干擾組TGF-β1 mRNA穩(wěn)定性較對照組明顯降低(P0.05)。因此我們推測HuR主要通過增加TGF-β1 mRNA穩(wěn)定性從而調控TGF-β1表達并進而影響α-SMA和膠原酶Ⅰ的表達。3.siRNA干擾TGF-β1后,CCK8檢測發(fā)現干擾組細胞增殖明顯降低,細胞內TGF-β1表達較對照組也降低了 23.8%,然而增加外源性TGF-β1(4ng/ml)刺激,HuR和COI-I表達恢復,說明TGF-β1參與調控HuR與COI-I的表達。4.OVA組小鼠肺組織內炎性反應較PBS組更顯著,OVA組誘導的小鼠BALF內細胞總數 7.56 ± 1.53 × 106/ml,其他細胞如 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 刺激組小鼠 BALF 內細胞總數 3.69±0.98×106/ml,相應細胞所占的比例依次:68.88±2.73%,6.25±0.95%,18.12±1.58%和3.02±0.63%。(P0.05)。Sirius Red Staining 發(fā)現 OVA 組小鼠肺間質內彌漫Ⅰ型和Ⅲ型膠原沉積。5.PCR、Western印記聯合免疫組織化學共同顯示OVA組小鼠肺組織內高表達HuR、TGF-β1等,在小鼠進一步證實了 HuR與TGF-β1對氣道重塑的作用。結論1.HuR與TGF-β1共同調控α-SMA和COI-I的表達,從而影響氣道重塑。2.HuR、TGF-β1和α-SMA在哮喘小鼠肺組織內高表達,肺間質內存在大量Ⅰ型和Ⅲ型膠原沉積。
[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.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R562.25

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