经典型瞬时感受器电位通道在鼻息肉中的作用及机制研究

发布时间：2018-05-10 22:12

本文选题：经典型瞬时感受器电位通道 + 鼻息肉　；参考：《山东大学》2013年博士论文

【摘要】：背景鼻息肉(nasal polyps)是上呼吸道常见疾病,发病率约为1-2%。现有的研究发现鼻息肉的病理生理基础是重度鼻粘膜组织水肿和慢性炎症反应,但其具体机制至今仍未阐明。近年来人们发现细胞内钙离子在免疫细胞的活化、增殖、分泌细胞因子和脱颗粒反应等功能中起关键作用,但是其具体调节机制尚未清楚。经典型瞬时感受器电位(canonical transient receptor potential, TRPC)通道是果蝇TRP蛋白的哺乳动物同系物。TRPC通道能被IP3激活而产生持续的钙内流。研究发现TRPC通道表达于中性粒细胞、嗜酸性细胞和T淋巴细胞等免疫细胞,介导免疫细胞钙库操纵性钙内流机制；而在TRPC6敲基因鼠研究发现TRPC6的缺失减少了由卵清蛋白激发的支气管嗜酸性细胞数量。然而TRPC通道在鼻息肉发生发展中的的作用及分子机制尚未深入研究。因此我们提出如下假说：鼻息肉组织中TRPC通道以TRPC5通道表达增高为主,高表达的TRPC5通道可能通过激活NF-κB信号转导通路增加嗜酸性粒细胞浸润和致炎介质的增加,从而在鼻息肉的发病机制中发挥重要作用。目的 1.观察鼻息肉组织中经典型瞬时型感受器电位通道家族表达情况； 2.探讨经典型瞬时型感受器电位通道与鼻息肉嗜酸性粒细胞浸润和炎症反应的关系； 3.探讨经典型瞬时型感受器电位通道与核转录因子NF-κB的关系。方法 1.鼻息肉患者的入选及标本收集：从2011年5月至2011年12月在山东大学齐鲁医院耳鼻咽喉科随机收集行息肉切除术患者58例和接受鼻中隔偏曲矫正术的患者的下鼻甲黏膜35例做正常对照。 2.应用实时荧光定量逆转录聚合酶链反应(real time RT-PCR)检测鼻息肉患者组织中经典型瞬时型感受器电位通道mRNA表达水平。 3.应用病理组织学和免疫组织化学技术检测鼻息肉组织标本中经典型瞬时型感受器电位通道的分布和嗜酸性粒细胞数量,分析它们之间的关系。 4.应用蛋白免疫印迹(Western Blot)技术检测鼻息肉患者组织中经典型瞬时型感受器电位通道的蛋白表达、炎症因子IL-6和核转录因子NF-κB表达,分析他们之间的关系。结果 2.1一般临床资料分析鼻息肉患者和鼻中隔偏曲患者在年龄和性别方面无显著性差异(P＞0.05)。 2.2TRPC通道mRNA和蛋白表达水平鼻.息肉组织中TRPC5通道mRNA表达水平明显高于正常对照组(P＜0.05),其他成员表达变化不明显。鼻息肉组织TRPC5通道蛋白表达水平明显高于对照组(P＜0.05)。 2.3鼻息肉组织中嗜酸性粒细胞数量嗜酸性粒细胞在HE染色成明显的嗜伊红特性,显示为胞浆内呈反光强的鲜红色。鼻息肉组织中嗜酸性粒细胞数量明显高于正常鼻甲粘膜(P＜0.01)。 2.4鼻息肉组织IL-6的蛋白表达水平鼻息肉患者IL-6蛋白表达水平明显高于正常鼻甲粘膜(P＜0.05)。免疫组化显示鼻息肉组织中IL-6蛋白阳性产物主要位于细胞浆和细胞间质,阳性区域所占面积也明显增高(P＜0.05)。 2.5鼻息肉组织NF-κB的蛋白表达水平 Western blot结果显示：鼻息肉组织NF-κB的非活性形式p65表达量与正常鼻甲粘膜无显著差别(P＞0.05),但是NF-κB的活性形式磷酸化水平的p65(p-p65)表达量比正常鼻黏膜显著增高(P＜0.05)。 2.6TRPC通道表达量与鼻息肉组织中各项指标的相关分析鼻息肉组织中TRPC5通道表达水平与嗜酸性粒细胞数量、致炎因子IL-6表达量和NF-κB的活性形式p-p65表达量成正相关(P＜0.01)。结论 1.鼻息肉组织中TRPC5通道表达增高； 2.高表达的TRPC5通道可能通过激活NF-κB信号转导通路增加嗜酸性粒细胞浸润和IL-6的表达,从而在鼻息肉的发病机制中发挥重要作用。背景鼻息肉(nasal polyps)是多种致病因素共同作用的结果,其中以变态反应和鼻粘膜的慢性炎症为其病理生理基础。许多炎症因子、化学介质和趋化因子参与,其中IgE作为主要的介质在嗜酸性粒细胞激活和脱颗粒过程中起关键性作用。本研究第一部分研究发现：鼻息肉组织中TRPC通道以TRPC5通道表达增高为主,高表达的TRPC5通道可影响鼻息肉中嗜酸性粒细胞浸润和IL-6,也可影响核转录因子NF-κB的活性,但是对于其在鼻息肉中的作用机制尚不明确。有研究发现鼻息肉患者外周血和鼻息肉组织中IgE水平增高,IgE可以刺激鼻息肉组织产生更多的组胺、白三烯类、PGD2等早期反应介质,导致鼻黏膜水肿和慢性炎症反应。也有研究发现IgE可以调节多种离子通道和细胞内钙离子浓度。最近TRPC5通道可以稳定表达于HEK293细胞,为研究TRPC5通道的功能调控机制提供了简单可行的手段。因此我们推测鼻息肉组织中IgE表达增高,高表达IgE引起细胞内钙离子和钙库依赖的钙内流,而TRPC5通道是IgE作用的途径,这为揭示TRPC5通道在鼻息肉中的作用机制提供了新的理论依据。目的 1.研究鼻息肉组织中IgE表达改变； 2.体外表达系统中TRPC5通道的功能； 3.探讨TRPC5通道是否介导了IgE诱导的细胞内钙离子调控。方法 1.应用免疫组织化学技术检测鼻息肉组织标本中IgE的表达情况。 2.稳定表达TRPC5通道的HEK293细胞系(HEK-TRPC5)的培养：培养条件：DMEM-F12,含10%胎牛血清、50u/ml青霉素和0.5mg/ml链霉素,37℃、5%CO2培养箱中。应用5μig/ml blasticidin和400μg/ml zeocin筛选。由于其携带tetracycline (Te)转录抑制子,需要添加1μg/ml Te刺激24h来诱导TRPC5通道表达。 2.钙离子成像：Ca2+荧光标记物Fura-2AM(2uM)37℃孵育1h,固定发射波长510nm,固定激发波长在340nm和380nm作双波长时间扫描,应用TILL Vision软件包采集图像,进行细胞内钙成像,340nm和380nm时荧光信号比值反映了钙离子的浓度。记录不同浓度IgE对稳定表达的TRPC5通道介导的钙离子内流和钙库依赖的钙内流的作用。 3.膜片钳电生理记录：室温下,将细胞培养在盖玻片上,置于灌流槽内,应用EPC-10膜片钳放大器和Pulse软件进行刺激脉冲发放和数据记录；采用全细胞膜片钳技术记录TRPC5通道的电流强度的改变及IgE10μg/ml对TRPC5通道电流的作用。结果 1.鼻息肉中IgE蛋白表达：与正常粘膜比较,免疫组织化学染色显示鼻息肉中IgE蛋白阳性区域所占面积明显增高(P＜0.01)。 2TRPC5通道介导的细胞内钙离子增加：在Te诱导的细胞(Te+),钙离子成像显示TRPC5通道激活剂Gd3+(100μM)可以诱导细胞内钙离子的增加(P＜0.05),这种反应可以被TRPC通道特异阻断剂T5E3Ab (50μg/ml)抑制。 3TRPC5通道介导的细胞内钙库依赖性钙内流：在Te诱导的细胞(Te+),首先在无钙外液中用Tg耗竭细胞内钙库,然后将液体换成含2mM Ca2+的标准细胞浴液(复钙),可以引起明显的钙离子内流,这种现象即钙库依赖性钙内流。而在Te未诱导的细胞(Te-),产生小幅度的钙库依赖性钙内流,比Te+细胞明显低。证明这种钙库依赖性钙内流是过表达的TRPC5通道介导的。 4.IgE对TRPC5通道介导的细胞内钙离子增加的影响：在Te诱导的细胞(Te+),钙离子成像显示不同浓度IgE(0.1μg/ml、1μg/ml、10μg/ml)可以诱导细胞内钙离子的增加(P＜0.05),这种反应可以被TRPC通道特异阻断剂T5E3Ab (50μg/ml)抑制(P＜0.05)。 5.IgE对TRPC5通道介导的细胞内钙库依赖性钙内流的影响：在Te诱导的细胞(Te+),Tg耗竭细胞内钙库后再复钙可以引起明显的钙离子内流(P＜0.05)。与未加IgE刺激的细胞比较,10μg/ml IgE预刺激30min的细胞,钙库依赖性钙内流明显增加(P＜0.05)。 6.IgE对TRPC5通道电流强度的影响：膜片钳记录显示TRPC5激活剂Gd3+(100μM)可以使稳定表达TRPC5通道的HEK293细胞产生明显的TRPC5通道电流(P＜0.05),10μg/ml IgE也可诱导TRPC5通道电流,TRPC5通道特异性阻断剂T5E3(50μg/ml)可以抑制IgE引起的TRPC5通道电流。结论 1.鼻息肉组织中IgE增高； 2.增高的IgE可以激活TRPC5通道产生细胞内钙离子增加和增强钙库依赖性钙内流,这可能是TRPC5通道在鼻息肉发生和发展中的作用机制。
[Abstract]:background
Nasal polyps (nasal polyps) are common diseases of the upper respiratory tract. The incidence of the disease is about 1-2%.. It is found that the pathophysiological basis of nasal polyps is severe nasal mucous tissue edema and chronic inflammatory response, but its specific mechanism has not been elucidated.
In recent years, the intracellular calcium ions have been found to play a key role in the activation, proliferation, secretion of cytokine and degranulation of immune cells, but the specific regulation mechanism is not clear. The canonical transient receptor potential (TRPC) channel is a mammal homologue of the TRP protein of Drosophila melanogaster The.TRPC channel can be activated by IP3 to produce continuous calcium influx. It was found that the TRPC channel was expressed in neutrophils, eosinophils and T lymphocytes, which mediate the mechanism of calcium influx in the immune cell calcium library. In the TRPC6 knockout mouse study, the deletion of TRPC6 decreased the eosinophilia stimulated by ovalbumin. However, the role of TRPC channel in the development of nasal polyps and its molecular mechanism have not yet been studied. Therefore, we suggest that the TRPC channel in nasal polyps is increased mainly by TRPC5 channel, and the highly expressed TRPC5 channel may increase eosinophil infiltration and cause by activating the NF- kappa B signal transduction pathway. Increased inflammatory mediators play an important role in the pathogenesis of nasal polyps.
objective
1. to observe the expression of classical transient receptor potential pathway in nasal polyps.
2. to explore the relationship between the classical transient receptor potential channel and eosinophil infiltration and inflammatory response in nasal polyps.
3. to explore the relationship between classical transient receptor potential channel and nuclear transcription factor NF- kappa B.
Method
1. patients with nasal polyps were selected and collected: from May 2011 to December 2011, 35 cases of inferior turbinate mucosa of 58 patients with random collection of polyposis in the Department of Otolaryngology, Qilu Hospital, Shandong University, and 35 cases of inferior turbinate mucous membrane were used as normal controls.
2. real time RT-PCR (RT time RT-PCR) was used to detect the expression level of the classical transient receptor potential channel in the tissues of patients with nasal polyps.
3. the distribution of potential channels and the number of eosinophils in the specimens of nasal polyps were detected by histopathological and immunohistochemical techniques, and the relationship between them was analyzed.
4. the protein expression of the classical transient receptor potential channel in the tissues of patients with nasal polyps, the expression of inflammatory factor IL-6 and nuclear factor NF- kappa B were detected by Western Blot, and the relationship between them was analyzed.
Result
2.1 general clinical data analysis
There was no significant difference in age and sex between patients with nasal polyps and deviated nasal septum (P > 0.05).
2.2TRPC channel mRNA and protein expression level
The expression level of TRPC5 channel mRNA in nasal polyps was significantly higher than that in the normal control group (P < 0.05), and the expression of other members was not obvious. The expression level of TRPC5 channel protein in nasal polyps was significantly higher than that of the control group (P < 0.05).
The number of eosinophils in 2.3 nasal polyps
Eosinophils stained with HE showed eosinophilic characteristics, showing strong reflection in the cytoplasm.
The number of eosinophils in nasal polyps was significantly higher than that in normal nasal mucosa (P < 0.01).
Protein expression level of IL-6 in 2.4 nasal polyps
The expression level of IL-6 protein in nasal polyps was significantly higher than that in normal nasal mucosa (P < 0.05).
Histochemical analysis showed that the positive products of IL-6 protein in nasal polyps were mainly located in cytoplasm and intercellular substance.
The area is also significantly increased (P < 0.05).
Protein expression level of NF- kappa B in 2.5 nasal polyps
The results of Western blot showed that the non active p65 expression of NF- kappa B in nasal polyps was not significantly different from that of normal turbinate mucosa (P > 0.05), but the expression of p65 (p-p65) in the activity form of NF- kappa B was significantly higher than that of normal nasal mucosa (P < 0.05).
Correlation analysis between 2.6TRPC channel expression and indicators in nasal polyps
The expression of TRPC5 channel in nasal polyps was positively correlated with the number of eosinophils, the expression of inflammatory factor IL-6 and the p-p65 expression of the active form of NF- kappa B (P < 0.01).
conclusion
The expression of TRPC5 channel was increased in 1. nasal polyps.
2. the high expression of TRPC5 channel may increase eosinophil infiltration and IL-6 expression by activating the NF- kappa B signal transduction pathway, thus playing an important role in the pathogenesis of nasal polyps.
background
Nasal polyps (nasal polyps) are the result of a variety of pathogenic factors, in which the allergic reaction and chronic inflammation of the nasal mucosa are the basis of its pathophysiology. Many inflammatory factors, chemical mediators and chemokines are involved, in which IgE plays a key role in the process of eosinophil activation and degranulation. The first part of the study found that the TRPC channel in nasal polyps is mainly expressed in TRPC5 channel, and the high expression of TRPC5 channel can affect eosinophil infiltration and IL-6 in nasal polyps, and also affect the activity of NF- kappa B in the nuclear transcription factor, but the mechanism of its role in nasal polyps is not clear.
It has been found that the levels of IgE in peripheral blood and nasal polyps in patients with nasal polyps are increased. IgE can stimulate nasal polyps to produce more histamine, leukotrienes, PGD2 and other early reaction mediators, leading to nasal edema and chronic inflammatory response. There are also studies found that IgE can regulate multiple ionic channels and intracellular calcium concentrations. Recently, TRPC The 5 channel can be expressed steadily in HEK293 cells and provides a simple and feasible means to study the functional regulation mechanism of the TRPC5 channel. Therefore, we speculate that the expression of IgE in the nasal polyps is higher and the high expression of IgE causes intracellular calcium and calcium library dependent calcium influx, while the TRPC5 channel is the pathway of IgE action, which is to reveal the TRPC5 channel in the nose. The mechanism of action in meat provides a new theoretical basis.
objective
1. the changes of IgE expression in nasal polyps were studied.
2. the function of TRPC5 channel in the in vitro expression system;
3. to explore whether TRPC5 channels mediate IgE induced intracellular calcium regulation.
Method
1. immunohistochemical staining was used to detect the expression of IgE in nasal polyps.
2. the culture of HEK293 cell line (HEK-TRPC5) that expresses the TRPC5 channel steadily: culture conditions: DMEM-F12, 10% fetal bovine serum, 50u/ml penicillin and 0.5mg/ml streptomycin, 37 C, 5%CO2 culture box. 5 micron blasticidin and 400 micron g/ml zeocin are used. As it carries the transcriptional inhibitor, it needs to add 1 mu to stimulate 24. H is used to induce TRPC5 channel expression.
2. calcium ion imaging: Ca2+ fluorescent labeling Fura-2AM (2uM) was incubated for 1h, fixed emission wavelength 510nm, fixed excitation wavelength in 340nm and 380nm for dual wavelength scanning, using TILL Vision software package to collect images, intracellular calcium imaging, 340nm and 380nm when the ratio of fluorescence signals reflected the concentration of calcium ions. Record different concentrations IgE pairs. Stable expression of TRPC5 channels mediates calcium influx and calcium dependent calcium influx.
The electrophysiological record of 3. patch clamp: at room temperature, the cells were cultured on the cover glass, placed in the irrigation slot, using the EPC-10 patch clamp amplifier and the Pulse software to carry out the stimulation pulse and data recording, and the whole cell patch clamp technique was used to record the changes of the current intensity of the TRPC5 channel and the effect of IgE10 mu g/ml on the TRPC5 channel current.
Result
The expression of IgE protein in 1. nasal polyps: compared with normal mucous membrane, immunohistochemical staining showed that the area of IgE protein positive area in nasal polyps was significantly increased (P < 0.01).
2TRPC5 channel mediated intracellular calcium increased: in Te induced cells (Te+), calcium ion imaging showed that TRPC5 channel activator Gd3+ (100 u M) could induce intracellular calcium increase (P < 0.05). This reaction could be inhibited by T5E3Ab (50 mu g/ ml) of TRPC channel specific blocker.
3TRPC5 channel mediated intracellular calcium library dependent calcium influx: in the Te induced cell (Te+), the calcium Library in the Tg exhausted cells in the calcium free fluid, and then the liquid into a standard cell bath containing 2mM Ca2+ (calcium compound), can cause obvious calcium ion inflow. This phenomenon is calcium library dependent calcium influx. In Te uninduced cells (Te-) a small calcium dependent calcium influx was found, which was significantly lower than that of Te+ cells. It is demonstrated that this calcium dependent calcium influx is mediated by an over expressed TRPC5 channel.
The effect of 4.IgE on intracellular calcium ion increase mediated by TRPC5 channel: in Te induced cells (Te+), calcium ion imaging showed that different concentrations of IgE (0.1 mu g/ml, 1 mu g/ml, 10 u g/ml) could induce intracellular calcium increase (P < 0.05). This reaction could be suppressed by TRPC channel specific blocker T5E3Ab (50 micron g/ml).
The effect of 5.IgE on intracellular calcium dependent calcium influx mediated by TRPC5 channel: in Te induced cells (Te+), and in Tg depleted cells after calcium library, re calcium could cause significant calcium influx (P < 0.05). Compared with those without IgE stimulation, 10 mu g/ml IgE prestimulated 30min cells, and calcium library dependent calcium influx increased significantly (P < 0.05).
The effect of 6.IgE on the current intensity of the TRPC5 channel: the patch clamp recording shows that the TRPC5 activator Gd3+ (100 mu M) can produce a clear TRPC5 channel current (P < 0.05) for the HEK293 cells that stably express the TRPC5 channel (P < 0.05), and the 10 mu g/ml IgE can also induce the TRPC5 channel current. Channel current.
conclusion
The IgE increased in 1. nasal polyps.
2. increased IgE can activate TRPC5 channel to produce intracellular calcium increase and increase calcium library dependent calcium influx, which may be the mechanism of TRPC5 channel in the occurrence and development of nasal polyps.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R765.25

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