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
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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。
Background
Nasal polyps (NP) are a common disease of the upper airway; the incidence rate of NP is about1%-2%. The main pathological changes were:severe edema of loose connective tissue and chronic inflammation, however its pathogenesis has not been clarified.
Recent studies have found that intracellular calcium plays a key role in immune cell activation, proliferation, secretion of cytokines and degranulation reaction function. However, its mechanism is not yet clear. TRPC channels are mammalian homologue proteins to transient receptor potential (TRP) of the Drosophila. TRPC can be activated by IP3and induce continuous calcium influx. Previous studies found TRPC channels were expressed in immune cells such as, neutrophils, eosinophils, and T lymphocytes, which mediated immune cell store-operated calcium influx. TRPC6deficiency inhibited specific allergic immune responses and eosinophil infiltration. However, the role of TRPC channels in nasal polyps development and mechanism are not in-depth study. So we put forward the following hypothesis:the expression of TRPC5channel in nasal polyps are increased, TRPC5channel might play an important role in the development of NP through the activation of NF-KB signal transduction pathway, increase of eosinophil infiltration and inflammatory cytokines.
Objectives
1. To assess the expression of classical transient receptor potential channel family in patients with nasal polyps;
2. To study the relationships between TRPC channel expression and eosinophil infiltration and inflammatory reaction in nasal polyps;
3. To study the relationship between TRPC channel expression and nuclear transcription factor NF-κB in nasal polyp.
Methods
1. The inclusion of patients with nasal polyps and specimen collection：From2011May to2011December in Department of Otolaryngology, Qilu Hospital of Shandong University, we randomly selected58patients with nasal polyps to undergo polypectomy and35patients who received correction of the deviation of nasal septum. Inferior turbinate mucosa specimens were considered as normal control.
2. Real-time RT-PCR was used to detect mRNA expression of TRPC channels in patients with nasal polyps.
3. Histopathology and immunohistochemistry techniques were used to detect distribution of TRPC channel, IL-6, eosinophil number and nuclear transcriptionfactor NF-κB in nasal polyp tissues.
4. Western blot was used to detect protein expression of TRPC channel, inflammatory factor IL-6and nuclear transcriptionfactor NF-κB in nasal polyp tissues. Analyze the relationship between them.
Results
1. Analysis of clinical data Nasal polyps patients and control groups did not differ in age and gender.
2. mRNA and protein expressions of TRPC channel in nasal polyps
TRPC5channel mRNA expression level was significantly higher than that of control group (P<0.01). Other members of TRPC were not obviously changed (P>0.05). Compared with normal mucusa, TRPC5channel protein expression was significantly increased in NP (P<0.01).
3. Esinophils infiltration analysis
Eosinophils showed obviously eosinophilic cytoplasm and a strong reflection of bright red granules in HE staining. Compared with normal mucusa, eosinophil number was significantly raised in NP tissues (P<0.01).
4. Protein expression of IL-6
Compared with normal mucusa, IL-6protein expression was significantly increased in nasal polyps (P<0.01). Immunohistochemistry showed that IL-6positive products were mainly located in the cytoplasm and intercellular substance, with buffy granules stain.
5. NF-κB expression
Werstern blot results also confirmed the expression of protein p65tion and normal control group no difference (P>0.05), but the phosphorylation level of p65expression was significantly higher than that in normal mucusa (P<0.01).
6. Correlation analysis between expressions of TRPC channel with characteristics in nasal polyps
TRPC5channel expression was positively correlated with eosinophil infiltration, inflammatory factor and the phosphorylation level of p65(P<0.01).
Conclusions
1. TRPC5channel is upregulated in nasal polyps;
2. TRPC5channel plays an important role in the development of nasal polyps through the activation of NF-κB signal transduction pathway, increase of eosinophil infiltration and inflammatory cytokines.
Background
Many factors participated in the pathogenesis of nasal polyps(NP). The patho-physiological basis is allergic reaction and chronic inflammation of nasal mucosa. There are a lot of inflammatory cytokines, chemical medium and chemokines in NP, including IgE, which plays a key role in the eosinophil activation and degranulation. We found that TRPC5channel was upregulated in NP and that TRPC5channel could affect eosinophil infiltration, inflammatory cytokines and activation of NF-κB signal transduction pathway. However, the mechanism of canonical transient receptor potential channel5in NP is unclear.
IgE levels are increased in peripheral blood and tissues in NP. IgE can stimulate the nasal polyps to produce more histamine, leukotrienes, PGD2, resulting in nasal mucosa edema and inflammatory reaction. Other studies have found that IgE can regulate the intracellular calcium concentration and ion channels. Recently, TRPC5channels can be stably expressed in HEK293cells, which provides a simple and feasible method to study the function and regulatory mechanism of TRPC5channel. So we hypothesized that expression of IgE was increased in nasal polyps, and that IgE induced intracellular calcium increasing and store operated calcium influx, which was medieated by TRPC5channel.
Objectives
1. To assess IgE expression in nasal polyps;
2. To study the function of TRPC5channel expressed in vitro;
3. To explore whether calcium regulation in cells induced by IgE is mediated by TRPC5channels.
Methods
1. Detect the expression of IgE in nasal polyp tissue specimens by immunohistochemical technique.
2. TRPC5stably expressed HEK293cells (HEK-TRPC5):Cells was incubulated in DMED-F12supplemented with10%fetal bovine serum,100μg/ml streptomycin,100U/ml penicillin and1.5%sodium bicarbonate at37℃,5%CO2and95%air. TRPC5expression was induced by1μg/ml tetracycline (Te), for HEK-TRPC5cells carried a tetracycline transcriptional repressor.
3. Calcium imaging:Cells was incubated with Ca2+fluorescent marker Fura-2AM (2μM) at37℃for1h. The fixed emission wavelength was510nm, time series of fluoresce were recorded by a dual wavelength scanning in340nm and380nm with TILL Vision image acquisition software. Fluorescent signal ratio between340nm and380nm reflects the concentration of intracellular calcium. To study the effect of different concentrations of IgE on intracellular calcium concentration mediated by TRPC5.
3The patch clamp electrophysiological recording:Cells were cultured on glass coverslips and placed in the perfusion chamber at room temperature. The recordings were made by the application of EPC-10patch clamp amplifier and Pulse software. TRPC5channel currents were recorded by whole-cell mode of patch clamp recording. To study the effect of10μg/ml IgE on TRPC5channel currents.
Results
1. Expression of IgE in NP:Compared with normal mucusa, IgE protein expression was significantly increased in nasal polyps (P<0.01).
2. Intracellular calcium increase mediated by TRPC5channel:in Te induced cells (Te+), calcium imaging showed that TRPC5channel activator Gd3+(100μM) increased intracellular calcium (P<0.05), which could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μg/ml).
3. Store-operated calcium influx mediated by TRPC5channel:In Te induced cells (Te+), thapsigargin (Tg) was used to deplete intracellular calcium stores in calcium-free solutions, and then the bath solution was changed to standard bath solution with2mM Ca2+, resulting in significantly intracellular calcium increasing which phenomenon was called store-operated calcium influx. In Te-cells, there was only a slighter store-operated calcium influx
4. Effect of IgE on intracellular calcium increase mediated by TRPC5channel:In the Te induced cells (Te+), calcium imaging showed that different concentrations of IgE (0.1μg/ml,1μg/ml,10μg/ml) increased intracellular calcium (P<0.05), which could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μ g/ml).
5. Effect of IgE on store-operated calcium influx mediated by TRPC5channel:In Te induced cells (Te+), there was store-operated calcium influx which could be strentherned by pre-stimulation with10μg/ml IgE for30min (P<0.05).
6. Effect of IgE on TRPC5channel currents:In the Te induced cells (Te+), patch clamp recordings showed that TRPC5channel activator Gd3+(100μM) induced TRPC5channel currents (P<0.05) and that10μg/ml IgE also induced channel currents (P<0.05), which both could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μg/ml).
Conclusions
1. IgE expression was increased in nasal polyps.
2. Increased IgE could activate TRPC5channels resulting intracellular calcium increase and store-operated calcium influx enhancement.
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。
Background
Nasal polyps (NP) are a common disease of the upper airway; the incidence rate of NP is about1%-2%. The main pathological changes were:severe edema of loose connective tissue and chronic inflammation, however its pathogenesis has not been clarified.
Recent studies have found that intracellular calcium plays a key role in immune cell activation, proliferation, secretion of cytokines and degranulation reaction function. However, its mechanism is not yet clear. TRPC channels are mammalian homologue proteins to transient receptor potential (TRP) of the Drosophila. TRPC can be activated by IP3and induce continuous calcium influx. Previous studies found TRPC channels were expressed in immune cells such as, neutrophils, eosinophils, and T lymphocytes, which mediated immune cell store-operated calcium influx. TRPC6deficiency inhibited specific allergic immune responses and eosinophil infiltration. However, the role of TRPC channels in nasal polyps development and mechanism are not in-depth study. So we put forward the following hypothesis:the expression of TRPC5channel in nasal polyps are increased, TRPC5channel might play an important role in the development of NP through the activation of NF-KB signal transduction pathway, increase of eosinophil infiltration and inflammatory cytokines.
Objectives
1. To assess the expression of classical transient receptor potential channel family in patients with nasal polyps;
2. To study the relationships between TRPC channel expression and eosinophil infiltration and inflammatory reaction in nasal polyps;
3. To study the relationship between TRPC channel expression and nuclear transcription factor NF-κB in nasal polyp.
Methods
1. The inclusion of patients with nasal polyps and specimen collection：From2011May to2011December in Department of Otolaryngology, Qilu Hospital of Shandong University, we randomly selected58patients with nasal polyps to undergo polypectomy and35patients who received correction of the deviation of nasal septum. Inferior turbinate mucosa specimens were considered as normal control.
2. Real-time RT-PCR was used to detect mRNA expression of TRPC channels in patients with nasal polyps.
3. Histopathology and immunohistochemistry techniques were used to detect distribution of TRPC channel, IL-6, eosinophil number and nuclear transcriptionfactor NF-κB in nasal polyp tissues.
4. Western blot was used to detect protein expression of TRPC channel, inflammatory factor IL-6and nuclear transcriptionfactor NF-κB in nasal polyp tissues. Analyze the relationship between them.
Results
1. Analysis of clinical data Nasal polyps patients and control groups did not differ in age and gender.
2. mRNA and protein expressions of TRPC channel in nasal polyps
TRPC5channel mRNA expression level was significantly higher than that of control group (P<0.01). Other members of TRPC were not obviously changed (P>0.05). Compared with normal mucusa, TRPC5channel protein expression was significantly increased in NP (P<0.01).
3. Esinophils infiltration analysis
Eosinophils showed obviously eosinophilic cytoplasm and a strong reflection of bright red granules in HE staining. Compared with normal mucusa, eosinophil number was significantly raised in NP tissues (P<0.01).
4. Protein expression of IL-6
Compared with normal mucusa, IL-6protein expression was significantly increased in nasal polyps (P<0.01). Immunohistochemistry showed that IL-6positive products were mainly located in the cytoplasm and intercellular substance, with buffy granules stain.
5. NF-κB expression
Werstern blot results also confirmed the expression of protein p65tion and normal control group no difference (P>0.05), but the phosphorylation level of p65expression was significantly higher than that in normal mucusa (P<0.01).
6. Correlation analysis between expressions of TRPC channel with characteristics in nasal polyps
TRPC5channel expression was positively correlated with eosinophil infiltration, inflammatory factor and the phosphorylation level of p65(P<0.01).
Conclusions
1. TRPC5channel is upregulated in nasal polyps;
2. TRPC5channel plays an important role in the development of nasal polyps through the activation of NF-κB signal transduction pathway, increase of eosinophil infiltration and inflammatory cytokines.
Background
Many factors participated in the pathogenesis of nasal polyps(NP). The patho-physiological basis is allergic reaction and chronic inflammation of nasal mucosa. There are a lot of inflammatory cytokines, chemical medium and chemokines in NP, including IgE, which plays a key role in the eosinophil activation and degranulation. We found that TRPC5channel was upregulated in NP and that TRPC5channel could affect eosinophil infiltration, inflammatory cytokines and activation of NF-κB signal transduction pathway. However, the mechanism of canonical transient receptor potential channel5in NP is unclear.
IgE levels are increased in peripheral blood and tissues in NP. IgE can stimulate the nasal polyps to produce more histamine, leukotrienes, PGD2, resulting in nasal mucosa edema and inflammatory reaction. Other studies have found that IgE can regulate the intracellular calcium concentration and ion channels. Recently, TRPC5channels can be stably expressed in HEK293cells, which provides a simple and feasible method to study the function and regulatory mechanism of TRPC5channel. So we hypothesized that expression of IgE was increased in nasal polyps, and that IgE induced intracellular calcium increasing and store operated calcium influx, which was medieated by TRPC5channel.
Objectives
1. To assess IgE expression in nasal polyps;
2. To study the function of TRPC5channel expressed in vitro;
3. To explore whether calcium regulation in cells induced by IgE is mediated by TRPC5channels.
Methods
1. Detect the expression of IgE in nasal polyp tissue specimens by immunohistochemical technique.
2. TRPC5stably expressed HEK293cells (HEK-TRPC5):Cells was incubulated in DMED-F12supplemented with10%fetal bovine serum,100μg/ml streptomycin,100U/ml penicillin and1.5%sodium bicarbonate at37℃,5%CO2and95%air. TRPC5expression was induced by1μg/ml tetracycline (Te), for HEK-TRPC5cells carried a tetracycline transcriptional repressor.
3. Calcium imaging:Cells was incubated with Ca2+fluorescent marker Fura-2AM (2μM) at37℃for1h. The fixed emission wavelength was510nm, time series of fluoresce were recorded by a dual wavelength scanning in340nm and380nm with TILL Vision image acquisition software. Fluorescent signal ratio between340nm and380nm reflects the concentration of intracellular calcium. To study the effect of different concentrations of IgE on intracellular calcium concentration mediated by TRPC5.
3The patch clamp electrophysiological recording:Cells were cultured on glass coverslips and placed in the perfusion chamber at room temperature. The recordings were made by the application of EPC-10patch clamp amplifier and Pulse software. TRPC5channel currents were recorded by whole-cell mode of patch clamp recording. To study the effect of10μg/ml IgE on TRPC5channel currents.
Results
1. Expression of IgE in NP:Compared with normal mucusa, IgE protein expression was significantly increased in nasal polyps (P<0.01).
2. Intracellular calcium increase mediated by TRPC5channel:in Te induced cells (Te+), calcium imaging showed that TRPC5channel activator Gd3+(100μM) increased intracellular calcium (P<0.05), which could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μg/ml).
3. Store-operated calcium influx mediated by TRPC5channel:In Te induced cells (Te+), thapsigargin (Tg) was used to deplete intracellular calcium stores in calcium-free solutions, and then the bath solution was changed to standard bath solution with2mM Ca2+, resulting in significantly intracellular calcium increasing which phenomenon was called store-operated calcium influx. In Te-cells, there was only a slighter store-operated calcium influx
4. Effect of IgE on intracellular calcium increase mediated by TRPC5channel:In the Te induced cells (Te+), calcium imaging showed that different concentrations of IgE (0.1μg/ml,1μg/ml,10μg/ml) increased intracellular calcium (P<0.05), which could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μ g/ml).
5. Effect of IgE on store-operated calcium influx mediated by TRPC5channel:In Te induced cells (Te+), there was store-operated calcium influx which could be strentherned by pre-stimulation with10μg/ml IgE for30min (P<0.05).
6. Effect of IgE on TRPC5channel currents:In the Te induced cells (Te+), patch clamp recordings showed that TRPC5channel activator Gd3+(100μM) induced TRPC5channel currents (P<0.05) and that10μg/ml IgE also induced channel currents (P<0.05), which both could be blocked by TRPC5channel specific inhibitor T5E3Ab (50μg/ml).
Conclusions
1. IgE expression was increased in nasal polyps.
2. Increased IgE could activate TRPC5channels resulting intracellular calcium increase and store-operated calcium influx enhancement.
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