CUHK electronic theses & dissertations collection

The airway epithelium plays a central role in respiratory physiology through its transport and immunological functions. Our previous study suggested that P2Y receptors are expressed in airway epithelia and play a significant role in regulating transepithelial ion transport. P2Y receptors belong to the family of purinergic receptors, which can be stimulated by nucleotides such as UTP and UDP. P2Y receptors are G protein-coupled receptors and classically signal through G[subscript q], resulting in an increase in intracellular Ca²⁺ concentration ([Ca²⁺]ᵢ) and thereby in the activation of Ca²⁺-dependent ion channels and downstream signalling pathway(s). Furthermore, P2Y receptors are involved in asthmatic inflammation.Estrogen (or E₂) is an important hormone in human physiology. In addition to the classical nuclear hormone receptors ERα and ERβ, a novel estrogen receptor, G protein-coupled estrogen receptor (GPER), was recently identified and found to be involved in both rapid signalling and transcriptional regulations. The action of GPER is unclear, but it has been implicated in mediating anti-inflammatory responses.In our experiments, both human bronchial epithelial cell line, 16HBE14o-, and primary normal human bronchial epithelial cells expressed GPER at mRNA and protein levels, as demonstrated by RT-PCR and western blotting, respectively. ERα and ERβ expression were also detected at mRNA and protein level. Expression of GPER receptors was localized in the human bronchial epithelial cells by immunofluorescence staining and western blotting of fractionated cell lysates.[Ca²⁺]ᵢ induced by nucleotides were monitored by calcium imaging technique using MetaFluor fluorescence ratio imaging system. Stimulation of epithelial cells with E₂ or with the specific agonist of GPER, G1, rapidly attenuated a UDP-, UTP- or ATPyS- evoked increase in [Ca²⁺]ᵢ in both 16HBE14o- cell line and primary cells. This inhibitory effect of E₂ and G1 were concentration dependent, while this effect was reversed by GPER specific antagonist, G15. To examine the effect of E₂ and G1 on UDP-activated intracellular Ca²⁺ release and influx, the epithelia were exposed to nominally Ca²⁺ -free solution in the presence or absence of G1 or E₂, and then stimulated with UDP. Subsequently, Ca²⁺ was added to the perfusate. Both E₂ and G1 could inhibit UDP-induced Ca²⁺ release. However, only E₂ but not G1 could inhibit UDP-induced Ca²⁺ influx.E₂ or G1 inhibited the secretion of two pro-inflammatory cytokines, interleukin (IL)-6 or IL-8, in cells stimulated by different nucleotides or the cationic protein, poly-L-arginie, as quantified by ELISA. CFP-Epac-YPF, an Epac-based polypeptide FRET reporter was used to monitor the real-time cAMP changes in 16HBE14o- cells. Both G1 and E₂ induced an increase in cAMP production. The transepithelial chloride (Cl⁻) secretion was measured using short circuit current technique in cells grown on permeable support. Cl⁻ secretion induced by apical UDP was partially inhibited by G1 in a concentration dependent manner.Our results provide the first evidence that human bronchial epithelia express GPER, which interact with the P2Y receptor-mediated calcium signalling pathway and cytokine secretion. Moreover, the anti-inflammatory role of GPER may be due to its opposing effect on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.气道上皮具有调节运输以及参与免疫反应等功能，在呼吸生理学研究中有着十分关键的意义。我们曾经的研究发现P2Y受体在气道上皮中表达并调节上皮细胞离子运输过程。P2Y受体属于嘌呤受体，可被三磷酸尿苷（UTP），二磷酸尿苷（UDP）等核苷酸激活。同时，P2Y受体也是一类G蛋白偶联受体，可通过活化G[subscript q]蛋白调控细胞内钙离子浓度而激活钙依赖性离子通道及其他下游信号通路。此外P2Y受体还参与哮喘炎症的调控。雌激素（或雌二醇，E₂）是人体一类十分重要的激素。除传统的核受体ERα与ERβ外，一类新型雌激素受体GPER已被发现和鉴定。GPER属于G蛋白偶联受体，可同时参与转录调控和非基因依赖的快速信号调节。尽管具体机理尚不明确，但研究发现GPER可介导抗炎症反应。实验结果显示，在支气管上皮细胞株16HBE14o-和原代人支气管上皮细胞中GPER都被检测到基因和蛋白水平的表达。GPER在人支气管上皮细胞中的定位也通过免疫荧光染色（immunofluorescence）和亚细胞组分蛋白质印迹（western blot of fractionated cells）得到鉴定。本研究中，荧光显微技术（fluorescence microscopy）被用于测定核苷酸介导的细胞内钙离子浓度（[Ca²⁺]ᵢ）。在16HBE14o- 和原代培养人支气管上皮细胞中，E₂和GPER特异性激动剂G1都可抑制核苷酸介导的 [Ca²⁺]ᵢ增加，且这种抑制作用呈浓度依赖。GPER特异性拮抗剂G15可抵消G1的抑制作用。进一步研究表明，E₂和G1都可抑制UTP诱导的胞内钙库释放，然而只有E₂抑制UTP诱导的胞外钙离子内流。除钙离子调节外，E₂和G1还可抑制支气管上皮细胞中核苷酸或聚精氨酸（poly-L-arginine）刺激介导的两种促炎症细胞因子，白介素6（IL-6）和白介素8（IL-8）的分泌。酶联免疫法（ELISA）被用于细胞因子的定量。同时，CFP-Epac-YPF作为一类多肽荧光共振能量转移（FRET）探针被转染入16HBE14o- ，探测细胞内腺苷-3',5'-环化一磷酸（cAMP）的实时变化。结果显示在人支气管上皮细胞中E₂和G1都可引导cAMP生成。此外，我们使用短路电流（short-circuit current, Isc）技术测定单层上皮细胞的氯离子（Cl⁻）分泌，并发现人支气管上皮顶膜面UDP诱导的Cl⁻ 分泌可被G1部分抑制，且抑制效果呈浓度依赖。本研究首次证明GPER表达于人支气管上皮, 且激活GPER对P2Y受体介导的钙离子信号通路以及细胞因子生成起到抑制作用。这些结果表明在气道炎症反应中，GPER可通过反向调节P2Y受体激活的促炎症作用，达到抗炎症的效果。Hao, Yuan.Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.Includes bibliographical references (leaves 187-211).Abstracts also in Chinese.Title from PDF title page (viewed on 03, November, 2016).Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only