【作者】 俞延芳；

【导师】 张连生；

【作者基本信息】 兰州大学 ， 血液病学， 2011， 硕士

【摘要】 P2Y14是一种抑制环磷酸腺苷(cyclic AMP, cAMP)合成的Gi (inhibitory adenylate cyclaes g protein, Gi)蛋白偶联受体,能够被尿嘧啶核苷-5’-二磷酸葡萄糖(UDP-glucose, UDPG), UDP一半乳糖(UDP-galactose)和UDP-N乙酰葡糖胺(UDP-N-acetylglucosamine)激活。最近的研究表明,P2Y14受体参与T淋巴细胞免疫功能的调节以及气道上皮中促炎因子白细胞介素-8(IL-8)的合成。同时,在凝血酶诱导的星型胶质细胞中观察到UDP-Glc的胞外释放,这表明在炎症的发生过程中P2Y14受体可能被激活。目的本研究旨在通过探讨P2Y14受体在人肺微血管内皮细胞(Human Lung Microvascular Endothelial Cells, HLMVEC)上的功能,从而揭示其在呼吸系统疾病及血液系统疾病中的发生与发展中的潜在机制。方法通过RT-PCR和免疫印记法(Western blot)来证实P2Y14受体在HLMVEC内的表达。同时,环磷酸腺苷(cyclic AMP, cAMP)实验表明P2Y14的特异性底物UDP-glucose可以抑制腺苷酸环化酶(adenylate cyclase, AC)特异性激活剂毛喉素(forskolin)诱导的cAMP产物的增加。而在HLMVEC细胞信号转导的研究中发现,给予P2Y14的特异性底物UDP-glucose处理后,激活了Raf-1, MEK-1/2和ERK1/2信号转导通路。并且,通过电子细胞基质阻抗判断(Electrical cell-substrate impedance sensing, ECIS)的方法来观察给予HLMVEC UDP-Glc预处理后对内皮细胞创伤愈合的促进作用。结果1.P2Y14受体在HLMVEC内高表达。2.P2Y14的特异性底物UDP-glucose可以抑制腺苷酸环化酶特异性激活剂毛喉素(forskolin)诱导的cAMP产物的增加。3.在HLMVEC细胞信号转导的研究中发现,给予P2Y14的特异性底物UDP-glucose处理后,激活了Raf-1, MEK-1/2和ERK1/2信号转导通路。4.通过电.子细胞基质阻抗判断(Electrical cell-substrate impedance sensing,ECIS)的方法观察到对HLMVEC给予P2Y14的特异性底物UDP-Glc (100μM,15min)预处理后激活了ERK1/2信号转导通路,并对内皮细胞的创伤愈合发挥促进作用。结论1.P2Y14受体广泛表达于胃,胎盘,脂肪组织,心脏以及各种免疫细胞中,本研究首次证实了其在肺血管内皮细胞中的功能性表达。这对在其他组织器官中发现P2Y14受体的表达以及功能提供了一个新的方向。2.P2Y14的特异性底物UDP-glucose可抑制forskolin诱导的cAMP产物的增加。而在肿瘤细胞中普遍存在着cAMP含量下降的现象。因此,P2Y14受体可能通过调节cAMP的含量来进一步影响肿瘤细胞的生长,分化。3.P2Y14的特异性底物UDP-glucose能够激活Raf-1, MEK-1/2和ERK1/2信号转导通路,从而促进细胞从由G0/G1期进入S期并影响细胞增殖、分化、凋亡等一系列生物学效应。4.血管内皮细胞的损伤及功能紊乱与多种疾病的发生密切相关。而P2Y14的特异性底物UDP-Glc通过激活ERK1/2信号转导通路,从而促进内皮细胞的创伤修复。更多还原

【Abstract】 The P2Y14 is a Gi-coupled receptor that inhibits the synthesis of cAMP, and is activated by UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine. Recent studies indicate the role of P2Y14 in modulating the immune function in T-lymphocytes and secretion of the proinflammatory cytokine IL-8 in airway epithelial cells. Furthermore, extracellular release of UDP-glucose was observed in thrombin-treated astrocytes suggesting that P2Y14 may be activated in inflammation.AIMThe aim of this study was to identify the functional expression of P2Y14 receptor in HLMVEC, and thus further demonstrated the mechanism of P2Y14 receptor in the occurrence and progressive of respiratory and blood diseases.METHODSWe used RT-PCR and western blot to investigate the mRNA and protein expression of P2Y14 receptor in the HLMVEC. The cAMP assay was used to identify the function of P2Y14 receptor under the P2Y14-specific substrate, UDP glucose, treatment in HLMVEC. Signaling studies show treatment of HLMVEC with UDP-glucose activates the Raf-1/MEK-1/ERKl/2 pathway. The ECIS (Electrical cell-substrate impedance sensing) was used to check the permeability of HLMVEC with UDP-glucose treatment.RESULTS1. Our data indicate for the first time that P2Y14 is expressed in endothelial cells (HLMVEC) as evidenced by RT-PCR, Western blot.2. The cAMP assay indicates that P2Y14 is functional in HLMVEC as an addition of the P2Y14-specific substrate, UDPglucose, inhibited the forskolin-induced cAMP production.3. Signaling studies show treatment of HLMVEC with UDP-glucose activates the Raf-1/MEK-1/ERK1/2 pathway. 4. The ECIS (Electrical cell-substrate impedance sensing) data indicate that pretreatment of HLMVEC with UDP-glucose (100μM,15min) stimulated wound healing suggesting the participation of P2Y14 in endothelial repair after acute lung injury.CONCLUSIONS1. P2Y14 receptor is widely expressed in stomach, placenta, adipose tissue, lung, heart and many kinds of immunocells. Our data indicate for the first time that P2Y14 is expressed in endothelial cells (HLMVEC). This result will benefit the other functional and expressional studies of P2Y14 receptor in many kinds of other cells and tissues.2. The cAMP assay indicates that P2Y14 is functional in HLMVEC as an addition of the P2Y14-specific substrate, UDPglucose, inhibited the forskolin-induced cAMP production. Therefore, tumor cell growth and differentiation may be regulated by P2Y14 receptor through the up- or down-regulation of cAMP in the cells.3. The treatment of HLMVEC with UDP-glucose activates the Raf-1/MEK-1/ERK1/2 pathway, and thus can promote the cells go from G0/G1 to S cycle and further affect the cell proliferation, differentiation and apoptosis and so on.4. The wound and function disorder of endothelial cells are close related many kinds of diseases. The pretreatment of HLMVEC with UDP-glucose stimulated wound healing suggesting the participation of P2Y14 in endothelial repair after acute injury.更多还原