TRPV通道在慢性低氧性肺动脉平滑肌细胞中的功能性表达,Functional Expression of TRPV Channels in Chronically Hypoxic Human Pulmonary Arterial Smooth Muscle Cells,首都医科大学 呼吸生理与病理生理研究室 首都医科大学 附属北京朝阳医院 王跃秀 王军 王辰,2,一、研究背景,肺动脉平滑肌细胞(PASMC)的增殖是低氧性肺动脉高压(hypoxic pulmonary hypertension , HPH)发生发展过程中的重要病理生理学改变，其与平滑肌细胞内钙离子密切相关。,3,细胞内钙稳态 Calcium Homeostasis,Ca 2+cyt,SR,VDCC,ROC,SOC,受体操纵性通道 ROC receptor-operated channel,电压依赖性钙通道 voltage-depended calcium channel,钙库操控性通道 store-operated channel, SOC,4,在PASMC上VDCC并不发达，其阻断剂不能完全阻止钙离子内流； 在离体情况下，阻断ROC的功能后，对慢性低氧诱导的PASMC的高Ca2浓度并没有明显作用。 低氧性肺动脉高压的PASMC细胞中，Ca2浓度增高不能完全被VDCC的阻断剂所阻断，但能被SOC的阻断剂明显抑制。,Remillard CV, Yuan JX. TRP channels, CCE, and the pulmonary vascular smooth muscle. Microcirculation. 2006,13:671-692.Cioffi DL, Wu S, Stevens T. Transient receptor potential cation channels and store-operated Ca channels. Boca Raton, FL: Taylor & Francis Group. 2005, 125-146.Landsberg JW, Yuan JX. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci . 2004, 19:44-50 .,5,SOC可能是低氧时肺动脉平滑肌细胞调节钙离子跨膜转运的重要途径。,目前研究认为：瞬时受体电位( transient receptor potential, TRP) 蛋白家族中的成员组成了SOC。,6,TRP通道的分类,TRPV,TRPC,TRPM,TRPP,TRPML,TRPA,TRPN,TRPV (Vanilloid receptor related TRP) Subtype : TRPV1-6,7,Firth AL, Remillard CV, Yuan JX. TRP channels in hypertension. Biochim Biophys Acta (2007) 1772:895-906,TRP通道的结构,TRP通道均为六次跨膜蛋白，其N末端和C末端均在胞内，由第五和第六跨膜结构域共同构成非选择性阳离子孔道。,8,大鼠的肺动脉和主动脉平滑肌上有TRPV通道家族成员(TRPV1-4)的分布; 在低氧下，人肺动脉内皮细胞中有TRPV1，TRPV2，TRPV4通道的表达; TRPV1在大鼠PASMCs表达并通过介导细胞外Ca2+内流而提高细胞内Ca2+水平。,Yang XR, Lin MJ, McIntosh LS, et al. Am J Physiol Lung Cell Mol Physiol. 2006, 290:L1267-1276. Fantozzi I, Zhang S, Platoshyn O, et al. Am J Physiol Lung Cell Mol Physiol. 2003, 285:L1233-1245. Minke B . Cell Calcium 2006, 40:261-275,TRPV通道在肺血管的研究,目前在人肺动脉平滑肌细胞上TRPV通道的研究未见报道,9,通过 RT-PCR, Real-time PCR检测 TRPV通道家族成员（TRPV1-6）的表达,人肺动脉平滑肌细胞 HPASMC,RT-PCR, Real-time PCR,细胞 计数,细胞内钙测定,低氧肺动脉高压平滑肌细胞模型的建立 3%O2, 72h,Western blot analysis,流氏细胞周期测定,二、技术路线,10,通过鉴别TRPV通道家族成员在人肺动脉平滑肌细胞中的表达以及观察低氧条件对其通道表达与功能的影响, 探讨TRPV通道在低氧性肺动脉平滑肌细胞增殖中的作用。 为进一步阐明低氧性肺动脉高压中平滑肌细胞增殖的机制以及寻找药物治疗的靶点提供实验依据。,三、研究目的及意义,11,Cell Culture. 三气恒温培养箱（Thermo） Hypoxic treatment. 三气恒温培养箱（Thermo） Conventional RT-PCR. PCR仪，凝胶成像系统（Bio-Rad） Real-time PCR. MX3000P Real-Time PCR（Stratagene） Western blot Analysis . 电泳仪（Bio-Rad） Cell count. Flow cytometry. 流式细胞仪EPICS XL（Coulter） Measurement of Intracellular Ca 2+ . 落射荧光显微镜（Nikon），DG4（Nikon）,四、研究方法,12,五、结果,RT-PCR,1、TRPV通道家族成员在人肺动脉平滑肌细胞上的表达,13,Real-time RT-PCR analysis of the relative expression of TRPVs mRNA in human PASMCs. n=6,Real-time PCR,五、结果,1、TRPV通道家族成员在人肺动脉平滑肌细胞上的表达,14,五、结果,2、TRPV通道家族成员在低氧性肺动脉平滑肌细胞上的表达,RT-PCR,15,Real-time RT-PCR analysis of the relative expression of TRPV14 mRNA in human PASMCs under normoxia (N) and hypoxia (HP). Values are mean SEM, n = 6, * P 0.05 vs. N.,五、结果,2、TRPV通道家族成员在低氧性肺动脉平滑肌细胞上的表达,Real-time PCR,TRPV1,TRPV2,TRPV3,TRPV4,0,50,100,150,200,250,300,350,400,450,N,HP,*,Relative Quanitity (dRn),16,Hypoxia increased the expression of TRPV1 protein in human PASMCs. (A) Western blot analyses of TRPV1 channel proteins in human PASMCs cultured in normoxia and hypoxia for 72 h. The molecular masses of TRPV1 and Actin are 90 and 43 kDa, respectively. (B) Summarized data showing the protein levels for TRPV1 normalized to the amount of Actin in human PASMCs cultured during normoxia and hypoxia for 72 h. The results are presented as the mean SEM, n=6, * P0.05 vs.N.,五、结果,2、TRPV通道家族成员在低氧性肺动脉平滑肌细胞上的表达,Western blot,17,五、结果,3、低氧对肺动脉平滑肌细胞增殖能力的影响,The capability of human PASMCs proliferation was significantly increased under hypoxic (HP) compared with normoxia condition (N). The results are presented as the mean SEM, n=20, *P0.001 vs.N,Cell count,18,Capsazepine inhibited hypoxia-induced human PASMCs proliferation in a dose-dependent manner. (A) Human PASMCs were treated for 72 h with SMBM (0.5% FBS) in the presence of 0 , 0.1 , 1 , 10 , and 100M capsazepine or methanol (meth, 100 M) under hypoxia. The results are presented as the mean SEM, n = 8, * P 0.001 vs. 0 group. (B) Dose-response curves of the effect of capsazepine on the growth of human PASMCs under hypoxia.,3、低氧对肺动脉平滑肌细胞增殖能力的影响,五、结果,Cell count,19,The effect of Capsazepine (1M) on cell cycle progression of S Phase. *P0.05 vs. N, P 0.05 vs. HP.,五、结果,3、低氧对肺动脉平滑肌细胞增殖能力的影响,Flow cytometry,20,五、结果,4、人肺动脉平滑肌细胞内钙水平测定,N=45,N=42,N=45,21,小结,22,TRPV1可能是通过SOC增加细胞内钙浓度而参与低氧性人肺动脉平滑肌细胞的增殖作用。,1. 第三届全国肺血管病学术会议暨首届国际肺循环病研讨会 （摘要） 2. 瞬时感受器电位通道在呼吸系统的表达与功能. 国际呼吸杂志，2008,283:72-375. 3. Functional expression of transient receptor potential vanilloid -related channels in chronically hypoxic human pulmonary arterial smooth muscle cells. Journal of Membrane Biology （Accepted）,六、结论,23,北京市教委基金(KM200710025002),本研究获得以下基金资助,教育部科学计数研究重点项目(208002),北京市自然基金(7082012),国家“十一五”科技支撑项目 (2006BAI01A06),24,Thank you!,