【作者】 张继伟；

【导师】 胡清华；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 第一部分细胞外钙敏感受体在缺氧诱导肺动脉平滑肌细胞胞浆钙浓度增加中的作用和机制目的：研究细胞外钙敏感受体(extracellular calcium-sensing receptor, CaSR)在缺氧诱导肺动脉平滑肌细胞胞浆内钙离子浓度([Ca2+]i)增加中的作用及机制。方法：原代培养SD大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells, PASMCs);免疫组织化学染色和western blot检测PASMCs上CaSR的表达情况；用不同浓度的细胞外Ca2+处理PASMCs检测CaSR功能；用钙离子荧光探针Fura-2/AM和荧光成像系统检测[Ca2+]i;用H2-DCFDA和mito-RoGFP检测细胞内活性氧(reactive oxygen species, ROS)生成；shRNA基因技术干扰CaSR、STIM1和RyR3的表达,并用western blot检测干扰效率。结果：(1)免疫组织化学染色和western blot均检测到PASMCs细胞膜和胞浆CaSR表达,western blot结果显示细胞膜和胞浆中CaSR的分子量分别为55-72kD和170kD左右。(2)用4、6、7.5mM细胞外Ca2+处理PASMCs,可诱导ERKl/2磷酸化水平逐渐增强,同时6mM和7.5mM细胞外Ca2+又可以引起[Ca2+]i显著增高,而此效应可被CaSR特异性阻滞剂Calhex231所抑制。(3)缺氧可诱导PASMCs ROS产生增加,用外源性H202校正并模拟,缺氧诱导ROS产生相当于15.6μM H2O2诱导ROS的产生。(4)缺氧以及15.6μM H2O2均能引起PASMCs [Ca24]i升高,同时Calhex231和shRNA-CaSR能抑制缺氧以及15.6μM H2O2引起的[Ca2+]i升高。(5)线粒体DNA剔除不仅抑制缺氧引起的ROS生成增多,也抑制缺氧引起的[Ca2+]i增加,这种效应能被外源性15.6μM H2O2所逆转,Calhex231和shRNA-CaSR又能够阻止H202对上述反应的逆转作用。同时PEG-catalase预处理PASMCs能够抑制缺氧引起的[Ca2+]i增加,而PEG-SOD预处理则无此作用。(5)100μM ryanodine抑制RyR功能,可以减弱缺氧和15.6μM H2O2引起的[Ca2+]i升高,但是IP3R的抑制剂20μM XeC却无此作用；若同时应用PKA抑制剂H-89,则100μM ryanodine不再抑制缺氧和H202引起的[Ca2+]i增加,20μM XeC却可以抑制此反应。储库操纵性钙离子进入(store-operated Ca2+entry, SOCE)抑制剂50μM SKF96365可显著抑制缺氧和15.6μM H2O2引起的[Ca2+]i升高。此外,STIM1和RyR3基因敲除也可减弱缺氧和H202引起的[Ca2+]i升高。结论：缺氧刺激引起PASMCs线粒体源性H202产生增加,增加的H202可以使CaSR敏感性增强,从而被细胞外Ca2+激活,进而引起RyR敏感性钙通道开放,钙库Ca2+释放,并激活SOCE,通过STIM1介导细胞外Ca2+内流,共同造成[Ca2+]i升高。第二部分细胞外钙敏感受体在离体和在体缺氧性肺血管收缩中的作用目的：探讨CaSR在离体和在体缺氧性肺血管收缩(hypoxic pulmonary vasoconstriction, HPV)中的关键作用。方法：慢病毒感染SD大鼠,下调CaSR蛋白表达；Power lab四导仪监测离体肺动脉张力及大鼠在不同条件下肺动脉压和心输出量。结果：(1)将慢病毒包装的CaSR特异性干扰质粒,从SD大鼠尾静脉注射72小时后,western blot检测CaSR蛋白表达水平明显下降。(2)离体实验中,缺氧和15.6μM H2O2均能引起肺血管张力增加,同时Calhex231能抑制缺氧以及15.6μM H2O2引起肺血管张力增加。此外,shRNA-CaSR可以抑制缺氧引起的肺血管张力增加。(3)在体实验中,缺氧可以引起SD大鼠肺动脉压力明显增加,同时Calhex231和shRNA-CaSR能抑制缺氧刺激引起的肺动脉压力增加。结论：CaSR介导缺氧诱导肺动脉平滑肌细胞[Ca2+]i的升高,最终导致缺氧性肺血管收缩和肺动脉高压。更多还原

【Abstract】 Part1The role and mechanism of the extracellular calcium-sensing receptor in hypoxia-induced cytoplasmic calcium concentration increase in pulmonary arterial smooth muscle cellsObjective:To study the role and mechanism of the extracellular calcium-sensing receptor (CaSR) in hypoxia induced [Ca2+]i increase in pulmonary artery smooth muscle cells (PASMCs).Methods:Primary culture of SD rat PASMCs were used in this study. The expression of CaSR in PASMCs was detected with immunohistochemistry and western blot. The function of CaSR was detected by using different concentrations of extracellular Ca2+to handle the PASMCs. The calcium fluorescence probe Fura-2/AM and fluorescence imaging system were used to detect [Ca2+]i change. H2-DCFDA and mito-RoGFP were used to detect the generation of intracellular reactive oxygen species (ROS). ShRNA gene technology was used to knock down the CaSR, STIM1and RyR3expression, and the interference efficiency was detected with western blot at the same time.Results:(1) Both immunohistochemistry and western blot have detected CaSR expression in PASMCs, and its distribution is reflected in within the cell membrane and the cytoplasm with molecular weights of55-72kD and170kD.(2) By using4,6and7.5mM extracellular Ca2+to handle PASMCs, the phosphorylation level of ERK1/2is enhanced significantly and gradually, then6mM and7.5mM extracellular Ca2+can induce [Ca2+]i significant increase, while this effect is inhibited by Calhex231(CaSR inhibitor).(3) With different concentrations of exogenous H2O2and hypoxia to stimulate PASMCs, and the varying levels of ROS were detected, it is found that15.6μM H2O2and hypoxia-induced ROS change has the same effect.(4) Both hypoxia and15.6μM H2O2can cause PASMCs [Ca2+]i rise, and Calhex231and CaSR gene knockdown can inhibit hypoxia and15.6μM H2O2-induced [Ca2+]i rise.(5)Mitochondrial DNA depletion can not only inhibit hypoxia-induced ROS generation, but also eliminate the hypoxia-induced [Ca2+]i increase, this effect can be reversed by exogenous15.6μM H2O2, and Calhex231and CaSR gene knockout are able to stop this reversal effect. PEG-Catalase pretreatment the PASMCs is able to inhibit hypoxia-induced [Ca2+]i increase, but PEG-SOD pretreatment does not have this effect.(5)100μM ryanodine inhibiting RyR function can reduce hypoxia and15.6μM H2O2caused [Ca2+]; rise, and IP3R inhibitor20μM XeC can be failed to reduce hypoxia and15.6μM H2O2caused [Ca2+]i rise, but if the application of PKA inhibitor H-89is able to make100μM ryanodine and20μM XeC produce opposite effect at the same time. SOCE inhibitor50μM SKF96365can inhibit hypoxia and15.6μM H2O2induced [Ca2+]i rise significant. STIM1and RyR3knockout can also reduce hypoxia and15.6μM H2O2induced [Ca2+]i rise.Conclusion:Hypoxia induced the production of mitochondria-derived H2O2increase in the PASMCs, the increased H2O2can sensitize CaSR, and CaSR activated by extracellular Ca2+, which caused the cytoplasm of Ca2+from intracellular calcium stores release by RyR, inducing SOCE opening and extracellular Ca2+inflow through STIM1, the combined action has induced [Ca2+]i rise in the PASMC. Part2The role of the extracellular calcium-sensing receptor in hypoxic pulmonary vasoconstriction in vitro and vivoObjective:To explore the key role of CaSR in hypoxic pulmonary vasoconstriction (HPV) in vitro and vivo.Methods:The lentiviral was used to infect SD rats, aiming to knock down the expression of CaSR protein. The Power lab four conductivity meters was used to monitor the tension of pulmonary artery and rat pulmonary artery pressure and cardiac output under different conditions. Results:(1) The lentiviral packaging of CaSR specific interference plasmid was injected into SD rat from tail vein,72hours later, western blot was used to detect the expression level CaSR protein, the expression level was significantly decreased.(2) In vitro experiments, both hypoxia and15.6μM H2O2can cause increased pulmonary vascular tone, the same time Calhex231can inhibit hypoxia and15.6μM H2O2cause increase in pulmonary vascular tone. In addition, the CaSR gene knockdown also can eliminate the increase in hypoxia-induced pulmonary vascular tone.(3) In vivo experiments, hypoxia can cause SD rat pulmonary artery pressure increased significantly, while Calhex231and CaSR knockout can inhibit hypoxia-induced increase in pulmonary artery pressure.Conclusion:The CaSR has involved in hypoxia-induced [Ca2+]i rise in pulmonary arterial smooth muscle cells, which resulted in hypoxic pulmonary vasoconstriction and pulmonary hypertension.更多还原