内毒素对肺动脉平滑肌细胞收缩能力的影响及机制研究

发布时间：2018-05-29 22:17

本文选题：脂多糖 + 肺动脉平滑肌细胞　；参考：《复旦大学》2010年博士论文

【摘要】： [目的]败血症休克情况下主要表现为体循环压力的下降和早期肺循环压力的升高,甚至肺动脉高压(pulmonary artery hypertension, PAH)。本实验旨在探讨高浓度的LPS是否导致肺动脉平滑肌细胞(pulmonary artery smooth muscle cell, PASMC)本身收缩能力的变化。[方法]通过急性消化酶分离法获得单个PASMC,在200倍倒置显微镜下,灌流含10μg/ml LPS的生理盐溶液,通过细胞长度的变化、完成收缩所需时间的变化观察LPS对PASMC的直接刺激作用。对LPS预处理的PASMC,分别给予80mM高钾溶液、苯肾上腺素及内皮素-1(endothelin-1, ET-1),观察LPS对PASMC的收缩能力的影响。[结果](1)本实验采取的急性消化酶法获得的PASMC存活率95%,PASMC纯度达97.7±1.78%。(2)10μg/ml的LPS不能直接引起PASMC收缩反应。(3)80mM的高钾等渗溶液可引起PASMC收缩,经10μg/ml LPS预处理10min,PASMC对80mM高钾等渗HBSS溶液的反应更敏感,表现为高钾刺激后2.5min(68.43±1.46 vs47.70±5.70,P0.001)、5min(75.42±0.87 vs 63.45±3.65,P0.01)、10min(80.23±0.57 vs 74.01±2.17,P0.05)时点收缩幅度较对照组明显增大。LPS预处理的PASMC完成50%(1.03±0.10 vs 2.38±0.36,P0.01)及90%(4.04±0.31 vs7.14±0.75,P0.001)总收缩长度所用时间较对照组显著缩短。(4)10μM及lmM的苯肾上腺素均不能引起PASMC收缩；LPS预处理后,10μM的苯肾上腺素也不能引起PASMC收缩。(5)10nM的ET-1作用于PASMC,约lmin左右,PASMC发生迅速而强烈的收缩。经10μg/ml LPS预处理10min,PASMC对10nM的ET-1的收缩反应没有明显的增强或抑制作用(P0.05)。[结论]10μg/ml的LPS不能直接引起PASMC收缩,经LPS预处理后对80mMK诱发的收缩反应表现为增敏作用,而对ET-1诱发的收缩反应无明显影响。 [目的]旨在探讨LPS对肺动脉平滑肌细胞(pulmonary artery smooth muscle cell, PASMC)内钙离子浓度([Ca2+]i)的影响及其与钙库操纵性钙通道(SOC)的活性和表达的关系。[方法]经典瞬时受体电位通道(transient receptor potential canonical, TRPC)被证实是SOC的主要分子基础,通过Real-time PCR检测正常及LPS刺激后SD大鼠肺动脉平滑肌上TRPC1、3、4、5、6 mRNA的表达。通过钙离子荧光探针及共聚焦显微镜技术观察LPS对血管收缩剂引起的PASMC细胞内[Ca2+]i变化、钙释放(calcium release)和钙内流(calcium entry)的影响。通过非选择性SOC阻滞剂SKF-96365、选择性SOC阻滞剂2-APB、电压操纵性钙通道阻滞剂硝苯地平和ET-1的受体阻滞剂BQ-123,观察SOC引起的钙内流在PASMC兴奋活动中的地位。[结果](1)TRPC1、3、4、5、6在肺动脉、颈动脉和尾动脉上均有表达,相互之间无显著性差异,P0.05。10μg/ml LPS孵育10min后,肺动脉上TRPC1(1.21E-05±0.01E-05 vs 3.82E-05±0.46E-05, P0.001)、TRPC3(1.34E-05±0.17E-05 vs 6.29E-05±0.15E-05, P0.001)、TRPC4(1.36E-05±0.15E-05 vs 7.46E-05±0.11E-05, P0.001)的表达显著性升高。(2)10μg/ml LPS刺激后,肺动脉上ETA-R(1.31E-05±0.23E-05 vs 5.26E-05±0.15E-05, P0.01)、ETB-R(1.44E-05±0.10E-05 vs 3.43E-05±0.20E-05, P0.001)的mRNA的表达明显升高；而α1-肾上腺素受体mRNA的表达显著降低(3.16E-05±0.10E-05 vs 0.72E-05±0.29E-05,P0.001)。(3)10μg/ml LPS预处理PASMC10min后,细胞内[Ca2+]i无可检测性变化(0.01±0.01 vs 0.01±0.02,P0.05),80mM高钾溶液(0.95±0.06 vs 1.25±0.10,P0.05)及10nM的ET-1(1.56±0.07 vs 1.98±0.09,P0.05)引起的PASMC细胞内[Ca2+]i增幅均较对照组明显增高。(4)10μg/ml LPS预处理的PASMC钙释放较对照组降低(1.36±0.07 vs1.66±0.06,P0.01),而钙内流较对照组增多(1.32±0.10 vs 1.06±0.07,p0.05)。(5)SOC通道依赖的钙内流在ET-1引起的PASMC激动作用占有重要作用,SOC通道的阻断剂SKF-96365和2-APB分别可阻断73.0%和70.8%的PASMC的激活作用。[结论]LPS预处理的PASMC细胞内[Ca2+]i无可检测性变化,但可使血管收缩剂引起的[Ca2+]i的增幅增大,[Ca2+]i增幅的增大主要来源于钙内流的增加。LPS可能通过上调SOC(即TRPC)通道的表达以及增敏SOC的活性改变PASMC的收缩能力。
[Abstract]:[Objective] the main manifestations of septic shock are the decrease of systemic circulation pressure and the increase of early pulmonary circulation pressure and even pulmonary hypertension (pulmonary artery hypertension, PAH). The aim of this experiment is to investigate whether the high concentration of LPS leads to the contractile ability of the pulmonary artery smooth muscle cells (pulmonary artery smooth muscle cell, PASMC) itself. [method] a single PASMC was obtained by the method of acute digestive enzyme separation. Under the 200 fold inverted microscope, the physiological salt solution containing 10 g/ml LPS was perfused. The direct stimulation effect of LPS on PASMC was observed through the changes of cell length and the time required for the completion of contraction. The PASMC of LPS pretreated, 80mM high potassium solution, benzene adrenaline respectively. The effect of LPS on the contractility of PASMC was observed by LPS and endothelin -1 (ET-1). [results] (1) the survival rate of PASMC obtained by the acute digestive enzyme method was 95%, the PASMC purity of 97.7 + 1.78%. (2) 10 micron LPS could not directly cause the PASMC contraction reaction. (3) the high potassium isosotic solution of 80mM could be contracted by 10 micron. The pretreated 10min, PASMC is more sensitive to the reaction of 80mM high potassium isosotic HBSS solution, which shows 2.5min (68.43 + 1.46 vs47.70 + 5.70, P0.001), 5min (75.42 + 0.87 vs 63.45 + 3.65, P0.01), 10min (80.23 + 0.57 vs 74.01 + 74.01). The duration of the total contraction length of.38 + 0.36, P0.01) and 90% (4.04 + 0.31 vs7.14 + 0.75, P0.001) was significantly shorter than that of the control group. (4) the phenylephrine of 10 mu M and lmM did not cause PASMC contraction; after LPS pretreatment, the phenylephrine of 10 mu did not cause PASMC contraction. (5) 10nM ET-1 acted on about, and was fast and strong. The contraction reaction of ET-1 was not obviously enhanced or inhibited by 10min after 10 g/ml LPS pretreatment (P0.05). [conclusion]10 u g/ml LPS could not directly induce PASMC contraction, and the contraction reaction induced by LPS preconditioning showed a sensitization effect on the contraction reaction induced by the LPS, but had no obvious effect on the contraction reaction induced by the LPS.
[Objective] to investigate the effect of LPS on the intracellular calcium concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (pulmonary artery smooth muscle cell, PASMC) and the relationship with the activity and expression of calcium channel manipulative calcium channel (SOC). [Methods] the classical instantaneous receptor potential (transient receptor potential) is proved to be a The main molecular basis was to detect the expression of TRPC1,3,4,5,6 mRNA on the pulmonary artery smooth muscle of SD rats after normal and LPS stimulation by Real-time PCR. A calcium ion fluorescence probe and confocal microscopy were used to observe the [Ca2+]i changes in PASMC cells induced by vasoconstrictor, calcium release (calcium release) and the shadow of calcium influx (calcium) by the calcium ion fluorescence probe. Sound. Through non selective SOC blocker SKF-96365, selective SOC blocker 2-APB, voltage controlled calcium channel blocker nifedipine and ET-1 receptor blocker BQ-123, observe the status of SOC induced calcium influx in PASMC excitatory activity. [results] (1) TRPC1,3,4,5,6 is expressed in the pulmonary artery, the carotid artery and the tail artery, and there is no one in each other. The expression of TRPC1 (1.21E-05 + 0.01E-05 vs 3.82E-05 + 0.46E-05, P0.001) was significantly higher in the pulmonary artery after P0.05.10 mu g/ml LPS was incubated for 10min. (2) the pulmonary artery (1.3) 1E-05 + 0.23E-05 vs 5.26E-05 + 0.15E-05, P0.01), the expression of ETB-R (1.44E-05 + 0.10E-05 vs 3.43E-05 0.20E-05) increased significantly, while the expression of alpha adrenoceptor decreased significantly. (3) after 10 micron pretreatment, there was no detection in the cells. The sex changes (0.01 + 0.01 vs 0.01 + 0.02, P0.05), 80mM high potassium solution (0.95 + 0.06 vs 1.25 + 0.10, P0.05) and 10nM ET-1 (1.56 + 0.07 vs 1.98 + 0.09, P0.05) increased significantly increased in PASMC cells than the control group. The internal flow was more than the control group (1.32 + 0.10 vs 1.06 + 0.07, P0.05). (5) the SOC channel dependent calcium influx played an important role in ET-1 induced PASMC agitation, and SOC channel blockers SKF-96365 and 2-APB could block the activation of 73% and 70.8% PASMC respectively. [conclusion]LPS pretreated PASMC cells have no detectable changes in PASMC cells. The increase in the increase of [Ca2+]i caused by vasoconstrictor, the increase of [Ca2+]i increase mainly from the increase of calcium influx,.LPS may change the contraction ability of PASMC by increasing the expression of SOC (TRPC) channel and increasing the activity of sensitized SOC.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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