本文選題：脂多糖 + 肺動脈平滑肌細(xì)胞��； 參考：《復(fù)旦大學(xué)》2010年博士論文

【摘要】： [目的]敗血癥休克情況下主要表現(xiàn)為體循環(huán)壓力的下降和早期肺循環(huán)壓力的升高,甚至肺動脈高壓(pulmonary artery hypertension, PAH)。本實(shí)驗(yàn)旨在探討高濃度的LPS是否導(dǎo)致肺動脈平滑肌細(xì)胞(pulmonary artery smooth muscle cell, PASMC)本身收縮能力的變化。[方法]通過急性消化酶分離法獲得單個PASMC,在200倍倒置顯微鏡下,灌流含10μg/ml LPS的生理鹽溶液,通過細(xì)胞長度的變化、完成收縮所需時間的變化觀察LPS對PASMC的直接刺激作用。對LPS預(yù)處理的PASMC,分別給予80mM高鉀溶液、苯腎上腺素及內(nèi)皮素-1(endothelin-1, ET-1),觀察LPS對PASMC的收縮能力的影響。[結(jié)果](1)本實(shí)驗(yàn)采取的急性消化酶法獲得的PASMC存活率95%,PASMC純度達(dá)97.7±1.78%。(2)10μg/ml的LPS不能直接引起PASMC收縮反應(yīng)。(3)80mM的高鉀等滲溶液可引起PASMC收縮,經(jīng)10μg/ml LPS預(yù)處理10min,PASMC對80mM高鉀等滲HBSS溶液的反應(yīng)更敏感,表現(xiàn)為高鉀刺激后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)時點(diǎn)收縮幅度較對照組明顯增大。LPS預(yù)處理的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)總收縮長度所用時間較對照組顯著縮短。(4)10μM及l(fā)mM的苯腎上腺素均不能引起PASMC收縮；LPS預(yù)處理后,10μM的苯腎上腺素也不能引起PASMC收縮。(5)10nM的ET-1作用于PASMC,約lmin左右,PASMC發(fā)生迅速而強(qiáng)烈的收縮。經(jīng)10μg/ml LPS預(yù)處理10min,PASMC對10nM的ET-1的收縮反應(yīng)沒有明顯的增強(qiáng)或抑制作用(P0.05)。[結(jié)論]10μg/ml的LPS不能直接引起PASMC收縮,經(jīng)LPS預(yù)處理后對80mMK誘發(fā)的收縮反應(yīng)表現(xiàn)為增敏作用,而對ET-1誘發(fā)的收縮反應(yīng)無明顯影響。 [目的]旨在探討LPS對肺動脈平滑肌細(xì)胞(pulmonary artery smooth muscle cell, PASMC)內(nèi)鈣離子濃度([Ca2+]i)的影響及其與鈣庫操縱性鈣通道(SOC)的活性和表達(dá)的關(guān)系。[方法]經(jīng)典瞬時受體電位通道(transient receptor potential canonical, TRPC)被證實(shí)是SOC的主要分子基礎(chǔ),通過Real-time PCR檢測正常及LPS刺激后SD大鼠肺動脈平滑肌上TRPC1、3、4、5、6 mRNA的表達(dá)。通過鈣離子熒光探針及共聚焦顯微鏡技術(shù)觀察LPS對血管收縮劑引起的PASMC細(xì)胞內(nèi)[Ca2+]i變化、鈣釋放(calcium release)和鈣內(nèi)流(calcium entry)的影響。通過非選擇性SOC阻滯劑SKF-96365、選擇性SOC阻滯劑2-APB、電壓操縱性鈣通道阻滯劑硝苯地平和ET-1的受體阻滯劑BQ-123,觀察SOC引起的鈣內(nèi)流在PASMC興奮活動中的地位。[結(jié)果](1)TRPC1、3、4、5、6在肺動脈、頸動脈和尾動脈上均有表達(dá),相互之間無顯著性差異,P0.05。10μg/ml LPS孵育10min后,肺動脈上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)的表達(dá)顯著性升高。(2)10μg/ml LPS刺激后,肺動脈上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的表達(dá)明顯升高；而α1-腎上腺素受體mRNA的表達(dá)顯著降低(3.16E-05±0.10E-05 vs 0.72E-05±0.29E-05,P0.001)。(3)10μg/ml LPS預(yù)處理PASMC10min后,細(xì)胞內(nèi)[Ca2+]i無可檢測性變化(0.01±0.01 vs 0.01±0.02,P0.05),80mM高鉀溶液(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細(xì)胞內(nèi)[Ca2+]i增幅均較對照組明顯增高。(4)10μg/ml LPS預(yù)處理的PASMC鈣釋放較對照組降低(1.36±0.07 vs1.66±0.06,P0.01),而鈣內(nèi)流較對照組增多(1.32±0.10 vs 1.06±0.07,p0.05)。(5)SOC通道依賴的鈣內(nèi)流在ET-1引起的PASMC激動作用占有重要作用,SOC通道的阻斷劑SKF-96365和2-APB分別可阻斷73.0%和70.8%的PASMC的激活作用。[結(jié)論]LPS預(yù)處理的PASMC細(xì)胞內(nèi)[Ca2+]i無可檢測性變化,但可使血管收縮劑引起的[Ca2+]i的增幅增大,[Ca2+]i增幅的增大主要來源于鈣內(nèi)流的增加。LPS可能通過上調(diào)SOC(即TRPC)通道的表達(dá)以及增敏SOC的活性改變PASMC的收縮能力。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363

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本文編號：1952657