【作者】 高山红；

【导师】 王怀良；

【作者基本信息】 中国医科大学 ， 药理学， 2003， 博士

【摘要】 目的 肺血管构型重建是肺动脉高压的基本病理改变。肺血管平滑肌细胞增殖和肥大是肺血管构型重建主要特征。5-HT在肺动脉高压的发病过程中有非常重要的作用。研究发现野百合碱所致肺动脉高压大鼠的肺血流中有5-HT水平升高并且肺血管平滑肌有5-HT_1B受体mRNA的表达增加。5-HT_1B受体基因敲除小鼠能抵抗缺氧诱导的肺动脉高压和肺血管构型重建。5-HT是一种重要的血管活性物质，5-HT不仅刺激肺动脉平滑肌细胞收缩还可造成其增生和肥大，并且能与多种细胞生长因子发生协同作用。很多研究表明5-HT的促有丝分裂作用是通过细胞表面的受体，但是5-HT对肺动脉平滑肌细胞的促有丝分裂作用机制涉及的受体亚型至今未能够完全阐明。为此我们假设5-HT_1B受体在肺动脉平滑肌细胞增殖和肺血管构型重建中有重要作用，并做以下研究： 1．建立大鼠血管平滑肌细胞体外培养方法，分析流式细胞术与³HTdR掺入法在观察细胞增殖指标上的相关性，为研究药物对细胞增殖和有丝分裂的影响和作用机制建立可靠的实验方法。 2．观察5-HT和5-HT_1B／1D受体激动剂舒马曲坦对肺动脉平滑肌细胞的促有丝分裂作用，研究选择性5-HT_1B受体拮抗剂SB₂₂₄₂₈₉和选择性5-HT_1D受体拮抗剂BRL₁₅₅₇₂对5-HT诱导的肺动脉平滑肌细胞增殖的影响，探讨5-HT对肺动脉平滑肌细胞促有丝分裂作用的5-HT_1B受体机制。探讨5-HT_1B受体和5-HT_1D受体在肺血管构型重建中的作用机制。 3．观察5-HT和5-HT_1B／1D受体激动剂舒马曲坦对肺动脉平滑肌细胞和主动脉平滑肌细胞的促有丝分裂作用的差异。比较选择性5-HT_1B受体拮抗剂和选择性5-HT_1D受体拮抗剂对5-HT诱导的肺动脉平滑肌细胞增殖和主动脉平滑肌细胞增殖的影响的差异，探讨5-HT对主动脉平滑肌细胞的促有丝分裂作用机制。根据主动脉和肺动脉平滑肌细胞对5-HT的促有丝分裂反应的差异性和5-HT_1B受体拮抗剂对主动脉和肺动脉平滑肌细胞增殖的影响的差异，探讨5-HT_1B受体拮抗剂成为高选择性的抗肺动脉高压新药先导物的可能性。方法 1.采用贴块法分离培养大鼠肺动脉平滑肌细胞和主动脉平滑肌细胞。用含20%FBS的DMEM培养液原代培养平滑肌细胞，用含5%FBS的DMEM培养液作Vehicle传代培养平滑肌细胞。用不同浓度的FBS刺激细胞生长，分析流式细胞术与’H一TdR掺人法在细胞增殖指标上的相关性。 2.施加5一HT、舒马曲坦和选择性5一HTIB受体拮抗剂SB224289、选择性5一HTID受体拮抗剂BRL15572等处理因素。用四哇盐（M，I’r）比色法计数平滑肌细胞增殖，观察5一HT、舒马曲坦、SB224289和BRL15572对平滑肌细胞增殖的影响。 3.用，H一胸腺嗜咙掺人法观察细胞DNA合成情况，比较实验组与对照组的cPm值，分析特异性的5一HT受体激动剂和5一HT受体拮抗剂对肺动脉和主动脉平滑肌细胞的DNA合成的促进和抑制作用，证明5一HTI。受体是5一HT诱导肺动脉平滑肌细胞有丝分裂的作用点之一。探讨5一HT对主动脉平滑肌细胞的促有丝分裂作用机制。研究选择性5一HT;B受体拮抗剂SB2242s9、选择性5一HT，。受体拮抗剂BRL15572对5一HT诱导的平滑肌细胞有丝分裂的影响。 4.用流式细胞术法分析细胞周期，配合M，rr检测观察细胞增殖，并探讨5一HT对细胞分裂周期的影响以及选择性5一HT受体拮抗剂影响平滑肌细胞有丝分裂所作用的细胞周期。结果 1.大鼠血管平滑肌细胞培养及细胞增殖的考察方法 流式细胞仪指标（PI和SPF）与，H一TdR掺人实验指标（cPm）有极为显著的相关关系。流式细胞仪技术与MTr活细胞染色技术联合应用考察细胞增殖和有丝分裂比单独应用一种指标更能准确反映细胞增殖和有丝分裂情况。 2.5一HT对肺动脉平滑肌细胞的促有丝分裂作用机制 （1）5一HT和舒马曲坦对PASMC的促有丝分裂作用 M竹法检测5一HT10一6 moFL一10一‘moFL可促进肺动脉平滑肌细胞的增殖，增殖率分别为256%士18%;228%土29%;201%土17%。舒马曲坦10一6 moFL一10一8 moFL也可促进肺动脉平滑肌细胞增殖，增殖率分别为从5%士2一%;220%士25%和199%士25%。5一HT从20一6一10一‘moFL和舒马曲坦从10一6一10一”moFL均可以使肺动脉平滑肌细胞的增殖率增加，并呈剂量依赖关系。 流式细胞仪分析5一HT10一5 moFL一10一7 moFL的细胞增殖指数（Pro-liferation Index）为23 .7%土1 .1%，22.6%土0.7%，20.5%土0.8%;S期细胞分数（S一Phase Cell Fraetion）为8 .5%土1 .3%，6.9%土0.4%，5.9%土0.5%。舒马曲坦10一，，10一6 moUL的增殖指数为23.9%土0.8%和21.2%士0.8%;S期细胞分数为8.8%土0.3%和6.6%士0.5%。空白对照组的增殖指数和S期细胞分数分别为17.59%士0.5%和4.68%土0.7%。5一HT和舒马曲坦能够促进肺动脉平滑肌细胞从Go/G，期进入S期，然后进人GZ/M期。5一HT对肺动脉平滑肌细胞的促有丝分裂作用可能有5-HT:二1。受体的参与。 （2）SB2242s9和BRL15572对5一HT诱导的PASMC增殖的影响 MTr法检测SB22428，从10一’moFL到10一‘“moFL的浓度能剂量依赖地抑制5一?更多还原

【Abstract】 ObjectiveSerotonin (5 - hydroxytryptamine, 5 - HT) plays an important role in pulmonary hypertension. It was reported that there was an increased expression of 5 - HT1B receptor mRNA in monocrotaline (MCT) induced pulmonary hypertension rats. Also, hypoxia increased pulmonary vascular remodeling in wild - type mice and this was reduced in the 5 - HT1B receptor knockout mice. Furthermore , hypoxia increased 5 - HT1 receptor - mediated constriction of pulmonary arteries from the wild - type mice and this was attenuated in the 5 - HT1B receptor knockout mice. These evidences suggested 5 - HT1B receptor plays an important role in the development of PHT. Serotonin is one of vasoactive substances and has been recognized to cause proliferation of a variety of cells. Several studies have shown that the mitogenic action of serotonin is initiated through its binding to cells surface receptors. Yet, the 5 - HT receptor mechanism of pulmonary artery smooth muscle cell proliferation and mitogenesis has not yet been fully e-licited. So, the objetive of the present study covers the following aspects.1. To investigate the mitogenetic effects of 5 - HT and, a 5 - HT1B/1D receptor agonist sumatriptan (Sum) on pulmonary artery smooth muscle cells (PASMC) , to identify the role of 5 - HT1B/1D receptors in PASMC proliferationand pulmonary remodeling.2. To observe the effects of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on PASMC proliferation induced by 5 - HT and sumatriptan, investigate the role of 5 - HT1B receptor in PASMC proliferation and pulmonary remodeling.3. To observe the effect of 5 - HT and 5 - HT1B/1D receptor agonist sumatriptan on ASMC proliferation and DNA synthesis, observe the difference reaction of PASMC and ASMC to 5 - HT and 5 - HT1B/1D receptor agonist. Study the effect of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on ASMC proliferation induced by 5 - HT, investigate the mitogenic mechanism of ASMC.4. According to the different mitogenic mechanism between ASMC and PASMC, research high selectivity and efficiency new medicine for the treatment of pulmonary remodeling and pulmonary hypertension, especially from the point of 5 - HT1B receptor and 5 - HT1B receptor antagonists.Methods1. PASMC and ASMC were isolated from rat pulmonary artery and aorta. DMEM culture media with 20% FBS were used in primary culture; DMEM culture media with 5% FBS were used as vehicle; 5-HT, sumatriptan, 5 - HT1Breceptor antagonist SB22489 and selective 5 - HT1D receptor antagonist BRL15572 were used as treatments.2. Proliferation of cultured PASMC and ASMC were evaluated by MTT assay , cells DNA synthesis were evaluated by 3 H - TdR incorporation, and cell cycle analysis, proliferation index (PI) , s - phase cell fraction (SPF) were performed by flow cytometry (FCM).3. All data were expressed as mean value standard deviation. Statistic a-nalysis was done by Statistical Program for Social Sciences (SPSS) Software. One Way ANOVA analyze multi - groups comparisons. Comparisons between groups were tested by LSD (least significant difference) test. P < 0. 05 wereconsidered to be statistical significant.Results1. SMC culture and evaluation of cell proliferation methodPI and SPF performed by FCM were closely correlated with cpm that was acquired from 3H -TdR incorporation tests. It is a better way to use MTT assay and FCM method in observing cells proliferation and mitogenesis than to use them alone.2. Mechanism of PASMC mitogenesis induced by 5 - HT(1) Effect of 5 - HT and sumatriptan on PASMCIt was found that 5 - HT 10-6 - 10-8 mole/L stimulated proliferation of PASMC by MTT assay. The proliferation rates ( PR) of 5 - HT 10 -6-10-8mol/ L treatments were 256%±18% ; 228%±29% and 201% ±17% respectively. Sumatriptan 10 -6-10-8mol/L mimicked the mitogenesis effect of 5 - HT. The proliferation rates of Sum were 245%±21% ; 220%±25% ; and 199%±25更多还原