【作者】 宋丹；

【导师】 王怀良；

【作者基本信息】 中国医科大学 ， 药理学， 2004， 硕士

【摘要】 前言 肺动脉高压（pulmonary arterial hypertension，PAH）是临床常见疾病。大量研究表明：肺血管收缩反应增强和肺血管重构（remodeling）是PAH形成的主要血管变化，人们发现血管重构是肺动脉压持续升高的主要因素，而肺动脉平滑肌细胞（pulmonary artery smooth muscle cells，PASMC）增殖则是血管重构的中心环节。近年来5-羟色胺（5-hydroxytryptamine，5-HT）在PAH形成和发展中的作用受到人们的重视。研究表明，它诱导PASMC的增殖与其特异性转载体介导的内在化有关。而且这一作用可被5-HT转载体（5-HT transporter，5-HTT）选择性抑制剂帕罗西汀、氟西汀剂量依赖性抑制。ERK／MAPK（extracellular signal-regulated protein kinase／mitogen-activated protein kinase，ERK／MAPK）信号转导通路与细胞生长分化密切相关，同时在其它类型的血管平滑肌细胞中5-HT诱导的增殖效应与MAPK信号转导通路的关系曾有过报道，但在PASMC中MAPK通路是否参与5-HT的信号转导尚未见报道。但有研究表明，在完整的细胞中，5-HTT介导的5-HT诱导的PASMC增殖与MAPK信号转导通路的激活有关。 为此本实验应用5-HTT选择性抑制剂氟西汀，以明确5-HTT在5-HT促进PASMC增殖过程的作用。并假设5-HTT所介导的PASMC增殖是通过ERK1／2 MAPKs信号转导通路实现的。首次应用针对ERK1／2 MAPK的反义寡核苷酸（antisense oligodeoxynucleotide，ASODN）抑制PASMC中ERK1／2的表达，使ERK1／2含量下调，观察其对PASMC的增殖情况及细胞周期的影响，以明确5-HTT介导的PASMC增殖与ERK1／2 MAPKs信号转导通路的关系。 方法 采用贴块法分离培养大鼠肺动脉平滑肌细胞。首先应用不同浓度的5-HTT抑制剂氟西汀(10^-5M-10^-7M)预作用细胞，进行MTT和流式细胞仪检测观察其对5一HT诱导的PASMC增殖情况及细胞周期的影响。应用脂质体介导ERKI/2 ASODN、正义ODN和随机ODN的细胞内转染，同样应用M竹和流式细胞仪方法进行检测，以观察不同类型ODN对5一HT诱导的PASMC增殖情况及细胞周期的影响。实验数据以Mean士SD表示，组间对比采用t检验，P<0.05认为差异显著，P<0.01认为差异非常显著。实验结果 1.氟西汀对5一HT诱导PASMC增殖的作用: 5一HT（20一6皿）显著促进玖sMe增殖。氟西汀（20一，一ro一’M）对5-HT诱导的PASMC增殖有抑制作用，且呈剂量依赖关系，10一SM浓度时抑制作用最强，而在10一7M浓度时则无抑制作用。因此选择10一6M为下一步实验中氟西汀的浓度。10一SM和10一6M浓度的氟西汀与5%FBs组间的细胞存活率无明显差异，说明氟西汀对血清诱导的细胞增殖无抑制作用。 2.ODN对5一HT诱导PASMC增殖的作用: 反义ODN可显著抑制5一HT诱导的PASMC增殖，正义和随机ODN对5一HT诱导的平滑肌细胞增殖无抑制作用。应用反义ODN组与5%FBS组间的细胞存活率存在显著差异。说明ASODN不仅能抑制5一HT诱导的细胞增殖作用，对血清诱导的细胞增殖也有抑制作用。 3.流式细胞仪细胞周期分析: 5一盯（10一6M）组中以sMe的sPF及H值较5%FBs组增加，而应用氟西汀（10一6M）预作用细胞可使两个指标较5一HT（10一6M）组下降。ASODN也可降低5一HT诱导的PASMC的SPF及PI值增加，且其下降幅度较氟西汀组要大。讨论 本实验应用5，HTT选择性抑制剂氟西汀预作用PASMC，可明显抑制5一HT的促细胞增殖效应。进一步证明了5一HTT在5一HT诱导PASMC增殖中的介导作用，也为研究5一H’I’细胞内信号转导提供了方向。同时，本实验首次通过ERKI/2 ASODN转染PASMC，有效地抑制了5一HT诱导的细胞增殖效应，表明ERKI/2 MAPK信号转导通路参与在以SMC中5-HT的信号转导。 近来的研究表明5一HT对PASMC的促增殖作用与低氧条件下细胞中5一H，I’r表达增加有关。而且，应用5一H竹选择性抑制剂及5一H竹基因敲除等方法可抑制低氧诱导的以H，证明5一HTT在PAH形成和发展中起到重要作用。本实验在细胞水平上，应用5一HTT选择性抑制剂氟西汀抑制PASMC在5一HT作用下的增殖反应，再一次证实了5一HTT的介导作用。有助于明确PAH的病理机制，同时也为PAH的治疗提供了新的靶点，应用5一H，IT抑制剂抑制血管平滑肌细胞增殖，可防止肺血管重构的发生。 本实验成功应用脂质体介导ERKI/2 ASODN转染PASMC。并观察到ERKI/2 ASODN能够显著抑制5一HT促细胞增殖，直接证实了ERKI/2MAPK信号转导通路参与5一HT在PASMC的信号转导。研究表明5一HT诱导的PASMC增殖过程中5一HTI’所介导的信号转导通路可能为:GAP酪氨酸磷酸化、RAS激活*NAD（P）H氧化酶激活产生ROS、激活·ERKI/2MAPK。因此应用5一HTT选择性抑制剂即可抑制5一HT诱导的ERKI/2MAPK信号转导通路的激活。 本实验的结果提示，5一HTT介导的5一HT内在化是PASMC发生增殖的重要机制，与PAH时肺血管重构关系密切，是PAH重要发病机制之一。同时，ASODN的应用也为PAH治疗提供了一个新的方案。结论 1.5一HTr选择性抑制剂氟西汀可抑制5一HT诱导的PASMC的增殖，明确了在PASMC中是5一HTr介导5一H更多还原

【Abstract】 Exposure to acute or chronic hypoxia leads to the development of pulmonary artery hypertension ( PAH). The cardinal features of PAH are persistent vaso-constriction and structural remodeling of the pulmonary vessels. Hyperplasia of pulmonary artery smooth muscle cells ( PASMCs) is the main component of pulmonary vascular remodeling which is associated with progressive elevation in pulmonary arterial pressure.Several studies have shown that 5 -hydroxytryptamine (5 - HT) plays an important role in the pathogenesis of PAH. 5 - HT exerts potent mitogenic and co - mitogenic effects on PASMCs, and these effects are associated with celluar internalization of 5 - HT mediated by 5 - HT transporter (5 - HTT). Therefore, inhibition of 5 - HTT might abolish the proliferative response of PASMCs to 5 -HT.Many stimuli resulting in cell growth, differentiation and vascular contraction may activate mitogen - activated protein kinase ( MAP kinase) - dependent signaling pathways. Among this family, the extracellular signal -regulated kina-ses (ERKs) are activated in response to growth and differentiation factors. Previous studies have reported that 5 - HT may induce the activation of ERKl/2 in the rat aortic smooth muscle cells and the rabbit isolated renal artery smooth muscle cells. However, whether the intracelluar signal pathway of 5 - HT in PASMCs is dependent on the activation of ERK is largely unconcerned.In this study, we first observed the effect of fluoxetine, an selective inhibitor of 5 - HT transporter on the proliferation of PASMCs in response to 5 - HT. Then, we hypothesized that 5 - HT induced the proliferation of PASMCs is de-pendent on the activation of ERK. We studied the effect of downregulation of ERK1/2, using antisense oligodeoxynucleotides (ODNs) , on the proliferation of 5 - HT stimulated PASMCs in vitro.MethodsLiposomal transfection was used to introduced ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate ( FITC) - laBcled antisense ODN in PASMCs. The effects of fluoxetine and ODNs on the proliferation of PASMC were evaluated by cells number counting and cell cycle analysis, and measured by Microculture tetrazolium (MTT) assay and Flow cytometry (FCM) separately. All the data are presented as means + SD, and assessed by ANOVA and t -test. P < 0.05 was considered significant.Results1. Uptake of ODNs by PASMCsAfter liposomal transfection 24 h and 48 h, FITC - laBcled antisense ODN were observed in the cytoplasm and the nuclei of PASMCs. There were more than 90% of the cells exhibited fluorescence.2. Effect of fluoxetine and ODNs on the proliferation of PASMCs induced by 5-HTMTT assay showed that 5 - HT ( 10-6M) could induce the proliferation of PASMCs. Pretreatment of the cells with fluoxetine (10-5M, 10-6M and 10-7 M) could produce a concentration - dependent reduction in proliferation rates ( PR) . According these results, we chose 10-6M as the concentration of fluoxetine used in other experiments.Pretreatment of PASMCs with ERK1/2 antisense ODN resulted in a significant inhibition of 5 - HT - induced cell proliferation and this effect is stronger than fluoxetine. In contrast, sense ODN and random ODN showed no effect.3. Effect of fluoxetine and antisense ODN on cell cycleFlow cytometric analysis of cell cycle distribution showed that the cells treated with 5 - HT had larger values of S - phase cell fractions ( SPF) and proliferation index ( PI) than control. While pretreatment with fluoxetine or antisense ODN could decrease these values. These results indicated that 5 - HT could promote the PASMCs from the G0/G1 phase of cell cycle into S phase and this effect could be inhibited by fluoxetine and antisense ODN.DiscussionOur result demonstrate that 5 - HTT plays a key role in the mitogenic effect of 5 - HT on PASMCs. Fluoxetine, a highly selective inhibitor of 5 - HT transporter inhibited the proliferation of PASMCs induced by 5 - HT in vitro. Meanwhile , we foun更多还原