【作者】 王爽；

【导师】 王怀良；

【作者基本信息】 中国医科大学 ， 药理学， 2005， 硕士

【摘要】 前言 肺动脉高压（pulmonary arterial hypertension,PHT）是临床常见疾病,肺血管收缩反应性增强、肺血管构型重建和肺循环凝血机制改变是其主要病理改变。其中肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMC）增殖是血管重构的中心环节。大量研究表明,5-HT可通过5-HT受体和（或）5-HT转载体（5-HT transporter,5-HTT）诱导PASMC增殖,引起PHT,但其具体机制仍不十分清楚。 关于5-HT促有丝分裂的作用机制报道很多。目前认为5-HT的促细胞增殖作用与5-HT_1B受体和（或）5-HTT有关,但5-HT引起PASMC增殖的确切作用机制尚有待进一步探讨。有研究发现,5-HT诱导的PASMC增殖的细胞内信号转导通路是通过激活ERKMAPK通路实现的。ERK1/2蛋白表达下调后,可明显抑制高压所致大鼠主动脉平滑肌细胞的增殖,直接证明了ERK1/2MAPK信号转导通路参与了平滑肌细胞增殖的细胞内信号转导。本实验分别、联合应用5-HT_1B受体选择性拮抗剂SB₂₂₄₂₈₉和5-HTT选择性抑制剂氟西汀（fluxetine）,观察它们对5-HT诱导的PASMC增殖情况和细胞周期的影响,明确5-FIT诱导PASMC增殖的机制。本实验还应用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的含量下调,观察其对5-HT和舒马曲坦诱导的PASMC增殖情况和细胞周期的影响,明确5-HT引起PASMC增殖的细胞内信号转导通路。 方法 采用贴块法进行大鼠PASMC的原代培养。细胞传代后,分别、联合应更多还原

【Abstract】 IntroductionPulmonary hypertension (PHT) is characterized by persistent vasoconstriction , structural remodeling of pulmonary vessels, inflammation and thrombosis. Hyperplasia of pulmonary artery smooth muscle cells (PASMCs) is the main component of pulmonary vessel remodeling that results in PHT. Many studies have shown that 5 - hydroxytryptamine (5 - HT) may be a key determinant of pulmonary remodeling, but the exact mechanism of proliferation of PASMCS has not been fully elicited.It was reported that there was an increased expression of 5 - HT1B receptor mRNA in monocrotaline (MCT) induced PHT rats. In wild - type mice, hypoxia increased right ventricular hypertrophy, pulmonary vascular remodeling, which was absent in 5 - HT1B receptor knockoutmice. 5 - HT transporter (5 -HTT) mediates internalization of indoleamine. Exposure of PASMCs to hypoxia results in a rapid increase 5 - HTT expression and activity, and this effect associated with potentiation of the mitogenic action of 5 - HT. It suggested that 5 -HT1B receptor and 5 - HTT play key role in pulmonary vessel remodeling.The present studies showed that 5 - HT induced the mitogen - activated protein kinases (MAPK) extracelluar signal -regulated kinase1 (ERK1) and ERK2 in rabbit renal artery smooth muscle cells. But in rat PASMCS, whether the intracelluar signal pathway of 5 - HT is dependent on ERKl/2 pathway is unconcerned. So, the aim of the present study was to investigate the receptor and the transporter mechanism of PASMCS proliferation and mitogenesis induced by 5 - HT and the intracelluar signal pathway of 5 - HT.Material and methodLiposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate ( FITC) - labeled antisense ODN in PASMCs. The effects of SB224289 - HT1B receptor selective inhibitor) , fluoxetine (5 - HIT selective inhibitor) and ODNs on the proliferation of PASMCs induced by 5 - HT and sumatriptan were evaluated by cells number counting and cell cycle analysis, and measured by Microculture tetrazolium (MTT) assay and Flow cytometry (FCM) respectively.Results1. Uptake of ODNs by PASMCs24 h after liposomal transfection, FITC - labeled antisense ODN were observed in both cytoplasm and the nuclei of PASMCs. There were more than 90% of the cells exhibited fluorescence.2. Microculture tetrazolium ( MTT) assay① The effect of SB224289, fluoxetine, SB224289 adding fluoxetine, ODNs on the proliferation of PASMCs induced by 5 - HT (1 μmol/L). The proliferation rate (PR) in response to 5 - HT increased from 130. 33% ±5. 24% to 195. 67% ±19.46%. 88224289(1 μmol/L, 100 nmol/L) , fluoxetine (1μmol/L, 100 nmol/L) , 86224289(1 μmol/L, 100 nmol/L) adding fluoxetine (1 μmol/L, 100 nmol/L) concentration - dependently inhibited the proliferation of PASMCs induced by 5 - HT( 1 μmol/L) , the PR decreased froml28.67% ± 13. 25% , 123.33% ±9.94%, 105.67% ±5.55% to 139.33% ±8.95%, 140.00% ± 3.21%, 124% ±7.64% respectively. Antisense ODN inhibited the proliferation of PASMCs significantly. And this ihibitory effect is similar to SB224289 adding fluoxetine. Sense ODN and random ODN showed no effect. ② Antisense ODN inhibited the proliferation of PASMCs induced by sumatriptan (1 μmol/L) in vitro from 164.67% ±6.67% to 76.67% ± 10.17%. Sense ODN and randomODN showed no effect.3. Flow cytometry (FCM)Flow cytometric analysis of cycle distribution showed that cells treated with SB224289, fluoxetine, SB224289 adding fluoxetine had litter values of SPF and PI than 5 - HT. The SPF decreased from 13% ±0.58% to 8.67% ±0.33% , 10. 33% ±0.33% , 6.33% ±0.33%. The PI decreased from 28.67% ±0.33% , to 25.67% ±0.33% , 26.33% ±0.88% , 23.67% ±0.33%. The increase of sumatriptan induced SPF and PI were significantly inhibited by ASODN with SPF from 11.67% ±0.33% to 3.33% ±0.33%, and PI from 27.33% ±0.33% to 22% ±0.58%.DiscussionThe present studies demonstrated that there is 5 - HT1B receptor mechanism and 5 - HTT mechanism on the proliferation of rat PASMCS induced by 5 - HT. We proposed the intracellular signal pathway of 5 - HT is ERK1/2 pathway dependent. Furthermore, 5 - HT activated ERKl/2 through 5 - HT1B receptor and 5-HTT.The data of present studies confirmed SB224289 ( a selective inhibitor of 5 -HT1B receptor) and fluoxetine (a selective inhibitor of 5 - HTT) inhibited the proliferation of PASMCs induced by 5 - HT. Keegan et al found that GR127935 ( a selective inhibitor of 5 - HT1B receptor) attenuated right ventricular pressure, right ventricular remodeling, vascular remodeling in CHPHT rats. The results of our studied proved sumatriptan induced the proliferation of PASMCs significantly. Some scholars assessed the role of 5 - HTT in mediating the mitogenic and comitogenic effects of 5 - HT in PASMCS. Precious studies demonstrated that in mice lacking the 5 - HTT gene and exposed to hypoxia, the remodeling of pulmonary vessels were decreased as wild - type controls. The evidence supported the key role for 5 - HT1B receptor and 5 - HTT in PASMCS proliferation and PHT.Activation of ERKl/2 pathway mediates cellular mitogenesis. In the present studies, the antisense ODN to ERKl/2 delivered by lipofectin proved an ef-更多还原