新型K-ATP开放剂埃他卡林对人肺动脉收缩与肺动脉平滑肌细胞增殖的影响教学内容.docx

此文档收集于网络，如有侵权请联系网站删除南京医科大学博士学位论文民!2pinR阴阳g1andinPinacidil前列环索，比那地尔PKCProtein恤JaSe C蛋白激酶CPH PVRB气SMR缸lOOal)hyper阳回R由ny vascularr回s回1臼pulmyarter臼lsmthmuscle 臼:l1s肺动脉高压 肺血管阻力 肺动脉平滑肌细胞SMSn刷地 muscle cells平滑肌掬月色SURSulfonylurea 阻碍阳磺酷腺受体2此文档仅供学习和交流南京医科大学博士学位论文论文独创性声明本论文堑型坠卫珏趑刻堪丝圭挞盟厶脏动然蝗缠皇眭动脉垩通 题细胞增殖的髭响是我个人在导师指导下进行的研究工作及取得的 研究成果。论文中除了特别加以标注和致谢的地方外，不包含其他人 或机构已经发表或撰写过的研究成果。其他同志对本研究的启发和所做的贡献均在论文中作了明确的声明并表示了谢意。专业：哩哩凼科作者签名：也!垒： 日期：坦呈!鱼!论文使用授权声明本人完全了解南京医科大学有关保留、使用学位论文的规定，即： 学校有权保留送交论文的复印件，允许论文被查阅和借阅；学校可以 公布论文的全部或部分内容，可以采用影印、缩印或其它复制手段保 存论文。保密的论文在解密后遵守此规定。作者签名：勉：导师签名：札日期：2竺!乡l埋南京医科大学博士学位论文中文摘要新型KATP开放剂埃他卡林对人肺动脉收缩 与肺动脉平滑肌细胞增殖的影响 研究生：朱煜明导师：王虹教授缺氧性肺动脉高压(Hypoxic pulmonary hypertension，HPH)作 为一类严重危害人类健康的疾病，是慢性阻塞性肺病(Chronic obstructivepulmonarydisease，COPD)的重要病理生理过程，也是发 展为慢性肺源性心脏病(Chronic cor pulmonale)的关键环节。尽管 在过去的十多年中，应用前列环素类药物、磷酸二酯酶一5抑制剂、内 皮素受体拮抗剂治疗肺动脉高压已经取得了一定的进展，但由于对 HPH的病因和发病机制尚未完全阐明，因而这些治疗并未从根本上 治疗肺动脉高压。因此，对缺氧性肺动脉高压形成机制的研究并研发 有效的针对致病靶标的防治药物显得尤为必要。近年来，国内外学者已经认识到肺血管平滑肌细胞钾通道在肺动 脉高压发生发展中的作用。钾通道功能下降，细胞内K+外流减少， 细胞膜去极化，电压依赖性钙通道开放，细胞外Ca2+内流，肌浆网内 ca2+释放，细胞内游离Ca2+浓度增加，肺血管平滑肌细胞收缩、增殖， 肺动脉压增高；另一方面肺动脉平滑肌细胞钾通道功能下降，细胞内 K+增多，细胞凋亡减少，进一步加重肺血管重构。有鉴于此，钾通道 开放剂对肺动脉高压的防治成为近年来国内外研究的热点和焦点。肺血管平滑肌细胞至少存在三种类型钾通道：(1)电压依赖性钾 (Kv)通道，(2)Ca2+激活性钾(Kca)通道，(3)ATP敏感性钾(KATP) 通道。细胞膜KATP通道是目前已知的唯一的可在机体缺血、缺氧等病理生理情况下代偿性开放的钾通道，是机体对抗缺血缺氧的重要自身保护机制，已经成为研发新型治疗肺动脉高压药物的重要靶标。 由于KATp通道调控人类肺动脉平滑肌细胞收缩、增殖的细胞信号及分子机制尚不明确，本文从器官、细胞和分子水平探讨KATP通 道调控人肺动脉平滑肌细胞收缩、增殖的细胞和分子生物学机制，系统评价我国学者自行研制的新型KATP开放剂埃他卡林(Iptakalim，南京医科大学博士学位论文IPT)对人肺动脉平滑肌细胞收缩和增殖的影响，为研究开发新型治 疗肺动脉高压的药物提供理论依据。本文主要围绕如下三个部分开展 工作：第一部分新型KATP开放剂埃他卡林对ET-1诱导的离体人肺动脉环收缩的影响目的：研究IPT对内皮素1(ET-1)诱导的人离体肺动脉环收缩的 影响及其机制。方法：分离正常离体人肺动脉条，置于含饱和混合氧 (9502和5C02混合气)和KrebsHenseleit(K-H)液(含molL吐： NaCl 119，KCl 47，MgS04 06，NaHC03 25，K_H2P04 12，CaCl2 25 和葡萄糖111，PH 74)的培养皿内，剪取3 mm长的血管环固定于含K-H液的浴槽内，持续小流量通入混合氧，静息张力下稳定60 min 后，通过张力换能器与十六道生理记录仪相连，记录血管环的张力变 化。按累积法给予7个浓度ET-1(O0550 nmolL以)，观察ET-1收缩 血管作用的量效关系，求出EC50；观察ET-1在EC50浓度的时效曲 线。在此基础上，以ET-1的EC50浓度使肺动脉环达到最大收缩幅度 时，按累积法加入IPTl0-13-10一t001Lo或Pinl0-1310一m01L，以等 容量溶剂做阴性对照，记录肺血管环收缩张力的变化，并以ET-l最 大收缩幅度为100，计算PT和Pin的舒张率。结果：在o0550 nmolL1浓度范围内，ET-1呈浓度依赖性地诱导离体人肺动脉环收缩， 其ECs0-q：L95为1019士126 nmolL一，bzV-Sb为0905+0186，r=091。在10-1310一nm01L。1浓度时，IPT呈浓度依赖地拮抗ET-1诱导的离体人肺动脉环收缩，其IC50为2711 nmolL1；Pin同样也呈浓度依赖性拮 抗ET-1 10nm01LJ诱导的动脉收缩，与口T组比较，二者无显著性 差异(PO05)。结论：IPT可拮抗ET-1诱导的人肺动脉环收缩， 其机制可能在于开放肺动脉平滑肌细胞上的KATP通道。妒T可以有效 舒张人肺动脉，表明其是一个富有潜力的治疗肺动脉高压的候选药 物。南京医科大学博士学位论文第二部分新型KATP开放剂埃他卡林对ET-1诱导的原代培养人肺动脉平滑肌细胞增殖的影响目的：研究IPT对ET-1诱导的原代培养的人肺动脉平滑肌细胞增殖 的影响及其机制。方法：以原代培养的人肺动脉平滑肌细胞为研究对 象，用ET-1诱导人肺动脉平滑肌细胞增殖，3H胸腺嘧啶核苷 (3I-ITdR)掺入法检测脱氧核糖核苷酸(DNA)合成；流式细胞仪 技术检测人肺动脉平滑肌细胞细胞周期。结果： ET。1(10 nM)使 原代培养的人肺动脉平滑肌细胞3HI。TdR掺入量增加，促进细胞由静 止期(GoG1期)进入DNA合成期(S期)和有丝分裂期(G2M期)； IPT呈浓度依赖性抑制ET1诱导的3I-ITdR掺入量增多，阻止人肺 动脉平滑肌细胞由静止期(GoG1期)进入DNA合成期(S期)和有 丝分裂期(G2M期)；Pin同样抑制ET1诱导的3HITdR掺入量增多 和细胞周期的改变，且与IPT比较二者无显著差异；格列本脲呈浓度 依赖性地逆转IPT与Pin对细胞增殖的抑制作用。结论：口T作为一种 新型的KATP开放剂，可以抑制人肺动脉平滑肌细胞的增殖，是一种 治疗肺动脉高压的侯选药物。第三部分新型KATP开放剂埃他卡林对ET-1诱导的原代培 养人肺动脉平滑肌细胞内Ca2+1c)，t及ERKl2磷酸化的影响目的：研究IPT对ET-1诱导的原代培养人肺动脉平滑肌细胞内游离 钙浓度(Ca2+cvt)及细胞外信号调节激酶1和2(ERKl2)磷酸化 的影响及其机制。方法：以原代培养的人肺动脉平滑肌细胞为研究对 象，分别用ET-1与PT处理人肺动脉平滑肌细胞后，应用激光扫描 共聚焦显微镜测定细胞内Ca2+cvt变化；使用Western blot检测ERKl2 磷酸化水平的改变。结果：ET一1诱导人肺动脉平滑肌细胞内游离Ca2+ 显著增加，IPT(10M)拮抗ET1诱导的人肺动脉平滑肌细胞内 ca2+】。vt升高；ET一1促使人肺动脉平滑肌细胞的ERKl2磷酸化，且 磷酸化ERKl2水平在加入ET-1后10 min达高峰，IPT呈浓度依赖 性抑制ET1诱导的肺动脉平滑肌细胞的ERKl2磷酸化。结论：IPT 作为一种特异性KATP开放剂，可能通过增强KATP通道的功能与通道南京医科大学博士学位论文蛋白的表达，使细胞内游离Ca2+减少，进而抑制丝裂原活化蛋白激酶 (MAPKs)的活化，最终导致肺动脉平滑肌舒张并抑制平滑肌细胞 的增殖。综合本文三个部分的研究结果，获得如下结论： 1IPT通过开放肺动脉平滑肌细胞上的KATP通道，拮抗ET-1诱导的人肺动脉环收缩，有效舒张人肺血管； 2IPT抑制细胞DNA合成，阻止细胞由静止期(GoG1期)进入DNA合成期(S期)和有丝分裂期(G2M期)，抑制人肺动脉平滑肌细 胞的增殖；3IPT通过增强KATP通道的表达，使细胞内游离Ca2+减少，进而抑 制ERKl2的磷酸化。i4总之，新型KArl,开放剂口T在整体、细胞、分子水平抑制人肺动 脉环的收缩与肺动脉平滑肌细胞增殖，是一个富有潜力的防治肺 动脉高压的候选药物。关键词：ATP敏感性钾通道；KATe开放剂；埃他卡林；肺动脉高压； 内皮素1；细胞增殖；丝裂原活化蛋白激酶南京医科大学博士学位论文The study on the effects of novel ATPsensitive potassium channel openeb iptakalim，on human pulmonaryo _ onl arterial C0nstrict0n and proliIeration oI DulmonarT arterialsmooth muscle cells PhDcandidate：rumhe ZHUMentors：ProfHong WANGSummaryHypoxic pulmonary hypertension(rWH)is the common cause of hospital admissionsHPH has been demonstrated to be important pathophysiological process and the key event in the process of chronic obstructive pulmonary disease(COPD)to chronic cor pulmonaleThetreatment of pulmonary hypertension(P两has improved significantlyover the past ten yearsCurrently available medications are prostanoids， phosphodiesterase-5 inhibitors，and endothelin receptor antagonistsHowever,since the primary etiology and mechanism of HPH remainunclear,the treatment has remained primarily palliativeIt is very important to elucidate the mechanism of HPH and search for new treatment options based on the causative factors of the pulmonaryhypertensionRecently，many experts have known that the plasmalemmal K十 channels in pulmonary arterial smooth muscle cells(PASMCs)might play an important role in the regulation of pulmonary vasoconstriction and vascular remodelingThe suppression of function and geneexpression of K+channels will cause a decrease in transmembrane K+current，which results in a less negative Em，leading to cellular membrane depolarization，activation of the Ltype ca十channels，and influx of Ca十in the cell down a 1 0000一fold gradientElevated【Ca十】is implicated instimulating vascular arterial smooth muscle cell proliferation and triggersvasoconstriction，leading to pulmonary hypertensionFurthermore，the7南京医科大学博士学位论文decreased activity of K+channel also helps to maintain a high concentration of K十in the cytoplasm and inhibit apoptosis，thereby promoting vascular remodelingTherefore，the therapeutic potent of K+ channel openers for pulmonary hypertension is worth to be further evaluatedThere are at least three types of K+channels in PASMCs：(1) voltagegated K+(踟)channels，(2)Ca2+_activated K+(a)channels，and (3)ATPsensitive K十(Kgav)channelsAmong them，KATP channels arenOW thought to play an important role as mediators of the response of vascular smooth muscle to a variety of pharmacological and endogenous vasodilatorsThey play important roles in the physiology and pathophysiology of vascular smooth muscle cells(SMCs)by coupling the metabolic state of the cell to its electrical activityTherefore，KA：IPchannels in PASMCs represent potential therapeutic targets for PHThe cellular signal mechanism of KATP channels on the proliferation and contraction of the human PASMCs is unclearIptakalim designed and synthesized by native researchers is a fatty paraamino compound with low molecular weightIt has been confirmed by substantial pharmacological，biochemical，and electrophysiological studies as well asreceptor-combining test as a newly selective Kgav channel opener (KAvpCO)In the present study,the effects of iptakalim on pulmonary hypertension were explored SO as to evaluate its therapeutic value and elucidate the mechanism of Kgav channels on the proliferation andconstriction of the human PASMCsThe study included following 3experimentsPart IThe effects of iptakalim on pulmonary artery ringconstriction induced by ET-1 in humanAIM：To investigate the effects of iptakalim on ET-1-induced constriction of huthuman smallnalpulmonarypulmonary arteries nnztgMETHODS：Humar S： man oulmonarvarteries(PA)were dissected free from connective tissues and cut into8南京医科大学博士学位论文cylindrical segments(23 rain in width)Then the PA rings were mounted on two stainless steel hooks suspended in KrebsHenseleit(K-固solution (composition in molL。1：NaCl 1 1 9，KCI 47，MgS04 06，NaHC03 25， KH2P04 12，CaCh 25 and Glucose 1 11，PH 74)oxygenated continuously with 9502-5C02 gas mixture at 37and connectedto a forcedisplacement transducer for tension recording with a resting tension of 800 mg and were equilibrated for l hr before the beginning of each experimentVascular responses to ET-1 were examined by cumulative application of increasing concentrations of ET-1 in the range 005-50 nmolL，and concentrationresponse curves were obtainedFor the study of dilatory response，PA rings were preconstricted witll ET-1， then exposed to cumulative concentrations of iptakalim or pinacidil (10以j1OmolLq)ThevasodilatoryresponsestoKATpCO(iptakalimor pinacidil)were expressed as：relaxation percentage of the preconstrictedvaluesRESULTS：The concentrationdependent human PA constrictionwas evoked by different ET-1 concentrations in the range O0550 nmolL，ECs0 values(95confidence limits)were 1019(893-1145) nmolLSlope B and standard error of B were 0905 and O1 86， respectivelyThe correlation coefficient of cumulative concentrationresponse curve was O9 1 Iptakalim antagonized vasoconstriction ind_uced bv ET-1 with the concentration of 1 0-131 03nmolLin a concentrationdependent mannerThe ICs0 value for dilating pulmonary arterial ringspreconstricted with ET-1 was 271 1 nmolL Pinacidil also antagonized vasoconstriction induced by ET-1 in a concentrationdependent mannerThere is no difference in the dilating effect between iptakalim and pinacidil(PO05)CONCLUSION： Iptakalim antagonized vasoconstriction induced by ET-1 via opening KAaVchannel of human PASMCsIptakalim could dilate effectively humanpulmonary arteryIt is a promising candidate for pulmonaryhypertension9南京医科大学博士学位论文Part IIThe effects of iptakalim Oil the proliferation of primary cultured human PASMCs induced by ET-1AIM：To explore the effects of iptakalim on the proliferation of primary culturedhuman PASMCs induced by ET-1 METHoDS：The experimental models of proliferation of primary cultured human PASMCs induced by ET-1 in vitro were establishedPHThymidine(31-11一TdR) incorporation was used to evaluate DNA synthesis and cell proliferation Cell cycle phase distribution was used to detect cell proliferation and analyzed by flow cytometryRESULTS：ET一1(1 0 nM)stimulated3HI-thymidine incorporation，propelled cell cycle from static phase(G0G1)to DNA synthesis(S)and mitotic phase(G2M)in humanPAsMcsIptakalim inhibited3HITdR incorporation of human PAsMCs and holdback cells from static phase(GoGI)to DNA synthesis(S)andmitotic phase(G2M)in a dosedependent mannerPinacidil also inhibited ET-1induced3HITdR incorporation and the transition of cell cycle phaseGlibenclimide，a selective KATP channel blocker，abolished the effect of iptakalim and pinacidil on the cell proliferation in aconcentrationdependent mannerCONCLUSION：Iptakalim acts as a specific KATP channel opener to antagonize the proliferative effect of ET-1 on the human PASMCsThis study provides further evidence that iptakalim may serve as another candidate drug to treat pulmonaryhypertensionPart HIThe effects of iptakalim on changes ofCa2+1cyt and phosphorylation of ERKl2 in primary cultured human PASMCs induced by ET-1AIM：To explore the effects of iptakalim on changes ofCa2+】c”andphosphorylation of ERK12 in primary cultured human PASMCs inducedby ET-1METHODS：The experimental models of primary culturedhuman PASMCs induced by ET-1 in vitro were establishedChanges in南京医科大学博士学位论文C一十】cyt were measured using laser scanning confocal microscopeThe phosphorylation of extracellular signalregulated kinases 1 and 2 (ERKl2)were analyzed by western blotRESULTS：The treatment with ET-1 resulted in a transient raise forCa2+cyt in human PASMCs and this raise could be blocked by iptakalim(10lxM)ET-1 induced phosphorylation of ERK 12 from 2 to 3 0 min with a peak response observed at 1 0 minIptakalim inhibited ET一1一induced ERK 12phosphorylation in a dosedependent manner CONCLUSION：Thus，we can presume that iptakalim may be capable of increasing K册expression and function，and that these processes cause membrane hyperpolarization， a decrease in caZ十 and inhibition of mitogen-activated protein kinases(MAPKs)activationThese actionspromote the vasorelaxation of PASMCs and result in the inhibition ofproliferation in PASMCsAltogether,following conclusion are demonstrated by the results in thepresent studies：1Iptakalim could antagonize vasoconstriction induced by ET-1 via opening KATP channel of human PASMCs and dilate effectively human pulmonary artery2Iptakalim inhibited rH卜TdR incorporation of human PASMCsand hold-back cells from static phase(G0G0 to DNA synthesis(S) and mitotic phase(G2M)Thus，iptakalim could inhibited the proliferation of human PASMCs3Iptakalim may be capable of increasing the expression and function of KATP channels，and that these processes cause a decrease in【Ca计】and inhibition of ERKl2 phosphorylation4The novel KATP channel openeriptakalim，is an promising candidate for the treatment of pulmonary hypertension because of its effects on both constriction of human pulmonary artery ring and proliferation of human PASMCs11南京医科大学博士学位论文Key words：ATPsensitive potassium channel；KATp channel openers； Iptakalim；Pulmonary hypertension；Endothelin一1；cell proliferation； Mitogenactivated protein kinases12南京医科大学博士学位论文前言“慢性阻塞性肺病(Chronic obstructive pulmonary disease， COPD)缺氧性肺动脉高压(Hypoxic pulmonary hypertension，HPH) 一陧性肺源性心脏病(Chronic cor pulmonale)是一组严重危害人蛳健康的慢性进行性疾病。根据国内近年统计资料，40岁以上人群该组疾病发病率为9左右，我国共约有4000万慢性阻塞性肺病患者。 该组疾病死亡率在我国农村和北方地区占第一位；据世界卫生组织估 计，COPD在全球疾病死亡原因当中，次于心脏病、脑血管病和急性 肺部感染，与艾滋病一起并列第四位。推测到2020年，全球该组疾 病的死亡率将跃升到第三位【l】o由于其患病人数多，死亡率高，社会 经济负担重，已经成为一个重要的社会公共问题。该组疾病的临床特 征为病程长，呈慢性经过，气流受限不完全可逆，呈进行性发展，病 人的生活质量急剧下降，也给家庭开口社会造成沉重的经济负担。目前 国内外对该病尚缺乏有效的治疗手段，迄今为止的治疗仅限于以缓解 症状为目的对症治疗。肺动脉高压(PH)在慢性肺源性心脏病的发病过程中起着举足轻 重的作用，是慢性阻塞性肺病发展成慢性肺源性心脏病的中心环节。 因此，对肺动脉高压的发生、发展及其实验治疗学的研究已经成为预 防和治疗慢，生肺源性心脏病的焦点，人们试图通过研究寻找针对该关 键环节的有效防治措施，以“切断自慢性阻塞性肺病向慢性肺源性心 脏病演变过程或延缓该过程的发展。通过这些研究，进一步探讨肺动 脉高压的发病机理，寻找针对该关键环节的有效防治措施。国内外学者对肺动脉高压的研究多集中在两个方面，一是探讨其 发病机制，二是寻找有效的治疗方法和药物。尽管在过去的十多年中， 应用前列环素类药物、磷酸二酯酶5抑制剂、内皮素受体拮抗剂治疗 肺动脉高压已经取得了一定的进展，但由于对肺动脉高压的病因和发 病机制尚未完全阐明，因而这些治疗并未从根本上治疗肺动脉高压。 因此，对肺动脉高压形成机制的研究并研发有效的针对致病靶标的防 治药物显得尤为必要。目前认为肺动脉高压的发病与肺血管阻力增加肺血管内皮 细胞功能不全、肺血管重构关系密切。其中，尤以内皮素1 (Endothelin。l，ET01)的作用得到普遍认同【2】。ET-1是血管内皮细胞南京医科大学博士学位论文分泌的，由21个氨基酸残基组成的缩血管活性肽。ET-1具有很强的 缩血管活性与致分裂