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}!2

pin
R阴阳g1andin

Pinacidil
前列环索

，比那地尔

PKC
Protein恤JaSe C
蛋白激酶C

PH PVR
B气SMα
R缸lOOal)'hyper阳回∞
R由n∞æy vascularr回s回1臼
pulm∞æyarter臼lsm∞thmuscle 臼:l1s
肺动脉高压 肺血管阻力 肺动脉平滑肌细胞

SMα
Sn刷地 muscle cells
平滑肌掬月色

SUR

Sulfonylurea 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 (95％02和5％C02混合气)和Krebs．Henseleit(K-H)液(含mol·L吐： NaCl 119，KCl 4．7，MgS04 0．6，NaHC03 25，K_H2P04 1．2，CaCl2 2．5 和葡萄糖11．1，PH 7．4)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(O．05～50 nmol·L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t001．Lo或Pinl0-13～10一m01．L～，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o．05～50 nmol·L1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10．19士1．26 nmol·L一，bzV-Sb为0．905+0．186，r=0．91。在
10-13～10一nm01．L。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27．11 nmol·L‘1；Pin同样也呈浓度依赖性拮 抗ET-1 10nm01．LJ诱导的动脉收缩，与口T组比较，二者无显著性 差异(P>O．05)。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-I]．TdR)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(Go／G1期)进入DNA合成期(S期)和有丝分裂期(G2／M期)； IPT呈浓度依赖性抑制ET．1诱导的[3I-I]．TdR掺入量增多，阻止人肺 动脉平滑肌细胞由静止期(Go／G1期)进入DNA合成期(S期)和有 丝分裂期(G2／M期)；Pin同样抑制ET．1诱导的[3HI．TdR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ERKl／2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(ERKl／2)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ERKl／2 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(10¨M)拮抗ET．1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ERKl／2磷酸化，且 磷酸化ERKl／2水平在加入ET-1后10 min达高峰，IPT呈浓度依赖 性抑制ET．1诱导的肺动脉平滑肌细胞的ERKl／2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： 1．IPT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； 2．IPT抑制细胞DNA合成，阻止细胞由静止期(Go／G1期)进入DNA
合成期(S期)和有丝分裂期(G2／M期)，抑制人肺动脉平滑肌细 胞的增殖；
3．IPT通过增强KATP通道的表达，使细胞内游离Ca2+减少，进而抑 制ERKl／2的磷酸化。 i
4．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 ATP—sensitive potassium channel openeb iptakalim，on human pulmonary
o _ ■ ‘o ■ ’·^ · n l ■ ’
arterial C0nstrict]0n and proliIeration oI DulmonarT arterial
smooth muscle cells Ph．D．candidate：rum／he ZHU
Mentors：Prof．Hong WANG

Summary

Hypoxic pulmonary hypertension(rWH)is the common cause of hospital admissions．HPH has been demonstrated to be important pathophysiological process and the key event in the process of chronic obstructive pulmonary disease(COPD)to chronic cor pulmonale．The
treatment of pulmonary hypertension(P两has improved significantly
over the past ten years．Currently available medications are prostanoids， phosphodiesterase-5 inhibitors，and endothelin receptor antagonists．
However,since the primary etiology and mechanism of HPH remain
unclear,the treatment has remained primarily palliative．It is very important to elucidate the mechanism of HPH and search for new treatment options based on the causative factors of the pulmonary

hypertension．
Recently，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 remodeling．The suppression of function and gene
expression of K+channels will cause a decrease in trans—membrane K+
current，which results in a less negative Em，leading to cellular membrane depolarization，activation of the L—type ca十channels，and influx of Ca十
in the cell down a 1 0000一fold gradient．Elevated【Ca十】is implicated in
stimulating vascular arterial smooth muscle cell proliferation and triggers

vasoconstriction，leading to pulmonary hypertension．Furthermore，the

7

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 remodeling．Therefore，the therapeutic potent of K+ channel openers for pulmonary hypertension is worth to be further evaluated．
There are at least three types of K+channels in PASMCs：(1) voltage—gated K+(踟)channels，(2)Ca2+_activated K+(＆a)channels，and (3)ATP—sensitive K十(Kgav)channels．Among them，KATP channels are
nOW thought to play an important role as mediators of the response of vascular smooth muscle to a variety of pharmacological and endogenous vasodilators．They 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 activity．Therefore，KA：IP
channels in PASMCs represent potential therapeutic targets for PH．
The cellular signal mechanism of KATP channels on the proliferation and contraction of the human PASMCs is unclear．Iptakalim designed and synthesized by native researchers is a fatty para—amino compound with low molecular weight．It has been confirmed by substantial pharmacological，biochemical，and electrophysiological studies as well as
receptor-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 and
constriction of the human PASMCs．The study included following 3
experiments．

Part I The effects of iptakalim on pulmonary artery ring
constriction induced by ET-1 in human

AIM：To investigate the effects of iptakalim on ET-1-induced constriction of huthuman smallnal‘pulmonarypulmonary arteries nnz"tg．METHODS：Humar． S： man oulmonarv
‘

arteries(PA)were dissected free from connective tissues and cut into

8

南京医科大学博士学位论文

cylindrical segments(2~3 rain in width)．Then the PA rings were mounted on two stainless steel hooks suspended in Krebs—Henseleit(K-固solution (composition in mol·L。1：NaCl 1 1 9，KCI 4．7，MgS04 0．6，NaHC03 25， KH2P04 1．2，CaCh 2．5 and Glucose 1 1．1，PH 7．4)oxygenated continuously with 95％02-5％C02 gas mixture at 37℃and connected
to a force—displacement transducer for tension recording with a resting tension of 800 mg and were equilibrated for l hr before the beginning of each experiment．Vascular responses to ET-1 were examined by cumulative application of increasing concentrations of ET-1 in the range 0．05-50 nmol·L～，and concentration—response curves were obtained．For the study of dilatory response，PA rings were preconstricted witll ET-1， then exposed to cumulative concentrations of iptakalim or pinacidil (10以j～1O—mol·Lq)．ThevasodilatoryresponsestoKATpCO(iptakalimor pinacidil)were expressed as：relaxation percentage of the preconstricted
values．RESULTS：The concentration—dependent human PA constriction
was evoked by different ET-1 concentrations in the range O．05～50 nmol·L～，ECs0 values(95％confidence limits)were 10．19(8．93-11．45) nmol·L～．Slope B and standard error of B were 0．905 and O．1 86， respectively．The correlation coefficient of cumulative concentration—response curve was O．9 1． Iptakalim antagonized vasoconstriction ind_uced bv ET-1 with the concentration of 1 0-13～1 0．3
nmol·L～in a concentration—dependent manner．The ICs0 value for dilating pulmonary arterial rings—preconstricted with ET-1 was 27．1 1 nmol·L～． Pinacidil also antagonized vasoconstriction induced by ET-1 in a concentration—dependent manner．There is no difference in the dilating effect between iptakalim and pinacidil(P>O．05)．CONCLUSION： Iptakalim antagonized vasoconstriction induced by ET-1 via opening KAaV
channel of human PASMCs．Iptakalim could dilate effectively human
pulmonary artery．It is a promising candidate for pulmonary

hypertension．

9

南京医科大学博士学位论文

Part II The effects of iptakalim Oil the proliferation of primary cultured human PASMCs induced by ET-1

AIM：To explore the effects of iptakalim on the proliferation of primary cultured human PASMCs induced by ET-1． METHoDS：The experimental models of proliferation of primary cultured human PASMCs induced by ET-1 in vitro were established．PH]Thymidine([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 cytometry．RESULTS：ET一1(1 0 nM)stimulated
[3HI-thymidine incorporation，propelled cell cycle from static phase
(G0／G1)to DNA synthesis(S)and mitotic phase(G2／M)in human
PAsMcs．Iptakalim inhibited[3HI—TdR incorporation of human PAsMCs and hold．back cells from static phase(Go／GI)to DNA synthesis(S)and
mitotic phase(G2／M)in a dose—dependent manner．Pinacidil also inhibited ET-1．induced[3HI．TdR incorporation and the transition of cell cycle phase．Glibenclimide，a selective KATP channel blocker，abolished the effect of iptakalim and pinacidil on the cell proliferation in a
concentration—dependent manner．CONCLUSION：Iptakalim acts as a specific KATP channel opener to antagonize the proliferative effect of ET-1 on the human PASMCs．This study provides further evidence that iptakalim may serve as another candidate drug to treat pulmonary
hypertension．

Part HI The effects of iptakalim on changes of[Ca2+1cyt and phosphorylation of ERKl／2 in primary cultured human PASMCs induced by ET-1

AIM：To explore the effects of iptakalim on changes of[Ca2+】c”and
phosphorylation of ERK1／2 in primary cultured human PASMCs induced
by ET-1．METHODS：The experimental models of primary cultured
human PASMCs induced by ET-1 in vitro were established．Changes in

南京医科大学博士学位论文

[C一十】cyt were measured using laser scanning confocal microscope．The phosphorylation of extracellular signal—regulated kinases 1 and 2 (ERKl／2)were analyzed by western blot．RESULTS：The treatment with ET-1 resulted in a transient raise for[Ca2+]cyt in human PASMCs and this raise could be blocked by iptakalim(10lxM)．ET-1 induced phosphorylation of ERK 1／2 from 2 to 3 0 min with a peak response observed at 1 0 min．Iptakalim inhibited ET一1一induced ERK 1／2
phosphorylation in a dose—dependent 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)activation．These actions
promote the vasorelaxation of PASMCs and result in the inhibition of
proliferation in PASMCs．

Altogether,following conclusion are demonstrated by the results in the
present studies：
1．Iptakalim could antagonize vasoconstriction induced by ET-1 via opening KATP channel of human PASMCs and dilate effectively human pulmonary artery．
2．Iptakalim inhibited rH卜TdR incorporation of human PASMCs
and hold-back cells from static phase(G0／G0 to DNA synthesis(S) and mitotic phase(G2／M)．Thus，iptakalim could inhibited the proliferation of human PASMCs．
3．Iptakalim 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 ERKl／2 phosphorylation．
4．The novel KATP channel opener'iptakalim，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 PASMCs．

11

南京医科大学博士学位论文

Key words：ATP—sensitive potassium channel；KATp channel openers； Iptakalim；Pulmonary hypertension；Endothelin一1；cell proliferation； Mitogen—activated protein kinases

12

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①肺血管阻力增加②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