【作者】 黄潋滟；

【导师】 邹飞；

【作者基本信息】 南方医科大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 背景神经胶质瘤是颅内常见恶性肿瘤,国际流行病学统计资料显示,脑胶质瘤的发病率居各种肿瘤的第9位;死亡率居第2位。具有发病率、复发率、死亡率高和治愈率低的特点,其预后极差。提高脑胶质瘤的综合治疗效果是国内外医学界长期以来努力探索的课题之一,迄今为止现代医学尚未找到令人十分满意的根治方法。因此,研究神经胶质瘤的发生发展机制,寻求新的治疗靶点已成为亟待解决的问题。离子通道的活动是细胞增殖存活的基础之一,其参与电信号的传导、肌肉收缩、激素分泌、免疫调节、细胞周期和代谢等生物活动过程,并与诸多病理过程密切相关。离子通道与恶性肿瘤的相关性研究正日益受到重视。大量的离子通道存在于各个类型的肿瘤细胞中,某些特定离子通道可调控肿瘤细胞的膜电压和Ca²⁺信号,调节胞质内离子浓度、pH和细胞体积,参与信号转导途径之间相互作用,调控肿瘤细胞的增殖和/或凋亡,是维持肿瘤细胞内环境稳定的重要因素。离子通道尤其是钾离子通道在恶性肿瘤的增殖及细胞周期的调节中发挥关键作用。然而,钾通道在此过程中的确切角色和具体的作用机制尚不明确。ATP敏感性钾通道(K_ATP通道)是一种重要的钾离子通道,其广泛分布于全身各个组织器官,如脑垂体、心肌、平滑肌、骨骼肌和胰腺的β细胞,参与了多种细胞功能的调控,同时也涉及神经元再生、死亡及细胞周期调控等过程。近年来,K_ATP通道在恶性肿瘤中的作用逐渐引起人们的关注。研究发现,K_ATP通道在某些恶性肿瘤中呈高表达,可影响抗脑肿瘤药物的血脑屏障通过率,并参与膀胱癌细胞的增殖调控。尽管K_ATP通道在肿瘤调控中的具体作用机制及信号转导通路尚不明确,但K_ATP通道以其在体内广泛性分布、在多种恶性肿瘤组织中高频率表达、可调控肿瘤细胞周期进展与细胞增殖的特点,已经成为无论是从基因水平还是蛋白水平的、极具价值的恶性肿瘤诊治新靶点。胶质瘤细胞的增殖受多条信号转导通路的调节,Ras/MAPK（mitogenactivated protein kinase）信号通路是其中最为重要的信号通路之一。ERK作为MAPK通路中介导细胞存活和增殖的重要分子,在肿瘤增殖过程中起到重要作用。因此,在研究K_ATP通道调控胶质瘤细胞增殖过程的分子机制时,我们首选ERK通路。本研究聚焦于K_ATP通道对胶质瘤细胞增殖调控的作用机制,分析K_ATP通道的功能改变与胶质瘤细胞增殖的关系,并探讨其调控胶质瘤细胞增殖的分子机制,为阐明K_ATP通道对肿瘤细胞的增殖调控机制提供新的理论基础,并为胶质瘤的治疗提供新的靶标。对开展以离子通道为靶点治疗神经胶质瘤提供重要的理论依据。目的以ATP敏感性钾通道(K_ATP通道)对胶质瘤细胞增殖的调控作用为切入点,从K_ATP通道的电生理学和分子生物学特性两方面入手,采用RNAi、免疫细胞化学、免疫印记、流式细胞术、激光共聚焦等技术,通过体内外实验,分析K_ATP通道的功能改变与胶质瘤细胞增殖的关系,并探讨其调控胶质瘤细胞增殖的分子机制,为阐明神经胶质瘤细胞增殖机制提供新的线索,为神经胶质瘤临床治疗提供新的治疗靶点。方法将胶质瘤细胞株U87与U251置于37℃培养箱内培养（5%CO₂、95%空气）,分别用K_ATP通道的激动剂二氮嗪（100 uM）,K_ATP通道的阻断剂甲糖宁（100 uM）或DMSO处理细胞;或MEK1的阻断剂U0126预处理30分钟后,再分别给予K_ATP通道的阻断剂甲糖宁及K_ATP通道的激动剂二氮嗪;或采用分子生物学的方法在胶质瘤细胞中过表达或干扰K_ATP通道。运用免疫细胞化学的方法观察K_ATP通道在细胞中的表达情况,运用MTT法及流式细胞术比较细胞的增殖情况;运用免疫印记方法检测K_ATP通道及ERK激酶在不同处理组中表达水平的差异。运用裸鼠成瘤技术观察K_ATP通道的活性改变对胶质瘤细胞在体内增殖情况的影响。运用激光共聚焦技术观察K_ATP通道的活性改变对细胞膜电位及胞内钙浓度的影响。结果数据表明,与正常的星形胶质细胞相比,在患者胶质瘤组织及胶质瘤细胞株中KATP通道均呈高表达（p＜0.01）。我们的实验还发现,K_ATP通道的活性改变可影响胶质瘤增殖:过表达K_ATP通道或激活K_ATP通道均可促进胶质瘤细胞的增殖（过表达组增殖率为11 8.33±3.21%,对照组增殖率为100±1.22%;第六天激动剂组U-87与U251增殖率分别为1393.66±30.36%,1567.40±34.36%;对照组增殖率分别为1051.16±67.96%,1163.54±115.49%）;使用RNAi技术降低K_ATP通道表达或抑制K_ATP通道活性均可抑制胶质瘤细胞的增殖（RNA干扰组相较于对照组,细胞的数目减少了75.48%～81.44%;第六天U-87与U251阻断剂组增殖率分别为889.08±27.76%,922.10±10.46%;对照组增殖率分别为1051.16±67.96%,1163.54±115.49%）（p＜0.01）。同时在在体成瘤实验中也得到相同的结果（U87细胞的成瘤实验中,阻断剂组、激动剂组及对照组的肿瘤重量分别为0.30±0.09g,0.93±0.21g,0.59±0.13g;在U251细胞的成瘤实验中,阻断剂组、激动剂组及对照组的肿瘤重量分别为0.32±0.05g,1.01±0.22g,0.62±0.16g）（p＜0.01）。流式细胞术结果显示,K_ATP通道阻断剂可将胶质瘤细胞的细胞周期阻滞在G0/G1期进而抑制胶质瘤细胞的增殖（U87细胞的流式细胞术结果显示:阻断剂组、激动剂组及对照组G0/G1期的百分比分别为63.71±1.56%,49.7±1.27%,56.96±3.34%;S期的百分比为1.96±0.16%,12.91±0.70%,6.21±1.91%;U251细胞的流式细胞术结果显示:阻断剂组、激动剂组及对照组G0/G1期的百分比分别为63.15±1.67%,50.71±1.30%,58.85±1.86%;S期的百分比为1.68±0.27%,13.12±0.31%,9.24±1.05%）。上述结果证明了K_ATP通道的确参与了胶质瘤细胞增殖过程的调节。为了探讨K_ATP通道调控胶质瘤细胞增殖过程的分子机制,我们选择了Ras/MAPK（mitogen activated protein kinase）信号通路ERK激酶作为研究的靶点。我们的实验结果表明,K_ATP通道可以通过影响ERK激酶的活性进而调控胶质瘤细胞的增殖:过表达K_ATP通道或激活K_ATP通道可以增加ERK的活性;使用RNAi技术降低K_ATP通道表达或抑制K_ATP通道活性均可降低ERK活性（p＜0.01）。同时,ERK激酶的阻断剂（U0126）可以阻断K_ATP通道对胶质瘤细胞增殖的促进作用,而ERK的结构性激活形式（constitutively activated,CA）可以逆转K_ATP通道阻断剂对胶质瘤细胞增殖的抑制作用（过表达CA组的细胞数目为对照组的110.99%）（p＜0.01）。这些结果表明,ERK激酶作为下游分子参与了K_ATP通道对胶质瘤细胞增殖的调控。为了进一步探讨K_ATP通道调节ERK通路的作用机制,我们检测了胶质瘤细胞的膜电位、胞内钙浓度以及细胞的旁分泌/自分泌功能的改变。实验结果表明,K_ATP通道的功能改变未能影响胶质瘤细胞的膜电位、胞内钙浓度以及细胞的旁分泌/自分泌功能（p＞0.05）。这些结果提示,K_ATP通道对ERK的调节作用并非通过其对细胞膜电学特性的调节,而可能通过细胞内通路（如调控线粒体膜电位及自由基产生等）完成。结论在调控胶质瘤细胞的增殖过程中,K_ATP通道通过影响ERK激酶的活性调控细胞的增殖。更多还原

【Abstract】 Background:Human malignant gliomas are aggressive tumors that are most common malignancy in brain tumor.Many patients with gliomas respond poorly to traditional radiation and chemotherapy,and recurrence rate of human malignant gliomas is 100%.Therefore,to understand the mechanism by which glioma develops is necessary for an efficient and specific inhibition of the progression of this form of cancer.Ion channels are found in a variety of cancer cells and necessary for cell cycle and cell proliferation.The roles of K⁺ channels in the process are,however,poorly understood.It is commonly accepted that cells require K⁺ channels to proliferate.In any case,it is intuitively acceptable that K⁺ channels,as key players in controlling membrane potential,are critical in proliferation processes.In addition,K⁺ channels also control cell volume to modulate cell proliferation.Adenosine triphosphate（ATP）-sensitive potassium channel(K_ATPchannel)was first discovered by Akinori Noma in cardiac myocytes and was subsequently found to be expressed in many other cell types,e.g.in cardiac and smooth muscle, pancreatic beta cells and various brain regions.K_ATPchannels are octameric proteins consisting of two different types of subunits:members of the Kir6 inwardly rectifying potassium channel family and sulfonylurea receptor（SUR）subunits,which are members of the ATP-binding cassette transporter superfamily.In functional channels, four pore-forming Kir6 subunits are joined together with four regulatory SUR subunits.In central nervous system,K_ATPchannels are composed of Kir6.2 and SUR1 subunits.Evidence showed that expression of KATP channels is up-regulated in human uterine leiomyoma cells for estrogenmediated cell proliferation,but the effects of K_ATPchannels on glioma cells are poorly understood.Mitogen-activated protein kinases（MAPKs）are a family of serine/threonine protein kinases that regulate a wide array of cellular processes.Extracellular signal-regulated kinases（ERK）are the member of MAPKs.ERK activation is associated with cell survival and cell proliferation in response to growth factors such as platelet-derived growth factor and epidermal growth factor,which makes ERK the principal candidate in the studies of cell proliferation.The classic pathway of ERK activation is characterized by the sequential phosphorylation of upstream kinases, namely,Raf-1 and mitogen-activated kinase kinase（MEK）.The aim of this study is to investigate the regulation mechanisms by which K_ATPchannels control glioma cells proliferation.Our results demonstrate that K_ATPchannels augment glioma cell proliferation via activating ERK and suggest that K_ATPchannels play a critical role in the development of human malignant gliomas.Aim:To study the mechanisms of ATP-sensitive potassium channels control glioma cells proliferation.Methods:Cell culture,Cell transfection,Immunocytochemistry,MTT assay, Western analysis,Cell cycle analysis,and Mice tumor model.Results:In the present study,we report that adenosine triphosphate （ATP）-sensitive potassium channel activity plays a critical role in the proliferation of glioma cells.The expression of K_ATPchannels in glioma tissues was greatly increased than that in normal tissues.Treatment of glioma cells with tolbutamide,K_ATP channels inhibitor,suppressed the proliferation of glioma cells and blocked glioma cell cycle in G0/G1 phase.Similarly,downregulation of K_ATPchannels by siRNA inhibited glioma cell proliferation.On the other hand,K_ATPchannels agonist diazoxide and overexpression of K_ATPchannels promoted the proliferation of glioma cells.Moreover,inhibiting K_ATPchannels slowed the formation of tumor in nude mice generated by injection of glioma cells.Whereas activating K_ATPchannels promoted development of tumor in vivo.The effect of K_ATPchannels activity on glioma cells proliferation is mediated by extracellular signal-regulated kinase（ERK） activation.We found that activating K_ATPchannel triggered ERK activation and inhibiting K_ATPchannel depressed ERK activation.U-0126,the mitogen activated kinase kinase（MEK）inhibitors blocked ERK activation and cell proliferation induced by diazoxide.Furthermore,constitutively activated MEK plasmids transfection reversed the inhibitory effects of tolbutamide on glioma proliferation,lending further support for a role of ERK in mediating this process.Conclusion:Our results suggest that K_ATPchannels control glioma cell proliferation via regulating ERK pathway.We concluded that K_ATPchannels are important in pathological cell proliferation and open a promising pathway for novel targeted therapies.更多还原