【作者】 李敏；

【导师】 王波；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 博士

【摘要】 【研究背景】卵巢癌是女性生殖器官常见的三大恶性肿瘤之一,恶性度高,早期诊断困难,多数患者就诊时已属晚期,死亡率居妇科恶性肿瘤首位。卵巢癌细胞具有高的增殖及转移潜力,然而对其恶性生物学行为机制尚不明确。因此研究卵巢癌的恶性生物学行为机制,寻找新的治疗靶点,成为亟待解决的任务。氯离子通道广泛分布于有机体的细胞膜及细胞器膜,其在细胞的容积调节、物质的跨膜转运、细胞电位、细胞器的酸化、细胞增殖分化等多种生理过程中发挥重要作用。氯离子通道在体内的调控受多种因素的的影响,根据功能及门控机制,氯通道可分为:电压门控性氯离子通道（Voltage-gated chloride channels,CLC）、容积调控性氯离子通道（Volume-regulated/sensitive anion/chloride channels,VRAC）、囊性纤维化跨膜传导调节体（Cystic fibrosis transmembrane conductanceregulator,CFTR）、钙激活性氯离子通道（Calcium-activated chloride channels,CLCA）、配体激活的氯离子通道（ligand-activated chloride channels）。配体激活的氯通道主要分布于神经系统。氯离子通道参与多种细胞的增殖,氯通道阻滞剂对诸如肝细胞、肺主动脉内皮细胞、淋巴细胞、血管平滑肌细胞等多种细胞的增殖具有抑制作用。引人瞩目的是:近年多项研究发现氯离子通道在多种肿瘤中表达异常,并且氯通道可能在肿瘤细胞的增殖、侵袭及转移等恶性生物学行为中起着重要作用。例如:CLC氯通道在脑胶质瘤组织和细胞中过表达,这有利于细胞大小和形状的改变,从而导致瘤细胞易于分裂和侵袭狭窄的细胞外脑间隙;容积调控性氯离子通道是调节鼻咽癌细胞通过G₁期限制点,促进细胞增殖的重要因子;还有研究报道其在肿瘤细胞中呈细胞周期依赖性改变。CLC-3是CLC氯离子通道的重要家族成员,并且CLC-3又是容积调控性氯离子通道最有可能的候选蛋白,所以其功能复杂多样,有研究报道前列腺癌细胞中抗凋亡调节因子呈剂量依赖性上调CLC-3蛋白表达水平,使细胞生存能力提高而抗凋亡。另有研究发现抑制CLC-3蛋白表达后,肿瘤细胞的侵袭转移能力降低。迄今为止,国内外尚未见氯离子通道在卵巢上皮性癌细胞中的表达情况及其对卵巢上皮性癌细胞增殖及细胞周期的影响。因此我们研究的目的是观察氯通道在卵巢上皮性癌细胞中的表达、探索氯离子通道在卵巢上皮性癌细胞增殖及细胞周期中的作用以及CLC-3单通道改变对卵巢癌细胞增殖的影响。第一部分氯离子通道在卵巢上皮性癌细胞中的表达及其在细胞增殖及细胞周期中的作用目的:观察氯离子通道在卵巢上皮性癌细胞株A2780中的表达,探讨氯离子通道在卵巢癌细胞株A2780增殖及细胞周期中的作用。方法:采用逆转录聚合酶链反应（RT-PCR）检测几种有代表性的氯离子通道mRNA在卵巢上皮性癌细胞中的基因表达;应用免疫细胞化学方法检测氯离子通道蛋白在A2780细胞中的表达。采用四甲基偶氮唑蓝（MTT）法,观察不同浓度及种类的氯离子通道阻滞剂阻断卵巢癌细胞氯离子通道后,卵巢癌细胞株A2780增殖活力的变化;采用流式细胞仪技术检测不同浓度及种类的氯离子通道阻滞剂阻断卵巢癌细胞氯离子通道后,卵巢癌细胞A2780细胞周期的变化。结果:RT-PCR检测提示卵巢癌细胞株A2780表达CLC-3及CFTR氯离子通道mRNA;但未检测到hCLCA-2 mRNA表达。免疫细胞化学染色显示卵巢癌细胞A2780表达CLC-3及CFTR氯离子通道蛋白,蛋白表达定位于细胞质膜。MTT检测表明不同浓度的氯通道阻滞剂NPPB、NFA、TAM作用细胞后明显抑制了A2780细胞的增殖活力。25μmol/L、50μmol/L、100μmol/L、200μmol/L的NPPB对A2780细胞的抑制率分别为23.4%、59.2%、90.8%及93.5%,各浓度组与对照组相比差异均有显著性（P＜0.05）。25μmol/L、50μmol/L、100μmol/L、200μmol/L的NFA对A2780细胞的抑制率分别为18.3%、40.9%、68.6%及75.1%,各浓度组与对照组相比差异均有显著性（P＜0.05）。10μmol/L、20μmol/L及30μmol/L的TAM对A2780细胞的抑制率分别为65.8%、73.4%及77.5%,各浓度组与对照组相比差异均有显著性（P＜0.05）。而相对特异性CFTR氯通道阻滞剂Glibenclamide作用细胞后对A2780细胞的增殖活力无明显影响。25μmol/L、50μmol/L、100μmol/L及200μmol/L的Glibenclamide对A2780细胞增殖的抑制率分别为2.1%、2.8%、3.7%及3.9%,各浓度组与对照组相比,差异无显著件（P＞0.05）。流式细胞仪检测发现不同浓度的氯离子通道阻滞剂NPPB、NFA、TAM作用A2780细胞72 h后,细胞周期的G₀/G₁和S期细胞分布均发生改变,细胞周期停滞于G₀/G₁期。而Glibenclamide作用A2780细胞后,细胞周期的G₀/G₁和S期细胞分布改变无统计学差异。1 NPPB对A2780细胞周期的影响50μmol/L、100μmol/L及200μmol/L的NPPB作用于A2780细胞后:细胞周期的G₁/G₁期细胞数由55.4±2.1%分别增加到73.1±3.9%、84.2±2.3%及86.5±2.6%,各浓度组与对照组相比,差异均有显著性（P＜0.05）。S期细胞数由34.0±2.5%分别下降至22.3±4.1%、12.1±2.3%及10.7±1.6%,各浓度组与对照组相比,差异均有显著性（P＜0.05）。2 NFA对A2780细胞周期的影响50μmol/L、100μmol/L及200μmol/L的NFA作用于A2780细胞后:细胞周期的G₀/G₁期细胞数由55.4±2.1%分别增加到69.3±3.5%、74.6±2.1%及79.5±3.6%,各浓度组与对照组相比,差异均有显著性（P＜0.05）。S期细胞数由34.0±2.5%分别下降至24.1±1.5%、18.7±1.9%及13.4±2.7%,各浓度组与对照组相比,差异均有显著性（P＜0.05）。3 TAM对A2780细胞周期的影响10μmol/L、20μmol/L及30μmol/L的TAM作用于A2780细胞后:细胞周期的G₀/G₁期细胞数由55.4±2.1%分别增加到68.7±2.4%、73.8±3.3%及79.4±3.1%,各浓度组与对照组相比,差异均有显著性（P＜0.05）。S期细胞数由34.0±2.5%分别下降至24.6±1.9%、20.3±2.2%及15.3±1.6%,各浓度组与对照组相比,差异均有显著性（P＜0.05）。4 Glibenclamide对A2780细胞周期的影响50μmol/L、100μmol/L及200μmol/L的Glibenclamide作用于A2780细胞后:细胞周期的G₀/G₁期细胞数由55.4±2.1%分别改变为55.8±2.9%、53.1±3.2%及54.2±3.5%,各浓度组与对照组相比差异均无显著性（P＞0.05）;S期细胞数由34.0±2.5%分别改变为33.7±2.6%、36.9±1.6%及35.6±2.8%,各浓度组与对照组比较差异均无显著性（P＞0.05）。结论:CLC及CFTR氯离子通道在卵巢癌细胞系A2780中表达。电压门控性氯离子通道及容积调控性氯离子通道可能在卵巢上皮性癌细胞的增殖及细胞周期中起着重要作用。第二部分CLC-3反义寡核苷酸对卵巢上皮性癌细胞增殖的抑制作用及其机制目的:观察电压门控性氯离子通道-3（CLC-3）反义寡核苷酸转染对卵巢上皮性癌细胞A2780增殖及细胞周期的影响。方法:以脂质体Lipofectamine介导的CLC-3反义寡核苷酸转染卵巢上皮性癌细胞株A2780,利用Western blot检测转染CLC-3反义寡核苷酸后A2780细胞CLC-3蛋白表达的变化;通过MTT法、流式细胞仪检测细胞增殖能力及细胞周期的变化。应用Western blot检测CLC-3反义寡核苷酸转染对卵巢癌细胞A2780细胞周期素D1（CyclinD1）表达的影响。结果:卵巢上皮性癌细胞A2780转染CLC-3反义寡核苷酸后,CLC-3的蛋白表达明显降低。50μg/ml和100μg/ml CLC-3反义寡核苷酸对CLC-3蛋白表达抑制率分别为62.5%及78.6%。而100μg/ml CLC-3正义寡核苷酸转染对CLC-3蛋白的表达未见明显影响。CLC-3反义寡核苷酸转染48 h后降低了A2780细胞的增殖能力,50μg/ml、100μg/ml反义CLC-3寡核苷酸对A2780细胞增殖的抑制率分别为48.6%及65.3%,与对照组相比差异有显著性（P＜0.05）;而100μg/ml CLC-3正义寡核苷酸转染对A2780细胞的增殖能力的影响与对照组相比差异无显著性（P＞0.05）。流式细胞仪检测结果表明CLC-3反义寡核苷酸转染A2780细胞48 h后,细胞周期分布发生改变,处于G₀/G₁细胞数目上升,S期细胞数下降。50μg/ml、100μg/ml的CLC-3反义寡核苷酸组G₀/G₁期细胞数由49.4±3.1%分别上升至70.3±3.4%和75.8±2.6%,与对照组比较有显著性差异（P＜0.05）;而S期细胞数由40.3±2.8%分别下降至23.5±2.1%和19.6±1.5%,与对照组比较有显著性差异（P＜0.05）。而转染正义寡核苷酸对A2780细胞的细胞周期无明显影响,100μg/ml CLC-3正义寡核苷酸转染A2780细胞后,G₀/G₁期细胞数由49.4±3.1%改变至50.2±3.6%,S期细胞数由40.3±2.8%改变至39.7±2.9%,与对照组相比差异均无显著性（P＞0.05）。Western blot检测显示CLC-3反义寡核苷酸抑制了A2780细胞的CyclinD1蛋白表达;而CLC-3正义寡核苷酸对A2780细胞CyclinD1的蛋白表达未见明显影响。结论:CLC-3反义寡核苷酸能够抑制卵巢上皮性癌细胞的增殖能力,CLC-3氯离子通道可能成为卵巢癌治疗的新靶点。CLC-3氯离子通道可能通过对CyclinD1的调控发挥其在卵巢上皮性癌细胞增殖及细胞周期中的作用。更多还原

【Abstract】 [Background]Ovarian cancer,one of the three common gynecological malignant genital tumors,is the leading cause of gynecological cancer deaths.Due to the lack of effective earlier diagnosis of ovarian cancer,most of patients are diagnosed with advanced-stage,with a resultant poor prognosis.Ovarian cancer cell is characterized by its high potential for unlimited proliferation and metastasis.The factors correlated with the malignant biological behavior of ovarian cancer,however,are poorly understood.There is an imperative need to explore new therapeutic targets and a better understanding of the mechanisms involved in the malignant biological behavior of ovarian cancer.Chloride（Cl^-） channels are ubiquitous transmembrane proteins which have been implicated in cell volume regulation,salt and fluid movements across epithelia, stabilization of membrane potential,intracellular organelle acidification,cell proliferation and differentiation.Chloride channels are regulated by various factors. According to their gating mechanisms,there are,from the functional point of view, five classes of chloride channel,including voltage-gated chloride channels（CLC）, volume/swell-regulated/sensitive anion/chloride channels（VRAC）,cystic fibrosis transmembrane conductance regulator（CFTR）,calcium-activated chloride channels （CLCA） and ligand-activated chloride channels which mainly forms synaptic channels.Chloride channels have been found in control of cell proliferation.Cl^- channel blockers inhibit cell proliferation in many types of cells,including liver cells, pulmonary artery endothelial cells,lymphoctes,vascular smooth muscle cells.Most attractively,recent studies indicate Cl^-channels express in a variety of tumor cells and support the essential role of plasma membrane chloride channels in the proliferation, invasion and migration of tumor cells.For example,CLC has been found to be specifically up-regulated in human glioma membranes,which in turn facilitate rapid changes in cell size and shapes and contribute to glioma cell division or invasion into narrow extracellular brain spaces.VRAC is one of the important factors that modttlate the passage of nasopharyngeal carcinoma cells through the G₁ restriction point and endow nasopharyngeal carcinoma cells with an enhanced proliferation ability.In addition,it is reported that VRAC displays cell cycle-dependent expression in tumor cells.CLC-3,an important member of the CLC superfamily,plays a crucial role in a variety of cellular processes.Moreover,CLC-3 has been considered the most likely molecular candidate of the VRAC.It is implicated that the antiapoptotic protein could up-regulate CLC-3 protein in a dose-dependent manner in the human prostatic carcinoma cells,which thereby protects cells against apoptotic stimuli and increases cell viability.Additionally,the present study demonstrates that inhibition of CLC-3 protein expression down-regulates invasion and migration ability of tumor cell.Up to date,there are no data available as to the expression of Cl^-channels and whether Cl^-channels are related to the cell proliferation and cell cycle progression in ovarian cancer cell.In this study,we aimed to observe the representative Cl^-channels expression,investigate the role of Cl^-channels in proliferation and cell cycle of ovarian cancer cells and study the effect of CLC-3 on human ovarian cancer cell proliferation. Objective:To investigate the expression and the roles of chloride channels in cell proliferation and cell cycle in human epithelial ovarian cancer cell line A2780.Methods:The expression of several representative chloride channels was detected by employing RT-PCR and immunocytochemical methods.The effects of chloride channels on proliferation and cell cycle of human ovarian cancer cell line A2780 were observed by means of MTT assay and flow cytometry.A variety of chloride channel blockers were used in order to observe the effects of chloride channels on human ovarian cancer cell line A2780.Results:CLC-3 and CFTR mRNA was detected by RT-PCR assay,and CLC-3 and CFTR proteins were found to express in A2780 cell plasma membrane by using immunocytochemical assay.The hCLCA mRNA,however,could not be detected in A2780 cells by using RT-PCR.Chloride channel blockers（NPPB,NFA and TAM） significantly inhibited the proliferation of A2780 cells as measured by MTT assay. Treatments with 25μmol/L,50μmol/L,100μmol/L and 200μmol/L NPPB caused inhibitory effects on A2780 cell proliferation potential,and the inhibitory rate was 23.4%,59.2%,90.8%,93.5%,respectively（vs control,P＜0.05）.After NFA-treatment, A2780 cell proliferation was greatly inhibited in the presence of 25μmol/L,50μmol/L, 100μmol/L and 200μmol/L NFA,and the inhibitory rate was 18.3%,40.9%,68.6% and 75.1%,respectively（vs control,P＜0.05）.TAM,a relatively specific blocker of volume-regulated chloride channels,also produced inhibitory effects on A2780 cell proliferation ability,and the inhibitory rate was 65.8%,73.4%,77.5%after treatment of A2780 cells with 10μmol/L,20μmol/L,30μmol/L TAM,respectively（vs control, P＜0.05）.In contrast,Glibenclamide,a relatively specific CFTR blocker,had no inhibitory effects on A2780 cell proliferation.The inhibitory rate was 2.1%,28%, 3.7%and 3.9%after treatment of A2780 cells with 25μmol/L,50μmol/L,100μmol/L and 200μmol/L Glibenclamide,respectively（vs control,P＞0.05）.In addition,chloride channel blockers（NPPB,NFA,TAM） also obviously affected the A2780 cell cycle,which increased the percentage of cells in G₀/G₁ phase,and reduced the percentage of cells in S phase.Glibenclamide,however,had no effect on the A2780 cell cycle.After treatment with 50μmol/L,100μmol/L and 200μmol/L NPPB,the proportion of G₀/G₁ phase of A2780 cells increased from 55.4±2.1%to 73.1±3.9%,84.2±2.3%and 86.5±2.6%respectively（vs control,P＜0.35）; Surprisingly,the proportion of S phase reduced from 34.0±2.5%to 22.3±4.1%, 12.1±2.3%and 10.7±1.6%,respectively（vs control,P＜0.05）.Effects of NFA on A2780 cell cycle progression were similar to those of NPPB.The proportion of G₀/G₁ phase increased from 55.4±2.1%to 69.3±3.5%,74.6±2.1%and 79.5±3.,5%, when 50μmol/L,100μmol/L and 200μmol/L NFA were used,respectively（vs control, P＜0.05）.Interestingly,the proportion of S phase reduced from 34.0±2.5%to 24 1±1.5%,18.7±1.9%and 13.4±2.7%,respectively（vs control,P＜0.05）.10μmol/L, 20μmol/L and 30μmol/L TAM also affected A2780 cell cycle progression,and the proportion of G₀/G₁ phase went up significantly from 55.4±2.1%to 68.7±2.4%, 73.8±3.3%and 79.4±3.1%,respectively（vs control,P＜0.05）.Whereas the proportion of S phase decreased from 34.0±2.5%to 24.6±1.9%,20.3±2.2%and 15.3±1.6%,respectively（vs control,P＜0.05）.After treatment with 50μmol/L, 100μmol/L and 200μmol/L Glibenclamide,the proportion of G₀/G₁ phase cells changed from 55.4±2.1%to 55.8±2.9%,53.1±3.2%and 54.2±3.5%,respectively （vs control,P＞0.05）;and the proportion of S phase cells changed from 34.0±2.5% to 33.7±2.6%,36.9±1.6%and 35.6±2.8%,respectively（vs control,P＞0.05）.Conclusions:Our results suggest that CLC-3 and CFTR are markedly expressed in human epithelial ovarian cancer cells.Chloride channels,CLC and VRAC especially, could play an important role in the proliferation and cell cycle of ovarian cancer cell. Objective:The aim of the present study was to observe the effect of CLC-3 antisense oligonucleotide on proliferation of human ovarian cancer cell line A2780.Methods:CLC-3 antisense oligonucleotides were transfected by lipofectamine into human epithelial ovarian cancer cell line A2780.Western blot was used to detect CLC-3 protein expression.The ability of cell proliferation was determined by using MTT assay,and the cell cycle was measured by employing flow cytometry（FCM）. Western blot were performed to detect the cyclinD1 protein expression in A2780 cells.Results:Western blot assay showed that CLC-3 antisense oligonucleotides inhibited the CLC-3 protein expression.Compared with the control group,the inhibitory rates of 50μg/ml,100μg/ml CLC-3 antisense oligonucleotides were 62.5%、78.6%, respectively;however,the CLC-3 sense oligonucleotides had no effects on CLC-3 protein expression of A2780 cells.MTT assay revealed that A2780 cell proliferation could be inhibited by CLC-3 antisense oligonucleotides.The inhibitory rates of 50μg/ml and 100μg/ml CLC-3 antisense oligonucleotides were 48.6%,65.3%, respectively（vs control,P＜0.05）.In contrast,the 100μg/ml CLC-3 sense oligonucleotides had no significant effect on cell proliferation（vs control,P＞0.05）. Moreover,transfection of A2780 cells with CLC-3 antisense oligonucleotides obviously arrested the progression of cell cycle,which increased the percentage of cells in G₀/G₁ phase,and reduced the percentage of cells in S phase.In 50μg/ml, 100μg/ml CLC-3 antisense-treatment groups,the proportion of G₀/G₁ cells increased from 49.4±3.1%to 70.3±3.4%,75.8±2.6%,respectively（vs control,P＜0.05）; while it decreased significantly from 40.3±2.8%to 23.5±2.1%,19.6±1.5%, respectively（vs control,P＜0.05） in the S phase.In contrast,CLC-3 sense oligonucleotides had no significant effects on cell cycle.After transfection with CLC-3 sense oligonucleotides,the cell population changed from 49.4±3.1%to 50.2±3.6%in the G₀/G₁ phase,from 40.3±2.8%to 39.7±2.9%in the S phase, respectively（vs control,P＞0.05）.Western blot assays indicated that CLC-3 antisense oligonucleotides inhibited cyclinD1 protein expression in A2780 cell line.However in cells treated with sense oligonucleotides,there exhibited no interference effects on cyclinD1 protein expression in A2780 cells.Conclusions:These results strongly indicate that CLC-3 may get involved in proliferation of human epithelial ovarian cancer cell and thus may be a useful therapeutic target.It is likely that the effects of CLC-3 on A2780 cell proliferation and cell cycle is mediated through downregulation of cyclinD1.更多还原