【作者】 高瑞；

【导师】 王泽华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 博士

【摘要】 在妇女生殖器肿瘤中，卵巢癌是最常见的三大恶性肿瘤之一，其五年生存率较其它妇科肿瘤明显降低。因此寻找有效的治疗方法，是提高卵巢癌患者生存率的关键。离子通道是几乎分布于机体每一个细胞膜上的亲水性微孔通道，它能够选择性的允许带电离子通过被动运输或主动扩散参与维持和调节机体的一系列基本活动。随着膜片钳技术和分子生物学的进展，离子通道与肿瘤之间的相关性已成为新的研究热点。目前关于离子通道的研究主要集中在钾离子通道、钙离子通道和氯离子通道等与肿瘤细胞增殖、耐药、转移的关系。最近有学者发现在肿瘤细胞的正常膜电位时存在有“窗口电压”，在此电压范围内由于钠离子通道的不完全失活或部分激活引起了的持续内向钠电流，导致细胞内离子稳态失衡或相关信号传导通路的异常变化。因此，钠离子通道在肿瘤发生发展中的作用也引起了人们的关注。电压门控钠通道(voltage-gate sodium channel，VGSC)是可兴奋组织和细胞产生电冲动的关键离子通道，由起主要功能的α亚单位和附属的β亚单位组成。根据基因结构和分布特点的差异，α亚单位可分为Nav1家族(Nav1.1至Nav1.9)和Nax。由于Nax和Nav1家族的结构相差较远，也没有发现其有实质性的功能，因此目前研究最多的主要是Nav1家族的九个成员。该家族的每个成员都由四个十分相似的结构域组成(D1-D4)，每个结构域含有六个跨膜片段(S1-S6)。选择性剪接是真核生物调控基因表达的重要方式之一。人类基因通过RNA的选择性剪接所产生的转录本能翻译成具有不同表型效应的蛋白质。但错误的选择性剪接却可能导致肿瘤的发生，甚至有些是肿瘤所特有的，通过剪接产生的变异体本身就可能成为肿瘤的治疗靶点。目前在肿瘤细胞中已发现有VGSC基因剪接变异体的功能性表达，尤其在D1∶S3 5’位置的剪接变异体还赋予肿瘤细胞转移的潜能，并且发现D1∶S35’剪接变异体随着个体的发育而迅速消失。因此，VGSC的D1∶S3 5’剪接变异体可能属于癌胚基因。目前已知的多种钠通道阻断剂对VGSC亚型并不是特异性的，在阻断目的亚型的同时也阻断了其他亚型。由于VGSC不同亚型参与不同肿瘤的发生发展，因此通过开发新的靶向VGSC亚型胞外肽段抗体来实现肿瘤的个体化治疗已经成为可能。第一部分电压门控钠通道Nav1家族在卵巢癌中表达的初步研究目的：通过研究卵巢癌细胞中电压门控钠通道(voltage-gated sodium channel，VGSC)的表达，探讨VGSC Nav1家族在卵巢癌转移过程中的作用。方法：以正常卵巢组织为对照，在卵巢癌细胞株Caov-3，SKOV3和Anglne中用RT-PCR检测VGSC Nav1家族mRNA的表达，用SBFI荧光探针法观察卵巢癌细胞内钠离子的分布，用MTT和Transwell小室法分别检测VGSC特异性阻断剂河豚毒素(tetrodotoxin，TTX)对卵巢癌细胞增殖活性、迁移性和侵袭性的影响。结果：在卵巢癌细胞中检测到有Nav1.1、Nav1.3、Nav1.4、Nav1.5和Nay1.7 mRNA的异常表达。钠离子主要分布于卵巢癌细胞的胞膜和胞浆中，尤其包膜中比较丰富。30μM TTX对卵巢癌细胞的增值活性没有影响，却能抑制卵巢癌高转移细胞(Caov-3和SKOV3)50％左右体外迁移和侵袭力，与处理前比较差异有统计学意义，P＜0.05；而TTX对卵巢癌低转移细胞Anglne的生物学特性没有明显影响。结论：电压门控钠通道Nav1家族的异常表达与卵巢癌的体外迁移和侵袭相关，可能成为新的肿瘤治疗靶点。第二部分电压门控钠通道亚型SCN5A／Nav1.5在卵巢癌中表达的研究目的：电压门控钠通道(voltage-gated sodium channel，VGSC)与多种恶性肿瘤的转移过程密切相关，其不同亚型参与不同肿瘤的发生发展。本研究主要探索VGSC亚型SCN5A／Nav1.5在人卵巢癌细胞和组织中功能性表达的意义。方法：以正常卵巢组织为对照，通过Real-time PCR、Western Blot、免疫细胞化学和免疫组织化学方法分别检测卵巢癌细胞Caov-3和上皮性卵巢癌肿瘤组织中SCN5A／Nav1.5在mRNA和蛋白水平的表达及其与卵巢肿瘤发生发展的相关性。结果在卵巢癌细胞Caov-3和上皮性卵巢癌组织中存在SCN5A／Nav1.5的异常表达。与正常卵巢组织相比较，Caov-3细胞和卵巢癌组织中Nav1.5 mRNA的相对表达量分别增加13.79±2.78倍和14.57±1.49倍，P＜0.05；而在良性卵巢肿瘤组织中没有发现Nav1.5 mRNA表达量的明显增加(1.02±0.26 vs 1.18±0.40)。Caov-3细胞和卵巢肿瘤组织中Nav1.5蛋白水平的表达变化与mRNA的表达变化基本一致。结论电压门控钠通道SCN5A／Nav1.5在卵巢癌的发生发展过程中起重要的作用，有可能成为卵巢癌治疗的靶标。第三部分电压门控钠通道亚型Nav1.5剪接变异体在卵巢癌中表达的研究目的：电压门控钠通道(voltage-gate sodium channel，VGSC)的选择性剪切被认为是钠通道蛋白形成多样性结构和功能的关键。本研究主要探索VGSC亚型Nav1.5剪接变异体在卵巢癌细胞Caov-3中的功能性表达。方法：以正常卵巢组织为对照，通过RT-PCR、DNA测序和Western blot分别检测卵巢癌细胞Caov-3中Nav1.5表达的剪接变异体类型。结果：Western blot和RT-PCR均检测发现在Caov-3细胞中有Nav1.5剪接变异体的异常表达，而在正常卵巢组织中却没有检测到；DNA测序发现卵巢癌细胞Caov-3中异常表达的Nav1.5主要D1∶S3 5’型剪接变异体。结论：在卵巢癌细胞中发挥主要作用的VGSC剪接变异体D1∶S3 5’型Nav1.5可能成为卵巢肿瘤新的标记物和治疗靶点。第四部分Nav1.5-E3抗体对卵巢癌细胞Caov-3迁移和侵袭力的抑制效应目的：本研究以人卵巢癌细胞Caov-3为研究对象，观察靶向电压门控钠通道(voltage-gated sodium channel，VGSC)亚型Nav1.5细胞外区域E3的特异性抗体(NaV1.5-E3抗体)对其体外迁移和侵袭性的影响。方法：通过激光共聚焦检测检Nav1.5-E3-抗体识别卵巢癌细胞Caov-3中Nav1.5的特异性，CCK-8试剂盒和Transwell小室法分别检测Nav1.5-E3抗体阻断Nav1.5活性后，卵巢癌细胞Caov-3的增值活性以及体外迁移和侵袭力的变化。结果：Nav1.5-E3抗体能特异性检测出Caov-3细胞上的Nav1.5：Nav1.5-E3抗体(16μg／ml)处理后，Caov-3细胞的增殖活性没有明显变化，但体外迁移和侵袭力减少为处理前的(64.14±8.39)％和(52.80±7.99)％，两组间比较差异有统计学意义，P＜0.05。结论：Nav1.5-E3抗体能显著抑制卵巢癌细胞Caov-3体外迁移和侵袭力，可能为卵巢肿瘤的治疗提供新的方法指导。更多还原

【Abstract】 Ovarian cancer is one of the three most common cancers among gynecologicalmalignancies in the world.Because of no significant symptom in the early stage, theoverwhelming majority of patients are found at the advanced stage and its five-year survivalrate is also the lowest in gynecological malignancies.It is necessary to find effective therapymethods to increase its survival rate.Ion channels are functionally expressed in amolst every tissue and cell as the mainsignaling molecules, and they also participate in determining many cellular basic behaviors.With the development of patch clamp technique and molecular biology, many researcheshave revealed that ion channels were associated with different aspects of carcinogenicprocess by involveing in cell proliferation, multidrug resistance, migration and invasion.The functional expression of potassium, calcium and chloride channels have been widelyinvestigated over the past few decades, but little is known about sodium channel intumorigenesis.Recently study have reported that there were a window of voltage at cancercellular normal membrane potential, where there is a little and continuous entry of sodiumbecause of the partial opening or closing of voltage-gated sodium channel (VGSC), and theentry sodium might be responsible for the disrupting intracellulare ion homeostasis andsome signaling pathways.Therefore, it is important to investigat the roles of VGSC incarcinogenic process.VGSC are manly expressed in excitable cells and play importantroles in the initial and propagate of action potentials, which are composed of principalαsubunit and auxiliaryβsubunit.As we have known, based on the differences of phylogeny,αsubunit consists of Nav1 family and Nax.Recently studies have reported that theincreased functional expressions of Nav1 family (Nav1.1-1.9) were involved in thedevelopment and progression of cancer.Nav1 family consists of 4 homologous domains(D1-D4) and every homologous domain having 6 transmembrane segments (S1-S6).Alternative splicing is the important and ubiquitous process in regulating multiplicitygene expression of eucaryote.It is important for cell differentiation, so aberrations of alternative splicing are important for tumorigenesis.VGSC genes also code diversity andplasticity functional splice variants protein thought alternative splicing.Among thesesplice variants, the D1:S3 5’ genomic splice have been found potentiation of cancermetastasis.In addition the splice variants of D1:S3 5’ were also expressed in neonatal orfetal rat, while it rapidly disappeared with the development of organism.Therefore, VGSCmight be embryonic gene, silent in mature cells and re-expressed in cancer cells.At present, the majority sodium channel blockers on sodium channel subtypes are notspecific, which not only inhibit the abnormal subtypes but the normal subtypes.Due to thedifferent subtypes of VGSC participating in the development of different cancers, it isnecessary to design new antibodies targeting extracellular peptide of VGSC.Part One Functionally Expressed of Voltage-gated SodiumChannel Nav1 Family in Human Ovarian CanerObjective: Voltage-gated sodium channel (VGSC) subtypes play important roles in thebionomics of many tumors.This study was to explore the effect of VGSC on metastasisbehaviors of human ovarian cancer cell lines.Methods: RT-PCR and SBFI confacol assayswere respectively used to detect the functional expression of VGSC Nav1 family in ovariancancer cells (Caov-3, SKOV3, Anglne) and normal ovary tissues; MTT and Transwellassays were respectively used to detect the effect of specific VGSC inhibitor tetrodotoxin(TTX) on cell proliferation, migration and invasion of ovarian cancer in vitro.Results: ThemRNA expression levels of Nav1.1, Nav1.3, Nav1.4, Nav1.5 and Nav1.7 wereup-regulation in ovarian cancer cell lines; the distribution of intracellular sodium wasmainly in cell membrane.30μM TTX could decrease the migration and invasion ofstrongly metastatic ovarian cancer cell Caov-3 and SKOV3 to 40%-60%; while TTX hadno obvious effects on weakly metastatic cell Anglne.Conclusion: The functionalup-regulation of Nav1 family in human ovarian cancer might participate in the matastasis of ovarian cancer and be a new therapeutic target.Part Two Functional Expressions of Voltage-gated SodiumChannel SCN5A/Nav1.5 in Human Ovarian CanerObjective: The different subtypes of voltage-gated sodium channel (VGSC) are known tobe correlated with the initiation and progression of many malignant cancers.This study wasto investigate the functional expressions and roles of SCN5A/Nav1.5 on human ovariancancer.Methods: Real-time PCR, Western Blot, Immunocytochemistry andImmunohistochemistry assays were respectively used to detect the expression ofSCN5A/Nav1.5 in mRNA and protein levels in epithelial ovarian tumor specimens andovarian cancer cells lines Caov-3.Results: SCN5A/Nav1.5 was over-expressed in ovariancancer specimens and ovarian cancer cells lines Caov-3.Compared with normal ovaryspecimens, their relative mRNA expression were increased by 13.79±2.78 fold and14.57±1.49 fold, P<0.05; while the Nav1.5 mRNA in benign ovarian tumor specimens haveno obvious up-regulation than that in normal ovary (1.02±0.26 vs 1.18±0.40).Their proteinlevels differences among Caov-3 cells and ovarian tumor specimens were similar to themRNA expression.Conclusion: SCN5A/Nav1.5 plays important roles in the progression ofovarian cancer, which might be a therapeutic target in anti-ovarian cancer research.Part Three Functional Expression of Voltage-gated SodiumChannel Nav1.5 Splice Variants in Ovarian CancerObjective: Aberrations of alternative splicing play important roles in tumorigenesis.Thisstudy was to explore the functional expression of VGSC Nav1.5 splice variants in ovarian cancer cell Caov-3.Methods: RT-PCR, DAN sequencing and Western blot assays wererespectively used to detect the alternative splicing of VGSC Nav1.5 in ovarian cancer cells.Results: DNA sequencing of RT-PCR products revealed that Nav1.5c, the D1:S3 5’ spliceform of Nav1.5 was the mainly splice variants existed in Caov-3 cells.Western blot assayrevealed that the Nav1.5 splice variants were up-regulation in Caov-3 cells but absent innormal ovary tissues.Conclusions: D1:S3 5’ Nav1.5 might play important roles in themetastatic process of human ovarian cancer and could serve as a therapeutic target incancer research.Part Four E3-targeted anti-Nav1.5 antibody inhibits themigration and invasion of ovarian cancer cells in vitroObjective: The purpose of this study was to investigate the effects of the third extracellulartargeted anti-Nav1.5 antibody (Nav1.5-E3 Ab) on in vitro metastatic cascades of ovariancancer cells Caov-3.Methods: Using confocal microscopy detect the specific and sensitiveof Nav1.5-E3-Ab in Nav1.5 expressed in Caov-3 cells; using CCK-8 kit and Transwellassays analyzed the effects of Nav1.5-E3-Ab on the migration and invasion of ovariancancer cell Caov-3 in vitro.Results: Nav1.5-E3-Ab could specific recognize the Nav1.5existed in Caov-3 cells and Nav1.5-E3 Ab (16μg/ml) reduced the migration and invasion ofCaov-3 cells to (64.14±8.39) % and (52.80±7.99) % with the same extent as tetrodotoxin(TTX), a specific blocker of VGSC.Conclusion: Nav1.5-E3-Ab could significantlysuppress the metastasis behaviours of ovarian cancer cell Caov-3 and it might be a newpromise for the treatment of ovarian tumors.更多还原