【作者】 张军辉；

【导师】 赵玉林；

【作者基本信息】 郑州大学 ， 耳鼻喉科， 2010， 博士

【摘要】 喉癌是耳鼻喉科最常见的恶性肿瘤,由于喉癌生长部位的特殊性,且手术、放射和化学治疗仍然存在并发症多、毒副作用大的问题,给患者带来较大的痛苦。因此,急需要在分子生物学的基础上提出新的诊断方法和简便、有效的治‘疗手段,这已成为当前研究的热点课题。虽然肿瘤发病机制迄今尚不明了,但可以肯定的是肿瘤的发生发展是多步骤、多阶段和多基因参与并异常表达的过程。很多科研机构研究结果发现肿瘤中存在很小比例的具有干细胞特性的肿瘤细胞,称其为瘤干细胞样细胞(Cancer stem cell-like cells, CSCLC),研究者认为这些CSCLC在肿瘤的起源和发展过程中起着关键的作用。虽然该学说目前的争议较大,但是有一点可以明确的是,肿瘤细胞中应该存在某些调控自我更新的关键基因的异常表达,这是保证肿瘤细胞失控的、异常的、单克隆无限制增殖的基础。生物体内有一系列信号分子维持着干细胞多能性,目前研究的最为广泛是OCT-4。OCT-4基因是POU转录因子家族的一个成员,在胚胎干细胞和成人干细胞上均有表达,参与胚胎发育过程中多向性分化的调节,而且可以控制其无限增殖的特性。目前研究发现OCT-4不仅在多能性胚胎干细胞和原始生殖细胞中表达,在某些恶性生殖细胞肿瘤如精原细胞瘤、胚胎性癌及实体瘤内也检测到了OCT-4的异常表达。有关在喉鳞状细胞癌中OCT-4的表达及其意义的研究,迄今国内外鲜有报道。为深入探讨OCT-4基因与喉癌恶性生物学行为的关系,明确OCT-4在喉癌发生、发展中的作用,寻求喉癌诊断、治疗和预后指标及寻找抑制喉癌发生、发展的有效方法,本研究检测了喉癌组织、癌旁正常组织及声带息肉组织中OCT-4的表达情况,探讨了OCT-4与喉癌恶性生物学行为的关系。并构建了携带OCT-4mRNA编码区cDNA序列和siRNA序列的真核表达载体来研究OCT-4对喉癌Hep-2细胞增殖及p-STAT3/STAT3和Survivin表达水平的影响；检测了丙戊酸钠(VPA)对喉癌Hep-2细胞OCT-4、p-STAT3/STAT3和Survivin表达水平的影响；通过裸鼠移植瘤实验进行体内试验,从多个方面深入探讨OCT-4和VPA对喉癌Hep-2细胞的影响,试图阐明OCT-4的表达在喉癌发生、发展中的意义；并进一步阐明OCT-4基因的抑制和VPA处理对喉癌Hep-2细胞增殖的抑制是否与下调STAT3蛋白磷酸化水平及Survivin的表达有关。为进一步探讨喉癌发生、发展的机制及寻找抑制喉癌发生、发展的治疗途径提供理论基础。本研究共分以下4章。第一章：OCT-4和Survivin在喉鳞状细胞癌组织中的表达及意义方法采用免疫组织化学、Real-time PCR和western的方法检测77例喉癌组织、18例癌旁正常组织及15例声带息肉组织之中OCT-4和Survivin的表达情况。结果喉癌组织OCT-4和Survivin阳性表达高于癌旁正常组织和声带息肉组织,三种喉组织表达差异有统计学意义(P<0.01);OCT-4阳性表达率与喉癌患者病理分级和淋巴结转移有关(P<0.01);Survivin阳性表达率与喉癌患者病理分级、淋巴结转移和T分期有关(P<0.01)。且喉癌组织中OCT-4和Survivin的表达相互之间呈明显的正相关(P<0.05)。第二章：OCT-4对喉癌Hep-2细胞影响的体外研究方法1.构建携带人OCT-4基因mRNA编码区全长cDNA序列和特异性siRNA序列的真核表达质粒载体,转染并筛选出阳性稳定转染细胞克隆。2.采用MTT和平板克隆法测定对照组和各转染组Hep-2细胞增殖和克隆能力。3.采用流式细胞术(FCM)测定对照组和各转染组Hep-2细胞周期和凋亡的情况。4.采用Real-time PCR和Western blot的方法检测对照组和各转染组Hep-2细胞p-STAT3/STAT3和Survivin表达的情况。结果1. pcDNA3.1-OCT-4和pSUPER-EGFP-OCT-4真核表达载体构建及转染Hep-2细胞成功。2.MTT实验结果显示：与对照组相比,pcDNA3.1-OCT-4-2组细胞增殖和克隆能力明显增强(P<0.01),而pSUPER-EGFP-OCT-4-c组细胞增殖和克隆能力明显减弱(P<0.01),pcDNA3.1和pSUPER-EGFP空质粒组则无明显差异(P>0.05)。3.FCM结果显示：与对照组相比,pcDNA3.1-OCT-4-2组G0/G1期细胞比例明显减少(P<0.01),S和G2/M期细胞比例升高(P<0.05); pSUPER-EGFP-OCT-4-c组G0/G1期细胞比例明显升高(P<0.01),S和G2/M期细胞比例减少(P<0.05),同时与各组相比其凋亡率增加；pcDNA3.1和pSUPER-EGFP空质粒组则无明显差异(P>0.05)。4. Real-time PCR和western结果显示：与对照组相比,pcDNA3.1-OCT-4-2组细胞p-STAT3蛋白、Survivin mRNA和蛋白表达升高,而在pSUPER-EGFP-OCT-4-c组表达降低(P<0.01), pcDNA3.1和pSUPER-EGFP空质粒组表达则无明显差异(P>0.05)。第三章：VPA对喉癌Hep-2细胞增殖作用的影响及机制研究方法1.采用MTT方法检VPA作用后Hep-2细胞增殖活性的改变,计算其抑制率。2.采用FCM检测经3mmol/L VPA作用0(对照组)、24、48、72h后Hep-2细胞细胞周期和凋亡情况。3.采用Real-time PCR和western方法检测经3mmol/L VPA作用0(对照组)、24、48、72h后Hep-2细胞OCT-4、p-STAT3/STAT3和Survivin表达的情况。结果：1.MTT结果显示：VPA对Hep-2细胞的生长抑制作用具有剂量和时间依赖性。2.FCM结果显示：经3mmol/L VPA作用后,随着时间的增加,G0/Gl期细胞比例逐渐升高(P<0.01),S期细胞比例逐渐降低(P<0.01),而G2/M期细胞比例无明显变化(P>0.05),细胞凋亡率呈时间依赖性逐渐增加。3. Real-time PCR和Western blot结果显示：经3mmol/L VPA作用后,随着时间的增加,OCT-4 mRNA和蛋白表达逐渐升高(P<0.01), p-STAT3蛋白及Survivin mRNA和蛋白的表达逐渐降低(P<0.01)。第四章：OCT-4和VPA对喉癌Hep-2细胞裸鼠移植瘤的影响及机制研究方法1.建立人喉癌裸鼠移植瘤模型。分为对照组、pcDNA3.1-OCT-4-2组、pSUPER-EGFP-OCT-4-c组和VPA实验组。每周测量瘤体大小,绘制肿瘤体积生长曲线。治疗终止时处死小鼠,剥除肿瘤称重,计算抑瘤率。2.采用TUNEL法检测各组裸鼠移植瘤组织中肿瘤细胞凋亡情况,统计细胞凋亡指数。3.采用Real-time PCR和western检测各组裸鼠移植瘤组织中OCT-4、p-STAT3/STAT3和Survivin表达情况。Results1. pcDNA3.1-OCT-4-2组裸鼠移植瘤体积显著大于对照组裸鼠移植瘤体积,组间比较差异具有统计学意义(P< 0.05); pSUPER-EGFP-OCT-4-c组和VPA组裸鼠移植瘤体积显著小于对照组裸鼠移植瘤体积,组间比较差异具有统计学意义(P<0.05)。2. TUNEL结果显示pSUPER-EGFP-OCT-4-c组和VPA组裸鼠移植瘤组织中凋亡细胞与对照组相比明显增多,组间比较差异具有统计学意义(P<0.01)。3. pcDNA3.1-OCT-4-2组和VPA组裸鼠移植瘤OCT-4 mRNA和蛋白表达显著高于对照组,组间比较差异具有统计学意义(P<0.01); pSUPER-EGFP-OCT-4-c组显著低于对照组,组间比较差异具有统计学意义(P<0.01)。pcDNA3.1-OCT-4-2组裸鼠移植瘤p-STAT3蛋白及Survivin mRNA和蛋白表达显著高于对照组,组间比较差异具有统计学意义(P<0.01)；pSUPER-EGFP-OCT-4-c和VPA组显著低于对照组,组间比较差异具有统计学意义(P<0.01)。STAT3蛋白在每一组都没有明显变化。结论1.OCT-4在喉癌组织中高表达,与喉癌的病理分级和淋巴结转移密切相关,与Survivin的表达相互之间呈明显的正相关；OCT-4促进喉癌Hep-2细胞的增殖和克隆,当抑制其表达时会导致细胞增殖和克隆能力的减弱,阻滞细胞于G0/G1期,诱导凋亡；其机制与抑制p-STAT3和Survivin表达密切相关。2.VPA可以抑制Hep-2细胞的增殖,阻滞细胞于G0/G1期,诱导凋亡。VPA可以下调p-STAT3和Survivin表达来抑制肿瘤的发生、发展。3.体内试验与体外实验结果相似,抑制OCT-4的表达或给与VPA处理均可通过下调p-STAT3和Survivin表达来抑制肿瘤的发生、发展。更多还原

【Abstract】 Laryngeal carcinoma is the most common tumor in ENT, due to the growth of parts of the particularity of laryngeal carcinoma, and there are still more complications, side effects problem of surgery, radiation therapy and chemotherapy, to bring greater suffering for patients. Therefore, an urgent need to proposed the new diagnostic methods and simple effective treatment based on molecular biology has become a hot topic of current research.Although the pathogenesis of cancer has so far not clear, but it is certain that the incidence of tumor development is the multi-step, multi-stage and multi-and abnormal expression of genes involved in the process. Studies in a lot of scientific research institutions found that existing a small percentage of tumor cells that have stem cell characteristics, called tumor stem cell-like cells (CSCLC), the researchers believe that these CSCLC in tumor origin and development process plays a key role. Although the doctrine of the current dispute to a larger, but one thing clear is that the tumor cells should be the existence of the abnormal expression of certain key genes regulating self-renewal, and this is to ensure that tumor cells out of control, abnormal, monoclonal unlimited proliferation basis.Creatures have a series of signaling molecules to maintain stern cell pluripotency, the most widely current study is about OCT-4. OCT-4 gene is a member of POU transcription factor family, expressed in embryonic stem cells and adult stem cells were, involved in embryonic development of multiple regulation of multiple differentiation, and can control the unlimited proliferation of features. Study found that OCT-4, not only expressed in pluripotent embryonic stem cells and primordial germ cells, in some malignant germ cell tumors such as seminoma, embryonal carcinoma and solid tumors also detected abnormal expression of OCT-4. About the research on OCT-4 expression in the laryngeal carcinoma and its significance, so far little has been reported at home and abroad.In order to thoroughly explore the OCT-4 gene and malignant biological behavior; of laryngeal carcinoma, study of the role of OCT-4 in laryngeal carcinoma occurrence and development for diagnosis, treatment and prognosis of laryngeal carcinoma indicators and look for effective methods to inhibit the development of laryngeal carcinoma, this study first detected the expression of OCT-4 in laryngeal carcinoma tissue, adjacent normal tissue and vocal nodules tissue, and explore the relationship between the OCT-4 and malignant biological behavior of in laryngeal carcinoma; Successfully constructed eukaryotic expression vector containing the OCT-4 mRNA code cDNA and the siRNA sequence to detect the impact of OCT-4 on human laryngeal carcinoma Hep-2 cells and Survivin, p-STAT3/STAT3 expression level; detected the impact of sodium valproate (VPA) on laryngeal carcinoma Hep-2 cells OCT-4, p-STAT3/STAT3 and Survivin expression. The final experiment was adoption of xenografts in nude mice in vivo. From several aspects analyzed OCT-4 and VPA impact on the laryngeal carcinoma Hep-2 cells in an attempt to clarify the OCT-4 expression in laryngeal carcinoma occurrence and development and it significance; And to further clarify the relation of OCT-4 gene inhibition and VPA treatment and the inhibition of Hep-2 cells proliferation whether by inhibiting phosphorylation of STAT3 protein and reducing tumor Survivin expression. To further explore the laryngeal carcinoma occurrence and development mechanism and to find inhibition methods for laryngeal carcinoma occurrence and development and provide a theoretical basis for therapeutic approaches. This study is divided into the following four chapters.The first chapter:the expression of OCT-4 and Survivin in laryngeal squamous cell carcinoma and it significanceMethods:The expressions of OCT-4 and Survivin protein were detected in 77 cases of laryngeal carcinoma tissue,18 cases of adjacent normal tissues and 15 cases of vocal nodules tissue by immunohistochemical, Real-time PCR and western.ResultsThe expression of OCT-4 protein and survivin in laryngeal carcinoma tissues were higher than in adjacent normal laryngeal tissues and vocal cords polyps tissues; The expression of OCT-4 had significant relations with histological grade and lymphatic metastasis (P<0.01); The expression of Survivin had significant relations with histological grade、lymphatic metastasis and T staging (P<0.01); There were significantly positive correlations among the expression of OCT-4 and Survivin in laryngeal carcinoma tissues (P<0.05).The second chapter:the impact of OCT-4 on human laryngeal carcinoma Hep-2 cells in vitroMethods:1. Constructed the eukaryotic expression vector with OCT-4 gene mRNA full-length cDNA code sequence and with siRNA sequence, transfected into and screened Hep-2 cells for positive stable transfected cell clones.2. The Hep-2 cell proliferative activity and clones capacity among the control group and the transfected group was observed by MTT and plate cloning method.3. The apoptosis rate and cell cycle distribution of the Hep-2 cells among the control group and the transfected group respectively analyzed by flow cytometry (FCM).4. The expression of p-STAT3/STAT3 and Survivin in Hep-2 cells of the control group and the transfected group was detected by Real-time PCR and Western,Results:1. The pcDNA3.1-OCT-4-2和pSUPER-EGFP-OCT-4-c eukaryotic expression vector were successfully constructed, transfected into Hep-2 cells.2. MTT and Plate cloning results showed that:Compared with the control group, the Hep-2 cell proliferative activity and clones capacity were markedly increased in pcDNA3.1-OCT-4-2 group (P<0.01); while decreased in pSUPER-EGFP-OCT-4-c groups (P<0.01); no significant difference in pcDNA3.1 and pSUPER-EGFP group (P> 0.05).3. FCM results showed that:Compared with the control group, in pcDNA3.1-OCT-4-2 group the proportion of cells in G0/G1 phase decreased (P<0.01), while cells in S and G2/M phase increased(P<0.05); in pSUPER-EGFP-OCT-4-c group the proportion of cells in G0/G1 phase was significantly higher (P<0.01), while cells in S and G2/M phase reduced (P<0.05), at the same time compared with each groups the apoptosis rate increased; pcDNA3.1 and pSUPER-EGFP empty plasmid group had no significant difference (P>0.05)4. Real-time PCR and western results showed that:Compared with the control group, the p-STAT3 protein and Survivin mRNA and protein were markedly increased in pcDNA3.1-OCT-4-2 group(P<0.01); while decreased in pSUPER-EGFP-OCT-4-c group(P<0.01); no significant difference in pcDNA3.1 and pSUPER-EGFP groups(P> 0.05).The third chapter:the impact of VPA on the Hep-2 cells proliferation and it mechanismMethods:1. The proliferative activity of the Hep-2 cells treated with VPA was observed by MTT method.2. The apoptosis rate and cell cycle distribution of the Hep-2 cells treated with 3mmoL/L VPA at 0 (control),24,48,72 h was detected by FCM.3. The expression of OCT-4 and Survivin in Hep-2 cells after treated with 3mmoL/L VPA at 0 (control),24,48,72 h were detected by Real-time PCR and Western.Results:1. MTT results showed that:the VPA inhibited Hep-2 cell growth in a dose-and time-dependent manner.2. FCM results showed that:After 3mmoL/L VPA treatment, along with the time increasing the cells in G0/G1 phase increased gradually (P<0.01), cells in S phase decreased gradually (P<0.01), while the cells in G2/M phase no significant change (P> 0.05), the rate of apoptosis was time-dependent increase gradually.3. Real-time PCR and western results showed that:After 3mmoL/L VPA treatment, along with the time increasing OCT-4 mRNA and protein expression gradually increased (P<0.01), while p-STAT3 protein Survivin mRNA and protein expression decreased gradually (P<0.01).The forth chapter:the study on the impact of OCT-4 and VPA on the Hep-2 cell xenografts in nude mice and its mechanismMethods:1. After establishment of human laryngeal carcinoma xenografts in nude mice were divided into control group, pcDNA3.1-OCT-4-2 group, pSUPER-EGFP-OCT-4-c group and the VPA experimental group, Weekly measureed each tumor size and nude body mass, drawn tumor volume growth curves. The mice were killed and removed tumor weighed to calculate tumor inhibition rate at termination of treatment.2. The apoptosis in xenografts tissue in nude mice of each group were detected by TUNEL.3. the expression OCT-4、p-STAT3/STAT3 and Survivin in tumor tissue of each nude mice group were detected by Real-time PCR and western.Results1. The volume of the tumors in pcDNA3.1-OCT-4-2 nude mice group was significantly higher than the control group, the difference between the two groups was statistically significant (P<0.05); The tumor volume in pSUPER-EGFP-OCT-4-c group and; the VPA Group were significantly smaller than the tumor volume in control group, the difference among the three groups were statistically significant (P<0.05).2. TUNEL results showed that the apoptotic cells of nude mice tumor tissue in pSUPER-EGFP-OCT-4-c group and VPA group significantly increased than control group, the difference among the three groups was statistically significant (P<0.01).3. The protein and mRNA expression of OCT-4 gene in pcDNA3.1-OCT-4-2 group and VPA group were significantly higher than the control group, the difference among the three groups was statistically significant (P＜O.01); while in pSUPER-EGFP-OCT-4-c group was significantly lower than the control group, the difference between the two groups was statistically significant(P<0.01). The expression of p-STAT3 protein and Survivin mRNA and protein in tumor of pcDNA3.1-OCT-4-2 group was significantly higher than the control group, the difference between the two groups was statistically significant (P<0.01); while in pSUPER-EGFP-OCT-4-c group and VPA group was significantly lower than the control group, the difference among groups were statistically significant (P<0.01); each group was no obvious STAT3 protein in the change.Conclusion:1. The expression of OCT-4 protein were higher in laryngeal carcinoma tissues; The expression of OCT-4 had significant relations with histological grade and lymphatic metastasis, and have significantly positive correlations with the expression of Survivin in laryngeal carcinoma tissues; The OCT-4 gene can promote the Hep-2 cell proliferation activity and cloning capability, while inhibiting the OCT-4 expression will lead to cell proliferation activity and cloning capability inhibition, block cells in the Go/Gi phase and induce apoptosis, its mechanism were related to inhibition of p-STAT3 and Survivin expression.2. VPA can inhibit the proliferation of laryngeal carcinoma Hep-2 cells, blocking cell in the Go/G1 phase and induce apoptosis; VPA can inhibit p-STAT3 and Survivin expression to participate in tumorigenesis and development.3. Results in vivo and in vitro experiments were similar that OCT-4 can regulate p-STAT3 and Survivin expression to participate in tumor occurrence and development.更多还原