【作者】 张晓英；

【导师】 孙善珍； 曲迅；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 背景和目的口腔鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)是头颈部最常见的恶性肿瘤之一。目前,口腔鳞状细胞癌的临床治疗方法仍然是以手术治疗为主,并辅以放射治疗和化学药物治疗。近年来,随着手术方式的不断改进以及辅助手段的加强,口腔鳞状细胞癌的治疗效果也逐步得以改善,但其五年生存率仍不足50%。随着分子生物学研究的不断深入,肿瘤的基因治疗已经成为恶性肿瘤治疗研究的新热点。由于基因治疗具有特异性强、损伤小、对原发灶及转移灶均有效的优点,已经成为继手术、放疗和化疗之后的新的有希望的治疗手段。因此,寻找可用于口腔鳞状细胞癌治疗的靶基因对于口腔鳞状细胞癌的治疗具有十分重要的意义生长抑制和DNA损伤基因(growth arrest- and DNA damage-inducible,Gadd)45基因家族由Gadd45α/Gadd45a, Gadd45β/Gadd45b/myd118及Gadd45γ/Gadd45g/cr6组成。其中,Gadd45a基因是DNA损伤后诱导表达的基因之一,也是抑癌基因p53和乳腺癌相关蛋白1(Breast Cancer Associated Protein 1,BRCA1)的下游靶基因。细胞DNA损伤后,Gadd45a基因可以通过p53依赖及非依赖两条途径被诱导表达升高,参与细胞周期监测点、细胞凋亡、DNA损伤修复及信号传导等重要细胞生命活动的调节,并由此介入了对肿瘤发生、发展的调控。已有研究发现,Gadd45a在乳腺癌、肺癌、前列腺癌等多种恶性肿瘤中均存在表达异常。但是,国内外关于Gadd45a与口腔鳞状细胞癌关系的研究尚未见报道。为了明确Gadd45a在人口腔鳞状细胞癌发生、发展中的作用,进一步以Gadd45a为靶点进行基因治疗提供实验依据,本论文从以下三个方面进行了研究：1. Gadd45a在口腔鳞状细胞癌组织中的表达及其临床意义2. Gadd45a基因沉默对舌鳞状细胞癌Tca8113细胞生物学性状的影响；3. Gadd45a基因沉默对舌鳞状细胞癌Tca8113细胞放疗敏感性的影响。实验方法1.探讨Gadd45a在口腔鳞状细胞癌中的表达及其临床意义1.1.病例的收集：收集山东大学齐鲁医院2003-2009年切除的原发性人口腔鳞状细胞癌手术标本106例,患者均行肿瘤局部扩大切除术及颈淋巴清扫术,术后病理明确诊断。患者的临床资料包括年龄、性别、肿瘤组织学分级、肿瘤临床分期及有无淋巴结转移等同时被收集整理。另外选取60例口腔鳞状细胞癌患者的癌旁组织作为对照。1.2.采用免疫组化方法检测Gadd45a在口腔鳞状细胞癌及癌旁组织中的表达及定位,并分析Gadd45a蛋白表达及定位与患者临床资料之间的关系。1.3. x2检验被用来分析Gadd45a在口腔鳞状细胞癌中的表达与患者年龄、性别、肿瘤组织学分级、临床分期及淋巴结转移之间的关系。2.观察Gadd45a基因沉默对Tca8113细胞生物学性状的影响2.1.采用免疫荧光技术观察Gadd45a蛋白在Tca8113细胞中的表达定位。2.2.采用RNA干扰技术沉默Tca8113细胞中Gadd45a基因的表达：设计并合成Gadd45a的特异性干扰序列及无意义对照序列,转染Tca8113细胞,分别于转染后48h及72h收集细胞,通过real time quantitive RT-PCR及Western Blot方法检测Gadd45a-siRNA的特异性及有效性。2.3.采用MTT法检测Gadd45a基因沉默对Tca8113细胞增殖能力的影响：将5×103个Tca8113细胞接种于96孔板,待细胞汇合度达40%时,将Gadd45a-siRNA及对照序列转染Tca8113细胞,并于转染后24h、48h及72h,用Biotek酶联免疫检测仪检测细胞在490nm的吸光度值。2.4.特异性靶向人Gadd45a的siRNA序列及无意义对照序列分别转染Tca8113细胞,并于转染后24小时收集细胞,利用流式细胞技术检测Gadd45a基因沉默对Tca8113细胞周期的影响。2.5.将转染Gadd45a-siRNA及无意义对照序列的Tca8113细胞以0.25%的胰酶消化、计数并接种于Transwell上层小室,置37℃培养箱培养16h,观察Gadd45a基因沉默对Tca8113细胞迁移能力的影响。3. Gadd45a基因沉默对Tca8113细胞放疗敏感性的影响3.1.单纯放射治疗对Tca8113细胞生物学性状的影响1)对数生长期的Tca8113细胞接种于6孔板,待细胞达到70%融和时,分别给予0Gy、4Gy、10Gy的射线照射,照射后将培养板放入37℃培养箱继续培养24h,通过倒置显微镜及细胞HE染色观察不同剂量射线照射对Tca8113细胞形态学的影响。2)对数生长期的Tca8113细胞接种于6孔板,待细胞达到70%融和时,分别给予0Gy、2Gy、4Gy、8Gy、10Gy的射线照射,照射后将培养板放入37℃培养箱继续培养24h,通过MTT实验观察不同剂量放射线对Tca8113细胞增殖的影响。3)对数生长期的Tca8113细胞接种于6孔板,待细胞达到70%融和时,分别给予0Gy、2Gy、4Gy、8Gy、10Gy的射线照射,照射后将培养板放入37℃培养箱继续培养24h,收集细胞,采用流式细胞技术检测放射线对Tca8113细胞凋亡的影响。4)对数生长期的Tca8113细胞接种于6孔板,待细胞达到70%融和时,分别给予0Gy、4Gy、10Gy的射线照射,照射后将培养板放入37℃培养箱继续培养24h,收集细胞,采用流式细胞技术分析放射线对Tca8113细胞周期的影响。3.2.放射治疗对Tca8113细胞Gadd45a基因表达的影响：将对数生长期的Tca8113细胞接种6孔板,待细胞达到70%融和时,分别给予OGy、4Gy、10Gy的射线照射,照射后将培养板放入37℃培养箱继续培养24h,通过免疫细胞化学、real time quantitive RT-PCR和Western Blot方法检测放射线对Tca8113细胞Gadd45a表达的影响。3.3.放射合并Gadd45a基因沉默对Tca8113细胞生物学性状的影响1)对数生长期的Tca8113细胞接种6孔板,待细胞达到40%融和时,将Gadd45a-siRNA及对照序列转染Tca8113细胞,并于转染后24h,分别给予0Gy、2Gy、4Gy、8Gy、10Gy的射线照射照射后将培养板放入37℃培养箱继续培养24h,利用MTT法观察Gadd45a-siRNA转染组与对照组经射线照射后细胞存活情况。2)对数生长期的Tca8113细胞接种6孔板,待细胞达到40%融和时,将Gadd45a-siRNA及对照序列转染Tca8113细胞,并于转染后24h,分别给予0Gy、4Gy、10Gy的射线照射,照射后将培养板放入37℃培养箱继续培养24h,流式细胞术分析Gadd45a-siRNA转染组与对照组经放射线照射后细胞凋亡情况。实验结果1. Gadd45a在口腔鳞状细胞癌中的表达及其临床意义口腔鳞状细胞癌及癌旁组织的免疫组化染色结果显示：口腔鳞状细胞癌及癌旁组织均高表达Gadd45a蛋白。并且,Gadd45a蛋白表达存在两种表达模式,一种是细胞浆和细胞核均着色,细胞核占优势,我们称之为细胞核模式；另一种是细胞浆着色明显,细胞核不着色或着色浅,我们称之为细胞浆模式。60例癌旁组织中,Gadd45a的表达均为细胞核表达模式。而在106例口腔鳞状细胞癌组织中,只有60例表现为细胞核模式,其余46例的阳性着色部位以细胞浆为主,细胞核着色浅或不着色,为细胞浆模式。近一步的统计分析表明,口腔鳞状细胞癌组织中Gadd45a表达模式的转换与患者的年龄、肿瘤的组织学分级、肿瘤临床分期及淋巴结转移之间存在密切的关系。年龄＜60岁的肿瘤患者组织中,Gadd45a的表达以细胞浆模式为主,而年龄＞60岁的肿瘤患者组织中,Gadd45a的表达以细胞核模式为主(p<0.05)。Gadd45a表达模式在不同组织学分级的口腔鳞状细胞癌之间存在显著的差异(p＜0.05),高分化及中分化口腔鳞状细胞癌组织中Gadd45a的表达分布以细胞核模式为主,而在低分化口腔鳞状细胞癌组织中Gadd45a的表达分布以细胞浆模式为主。72.2%的Ⅰ、Ⅱ期肿瘤患者组织中Gadd45a的表达以细胞核模式为主,而在Ⅲ、Ⅳ期肿瘤患者中以细胞核模式表达Gadd45a的组织标本只占48.6%,二者间存在显著的差异(p＜0.05)。在存在淋巴结转移的肿瘤患者组织标本中,以细胞浆模式表达Gadd45a的比例明显高于无淋巴结转移的患者(p＜0.05)2. Gadd45a基因沉默对Tca8113细胞生物学性状的影响2.1. Gadd45a在Tca8113细胞中的表达：为了研究Gadd45a基因在口腔鳞状细胞癌发生、发展中的作用,我们选择高分化的舌鳞状细胞癌细胞系Tca8113作为研究对象。细胞免疫荧光染色结果显示,Gadd45a在Tca8113细胞中广泛表达,其着色部位主要在细胞核,与高分化口腔鳞状细胞癌组织标本中Gadd45a的表达模式基本一致。2.2. Gadd45a-siRNA干扰效率的检测：为了证明Gadd45a-siRNA的特异性及有效性,real time quantitive RT-PCR及Western Blot方法被用来检测Gadd45a-siRNA转染组和对照组Tca8113细胞中Gadd45a的表达,结果显示,靶向Gadd45a的siRNA有效抑制了Tca8113细胞中Gadd45a mRNA及蛋白水平的表达。2.3. Gadd45a基因沉默对Tca8113细胞增殖的影响：我们利用MTT法检测了Gadd45a-siRNA转染组与对照组Tca8113细胞的增殖能力,结果显示,转染Gadd45a-siRNA的Tca8113细胞在转染后48h及72h的增殖能力较对照组明显增强。2.4. Gadd45a基因沉默对Tca8113细胞周期的影响：利用siRNA技术将Tca8113细胞中的Gadd45a基因沉默以后24h,收集细胞通过流式细胞仪检测细胞周期的变化,结果显示,Gadd45a基因沉默导致Tca8113细胞G2/M期监测点异常,处于G2期的细胞比例明显减少,而S期细胞的比例显著增多(p＜0.05)2.5. Gadd45a基因沉默对Tca8113细胞迁移能力的影响：Gadd45a-siRNA转染组及对照组Tca8113细胞接种于Transwell上层小室,37℃培养箱培养16h,计数迁移的细胞数,观察Gadd45a基因沉默对Tca8113细胞迁移能力的影响,结果显示,Gadd45a-siRNA转染组迁移的细胞数明显高于对照组(p＜0.05),表明Gadd45a基因沉默增强了Tca8113细胞的迁移能力。3. Gadd45a基因沉默对Tca8113细胞放疗敏感性的影响3.1.单纯射线照射对Tca8113细胞生物学性状的影响1)我们通过倒置显微镜及HE染色技术观察了不同剂量射线照射对Tca8113细胞形态的影响,结果发现Tca8113细胞经10Gy射线照射后24h,细胞体积变大,细胞间隙变宽,凋亡及坏死漂浮的细胞增多,细胞密度明显低于未照射或低剂量照射组细胞。2) MTT实验被用来观察放射线对Tca8113细胞增殖的影响,结果显示,4-10Gy放射线有效抑制了Tca8113细胞的增殖,且呈剂量依赖性3) Annexin V-PI细胞凋亡双染试剂盒被用于检测放射线诱导的Tca8113细胞的凋亡,结果显示,4-10Gy射线照射明显诱导了Tca8113细胞的凋亡,且呈剂量依赖性。4)细胞周期PI染色显示,10Gy射线照射导致了Tca8113细胞G2/M期的阻滞,使处于G1及S期的细胞明显减少3.2.放射线照射诱导了Tca8113细胞Gadd45a基因的表达上调：为了研究Gadd45a在口腔鳞状细胞癌放疗中的作用,我们首先通过real time quantitive RT-PCR和Western Blot观察了Tca8113细胞在接受射线照射前后Gadd45a表达的变化情况,结果发现,Tca8113细胞在接受4Gy或10Gy射线照射后24小时,Gadd45a mRNA水平较照射前明显升高(0.000965±0.00005 vs 0.000387±0.00002；0.001644±0.000065 vs 0.000387±0.00002)。并且10Gy射线照射亦显著诱导了Tca8113细胞Gadd45a蛋白的表达上调(p=0.0028)。3.3. Gadd45a基因沉默合并射线照射对Tca8113细胞生物学性状的影响：为了检测Gadd45a基因沉默对Tca8113细胞放疗敏感性的影响,我们首先利用real time quantitive RT-PCR和Western Blot检测了Gadd45a-siRNA的有效性,结果显示Gadd45a-siRNA有效抑制了Tca8113细胞中Gadd45a基因及蛋白水平的表达(p＜0.05)。随后我们采用MTT及流式细胞技术观察了siRNA实验组及对照组Tca8113细胞在接受不同剂量射线照射时细胞存活分数及凋亡情况的变化。MTT结果显示,Tca8113细胞在受到4Gy及以上剂量射线照射时,siRNA实验组细胞存活分数明显高于对照组(p＜0.05)。流式细胞技术检测结果也显示Tca8113细胞在受到4Gy及10Gy射线照射时,Gadd45a基因沉默组细胞的凋亡受到明显抑制,细胞凋亡分数较对照组明显下降(p＜0.05)结论1. Gadd45a在人口腔鳞状细胞癌中存在细胞浆-细胞核表达模式的转换,并且Gadd45a的表达模式与口腔鳞状细胞癌的组织学分级、TNM分期及淋巴结转移密切相关。2. Gadd45a在人舌鳞状细胞癌细胞中的表达缺失促进了癌细胞的生长和迁移,其机制可能是通过干扰细胞周期调控以及抑制细胞发生凋亡。3.放射诱导的人舌鳞状细胞癌细胞中Gadd45a基因表达升高促进了细胞凋亡,增强了人舌鳞状细胞癌细胞对放疗的敏感性。创新性和意义1.本研究的创新点是在国内外首次发现Gadd45a在人口腔鳞状细胞癌中的表达存在细胞浆-细胞核表达模式的转换,并且与患者的组织学分级及TNM分期有关。并通过体外实验进一步证实,Gadd45a基因表达缺失促进了舌鳞状细胞癌细胞的生长及迁移。为以Gadd45a为靶点进行基因治疗提供了理论依据。2.本研究首次采用RNA干扰技术揭示,放射诱导的舌鳞状细胞癌细胞中Gadd45a的表达升高增强了人舌鳞状细胞癌细胞对放疗的敏感性,为探索口腔鳞状细胞癌放疗增敏的靶基因提供了新的思路和方向。更多还原

【Abstract】 Background and ObjectiveOral squamous cell carcinoma is the most common tumor that arises in the head and neck. To date, the major clinical therapy methods are still operation, supplemented with radiotherapy and chemotherapy. In recent decades, although the treatment technology is gradually improving, the patient’s five-year survival rate is still less than 50%. With the deepening of molecular biology, cancer gene therapy research has become a new hot spot for cancer treatment. Therefore, studies of the molecular biology of the tumor are key issues to get insight into the mechanisms underlying the pathogenesis of oralsquamous cell carcinoma and, in turn, leading to more effective therapeutics finall y.The growth arrest-and DNA damage-indueible (gadd) 45 gene family, comprising Gadd45α/Gadd45a, Gadd45β/Gadd45b/myd118, and Gadd45γ/Gadd45g/cr6, is widely expressed in mammalian cells responding to stress stimuli.Gadd45a was the first stress-inducible gene found to be activated by the p53 tumour suppressor. Subsequently, Gadd45a was also shown to be a target of the BRCA1 (Breast Cancer Associated Protein 1). Gadd45a is a p53-regulated growth arrest and DN A-damage-inducible gene that is also regulated in a p53-independent manner. Gadd45a has been demonstrated to link to many important cellular processes such as DNA repair, chromatin accessibility, cell cycle checkpoints and genome stability. Whether Gadd45a plays a direct role in the progression and prognosis of oral squamous cell carcinoma remains unclear.In present study, we elucidate the significance of Gadd45a in the progression, treatment and prognosis of oral squamous cell carcinoma from three main points:1. The expression state and clinical significance of Gadd45a in human oral squamous cell carcinoma;2. To investigate the effect of Gadd45a-siRN A on cell growth and invasion of Tca8113 cells;3. To investigate the effect of Gadd45a-siRNA on radiosensitivity of Tca8113 cells.Materials and methods1. The expression status and clinical significance of Gadd45a in human oral squamous cell carcinoma1.1. The study included 106 patients with OSCC who underwent primary surgical resection at Qilu Hospital of Shandong University between 2003 and 2009. Sixty specimens of adjacent oral mucosa tissue were collected as control samples. The clini copathological information, including sex, age, tumor stage, and histological grade, was obtained from the clinical records.1.2. The Gadd45a protein expression in 106 cases of OSCC and 60 cases normal oral mucosa tissue were detected by immunohistochemistry.1.3. The relationship between the Gadd45a expression and the pathological characteristics was analyzed by SPSS statistical software. Chi-square test was used to analyse the data. Differences were considered significant at p< 0.05.2. The effect of Gadd45 a-siRN A on cell growth and invasion of Tca8113 cells2.1. The Gadd45a protein expression in Tca8113 cells was analyzed by immunofluorescence techniques.2.2. Examining the interference rate of siRNAs targeting Gadd45a: short interfering ribonucleic acid (siRNA) targeting Gadd45a or an irrelevant mRNA was chemically synthesized. Tca8113 cells were divided into three groups for transfection:lipofectamineTM 2000 only (mock), nonsense si-RNA and Gadd45a-siRNA. Mock and nonsense si-RNA were regarded as control groups. The constructed siRNAs were transfected into Tca8113 cells. Gadd45a expression was determined by real time quantitive RT-PCR and Western Blot techniques.2.3. The effect of Gadd45a gene silencing on proliferation of Tca8113 was tested by MTT after silencing gene of Gadd45a. The growth curve was draw to assess Tca8113 cell proliferation activity.2.4. The effect of Gadd45a gene silencing on cell cycle distribution of Tca8113 was analysed with flow cytometry at 24h after silencing Gadd45a gene. 2.5. The migration ability of Tca8113 after silencing Gadd45a gene was tested by Transwell chamber assays.3. The effect of G add45a-siRN A on rad iosensitivity of Tca8113 cells3.1. Irradiated with 0,2Gy,4Gy,8Gy, 10Gy ionizing radiation (IR), the survival fraction of Tca8113 cells were ascertained by MTT assays. The apoptosis and cell cycle of Tca8113 cells were detected by flow cytometry.3.2. Tca8113 cells were cultured in six-well plates until 70% confluence and then exposed to OGy,4Gy, lOGy IR respectively. The effect of IR on Gadd45a expression in human Tca8113 cell lines was examined by real time quantitative RT-PCR and Western Blot analysis.3.3. Tca8113 cells transfected with the Gadd45a-siRNA and the control cells were irradiated in the dose range from 0 to 10 Gy. After 24 h exposed to IR, MTT colorimetric assay was used to analyse the viability of the cells and flow cytometry measurement was used to quantify the percentage of apoptotic cells in the total cell population.Results1. The expression status and clinical significance of Gadd45a in human oral squamous cell carcinomaThe Immunohistochemistry results showed that Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns. All of 60 cases showed significant nucleus-dominant expression pattern in adjacent tissue. But in oral squamous cell carcinoma,60 out of 106 cases showed Gadd45a expression with nucleus-dominant patterns. The expression patterns of Gadd45a showed a significant difference in age, clinical stage, histological grade and lymph node metastasis (p<0.05).2. The effect of Gadd45a-siRNA on cell growth and invasion of Tca8113 cells2.1. Tca8113 cells are well-differentiated tongue squamous cell cancer cell lines and the expression of Gadd45a protein was found to mainly locate in nucleus of Tca8113 cells.2.2. Real time quantitive RT-PCR and Western Blot showed that Gadd45a expression was blocked efficiently by using siRNAs in Tca8113 cells.2.3. MTT assay showed that the proliferation of Tca8113 cells two days later after RNAi was significantly higher than that of the control (p<0.05).2.4. Flow cytometry detection confirmed that the percentage of G2/M phase cell in Gadd45a-siRNA group significantly decreased compared with the control groups (p<0.05).2.5. The migrating ability of Tca8113 cells in the group transfected with Gadd45a-siRNA increased dramatically in comparison to the other two groups(p<0.05).3. The effect of Gadd45a-siRNA on radiosensitivity of Tca8113 cells3.1. The effect of IR on biological behavior of Tca8113 cells. We observed the effect of IR on the morphology of Tca8113 cell by HE staining and inverted microscope. We found that Tca8113 cells irradiated with high doses of IR became larger and the space between cells became wider.3.2. Basal and IR-induced Gadd45a expression in Tca8113 cells. We initially examined the effect of IR on Gadd45a expression in human Tca8113 cell line by real-time quantitative RT-PCR and Western Blot analysis. Gadd45a induction was assessed by comparing the basal level to that present 24 h following treatment with 4Gy or 10Gy IR. We showed IR obviously induced Gadd45a mRNA expression compared with the basal level (0.000965±0.00005 vs 0.000387±0.00002 and 0.001644±0.000065 vs 0.000387±0.00002) in the dose range examined. Consistently, Gadd45a protein level detected by Western Blot was also induced by lOGy IR (P=0.0028) although Gadd45a protein level observed in Tca8113 cells treated with 4Gy IR had no statistical significance in comparison with the basal level (P=0.0566).3.3. The effect of Gadd45a gene silencing combining IR on the biological behavior of Tca8113 cells. To research the the influence of Gadd45a gene silencing on the radiotherapy sensitivity of Tca8113 cells, we firstly detected the effectiveness of Gadd45a-siRNA using real-time quantitative RT-PCR and Western Blot analysis. The results showed that Gadd45a siRNA could effectively inhibit the expression of Gadd45a at the mRNA and protein level in Tca8113 cells (p<0.05). Then using MTT and Flow Cytometry, we inspected the surviving fraction and apoptosis status of Tca8113 cells when receiving different dose of IR. The result showed that the surviving fraction of Gadd45a-siRNA group was obviously higher than the control group (p<0.05) when the Tca8113 cells receiving 4Gy or more dose of irradiation. The result indicated that Gadd45a-siRNA impeded the down of surviving fraction of Tca8113 cells induced by IR. The Flow Cytometry result showed that when receiving 4Gy and10Gy IR, the Gadd45a-siRNA group’s apoptosis was inhibited and the apoptotic fraction was obviously descended (p< 0.05). Conclusion1. Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns in human oral squamous carcinoma, and the expression patterns of Gadd45a showed a significant difference in age, clinical stage, histological grade and lymph node metastasis.2. Gadd45a gene silencing could distinctly enhance the proliferation and migrating ability of Tca8113 cells. The mechanism might be the interference of cell cycle and inhibition of apoptosis.3. Gadd45a overexpression induced by IR could enhance the radiotherapy efficacy of human oral squamous cell carcinoma.Originality1. We demonstrate, for the first time, that Gadd45a was expressed with cytoplasm-dominant and nucleus-dominant patterns in human oral squamous carcinoma, and the expression patterns of Gadd45a showed significant difference in age, clinical stage, histological grade and lymph node metastasis. And we found that Gadd45a gene silencing could distinctly enhance the proliferation and migrating ability of Tca8113 cells.2. We demonstrate, for the first time, that Gadd45a overexpression induced by IR could enhance the radiotherapy efficacy of human oral squamous cell carcinoma.更多还原