【作者】 赵雪柠；

【导师】 潘新良；

【作者基本信息】 山东大学 ， 耳鼻咽喉科学， 2012， 博士

【摘要】 前言下咽鳞状细胞癌是耳鼻咽喉头颈外科最常见的恶性肿瘤之一,具有发生位置隐蔽、浸润性极强、易粘膜下扩散、原发病变呈多中心生长的特点。因早期无明显的症状、缺乏特异性体征和可靠的诊断措施,所以,多数患者在临床确诊并接受治疗时,已属于晚期,病变常累及喉腔、颈段食道和舌根等器官；同时,由于喉咽部淋巴管丰富,极易发生颈淋巴结转移和包膜外扩散,并侵犯周围组织器官,如颈动脉等重要的血管,所以,下咽鳞癌是头颈部预后最差的恶性肿瘤之一。由于下咽癌生长部位的特殊性,手术治疗后,可能导致语言、呼吸及吞咽器官的功能障碍,严重影响患者的生存质量；而放疗和化疗又存在治疗不彻底、并发症多、患者痛苦较大等问题。因此,尽快在分子生物学的基础上提出新的诊断方法和简便有效、毒副作用小的治疗手段就成为亟须解决的课题。生物膜是细胞生命活动的主要结构基础,与细胞的恶性转化及肿瘤的演进密切相关。上个世纪50年代,Yamada用电子显微镜发现并命名了细胞质膜微囊—Caveolae;1972年,Singer和Nicolson提出“液相双层脂质镶嵌模型”；20世纪90年代研究发现：细胞膜的双层脂质结构是不均一的,结构蛋白Caveolin与某些富含胆固醇和鞘脂的微区域的结构变化密切相关,这些区域被称为脂筏,无结构蛋白时呈平板状,脂筏与Caveolin结合后形成的烧瓶状膜结构即Caveolae。Caveolin-1(CAV1)是Caveolae的标志性蛋白,为分子量约22kd的膜内蛋白质,其基因位于7q31.1,是形成烧瓶状结构Caveolae的必需成分。目前,CAV1与肿瘤形成之间的关系已成为研究的热点。研究证实：在大多数肿瘤细胞中,如乳腺癌、肺癌、宫颈癌、卵巢癌、肉瘤,CAV1表达下降,为抑癌基因；但在某些肿瘤,如甲状腺滤泡状癌中没有CAV1的表达；而在前列腺癌、胰腺导管腺癌及食道鳞状细胞癌中其表达水平上调,提示：在这些癌的形成过程中,CAV1是作为肿瘤促进因子。因此,CAV1在肿瘤中的作用可能与组织相关。在本课题中,我们成功构建了CAV1-小干扰RNA-慢病毒载体,通过感染下咽癌FaDu细胞株,进而检测CAV1对该细胞株体外生长的作用；并成功构建下咽鳞状细胞癌FaDu细胞株移植瘤动物模型,通过定期瘤体注射CAV1-小干扰RNA-慢病毒载体,以及后期对肿瘤组织的检测来研究CAVl对FaDu细胞株体内生长的作用。目的构建人CAV1-小干扰RNA-慢病毒载体(CAV1-RNAi-lentivirus)并转染下咽鳞状细胞癌FaDu细胞株,筛选获得稳定干扰CAV1表达的FaDu细胞。进而探讨CAV1-RNAi-lentivirus对人下咽鳞状细胞癌FaDu细胞株体内和体外生长的影响。方法1.构建人CAV1-RNA干扰慢病毒载体。设计针对CAVl的shRNA序列,Age Ⅰ和EcoRⅠ进行载体质粒双酶切,应用基因重组技术插入pGCSL-GFP慢病毒表达载体中,RT-PCR和DNA测序鉴定重组克隆。重组病毒质粒及其3种辅助包装原件载体质粒通过Lipofectamine TM2000共转染293T细胞,培养48h后,收集细胞培养上清液,将其浓缩后在293T细胞中测定病毒滴度,并进行大量包装和备用细胞转染。2. CAV1-RNAi-lentivirus对FaDu细胞体外生长的作用。应用CAV1-RNAi-lentivirus转染FaDu细胞,实验设立pGCSL-GFP-空载体对照组(GFP-lentivirus)和空白对照组。确定G418的最佳筛选浓度,于转染24h后,加入最佳筛选浓度的G418筛选稳定干扰CAV1表达的细胞。待细胞生长成肉眼可见的克隆后,使用浸有胰酶的无菌滤纸片消化挑取克隆,扩增培养。荧光倒置显微镜下观察并计算CAV1-RNAi-lentivirus的转染效率。采用RT-PCR、Western-blot方法检测细胞中CAV1的mRNA和蛋白水平的敲除效率。应用原位末端标记法(TdT-mediated dUTPnick end labeling, TUNEL)检测转染CAV1-RNAi-lentivirus后FaDu细胞的体外凋亡情况。3. CAV1-RNAi-lentivirus对FaDu细胞体内生长的作用。构建BALB/c-nude裸鼠下咽鳞状细胞癌FaDu细胞株移植瘤模型,待肿瘤长至一定时间和体积时,将所有动物随机分组。实验设立GFP-lentivirus阴性对照组和空白对照组。给药方法采用瘤周多点注射。实验组动物每周定期瘤周注射CAV1-RNAi-lentivirus(1×107TU,0.1m1),阴性对照组动物注射GFP-lentivirus(1×107TU,0.1ml),空白对照组注射同剂量的PBS,并每隔4d测量各组所有动物的肿瘤最长径与最短径,计算肿瘤体积变化和抑制率。自注射时间起32d后,采用脱臼法处死实验动物并取出移植瘤,测量标本体积并称重。以RT-PCR.免疫组化等方法检测三组肿瘤组织中目的基因CAV1的mRNA和蛋白表达变化情况,从而对CAV1-小干扰RNA-慢病毒载体在FaDu细胞株体内生长的作用进行评价,并探讨其可能的作用机制。结果1.RT-PCR和DNA测序鉴定重组克隆结果显示,实验采用基因重组技术成功构建CAV1-小干扰RNA-慢病毒载体,命名为CAV1-RNAi-lentivirus,测定滴度为1×108U/ml。2.将CAVl-小干扰RNA-慢病毒载体转染下咽癌FaDu细胞,经G418筛选,30天后获得稳定干扰CAV1表达的细胞,细胞转染效率>90%。运用RT-PCR检测方法证实,与空白对照组(1.297±0.068)和阴性对照组(1.183±0.093)相比,RNA干扰组细胞的CAV1的mRNA表达明显降低(0.47±0.050,P<0.05),差异有统计学意义。Western-blot检测结果表明,与空白对照组(0.973±0.117)和阴性对照组(0.83±0.091)相比,CAV1-RNAi-Lentivirus转染组细胞的CAV1蛋白表达水平(0.43±0.098,P<0.05)明显降低,差异有统计学意义。TUNEL检测结果证实,与空白对照组(AI=5.55±0.47%)和阴性对照组(AI=7.52±1.90%)相比,CAV1-RNAi-Lentivirus转染组(AI=22.39±2.61%,P<0.05)细胞的凋亡指数明显增加,差异有统计学意义。3.成功构建BALB/c-nude裸鼠下咽鳞状细胞癌FaDu细胞株移植瘤模型。定期注射CAV1-RNAi-Lentivirus组的裸鼠移植瘤平均重量和体积(1.24±0.42g,1.40±0.49cm3),均低于阴性对照组裸鼠移植瘤(1.79±0.42g,1.97±0.45cm3)和空白对照组移植瘤(1.88±0.54g,2.10±0.55cm3,P<0.05)。抑瘤率分别为30.8%和34.0%。对各组移植瘤进行的RT-PCR检测结果证实,与空白对照组(1.348±0.031)和阴性对照组(1.175±0.055)相比,CAV1-RNAi-Lentivirus注射组肿瘤组织的CAV1mRNA(0.671±0.070,P<0.05)表达水平降低,条带亮度明显减弱,差异有统计学意义。免疫组化染色结果显示,虽然治疗组移植瘤CAVl蛋白表达下降,但差异没有统计学意义(P>0.05)。结论本研究结果表明,重组CAV1-RNAi-lentivirus在体外转染下咽鳞状细胞癌FaDu细胞株后,G418筛选30天后可以获得稳定干扰CAV1表达的细胞,RT-PCR和Western-blot检测方法验证了基因的敲除效率。TUNEL实验结果显示,体外培养过程中,下调CAV1基因的表达,FaDu细胞的凋亡比对照组明显增加。实验成功构建BALB/c-nude裸鼠下咽鳞状细胞癌FaDu细胞株移植瘤模型,待肿瘤长至一定体积后,应用CAV1-RNAi-lentivirus定期瘤内多点注射,可以减缓肿瘤组织的生长,即FaDu细胞在体内的生长增殖受到抑制。以上实验结果提示,CAV1在下咽鳞状细胞癌FaDu细胞株的生长过程中可能起肿瘤促进因子的作用,下调CAV1的表达可能成为FaDu细胞形成的下咽癌的治疗方法之一。更多还原

【Abstract】 IntroductionHypopharyngeal squamous cell carcinoma is one of the most common malignant cancer in the Department of Otolaryngology Head and Neck Surgery, with the characters of hidden location, high infiltration, easy submucosal spread of the primary lesion and multi-center growth. Because of the lack of obvious symptoms, specific signs and reliable diagnostic measures in early stage, the majority of patients accept the clinical diagnosis and treatments are already advanced, often involving the larynx, cervical esophagus and tongue and other organs. As hypopharynx is lymphatic-rich area, it is easy to occur cervical lymph nodes metastasis, even outside spread of the capsule and invading of surrounding tissues and organs, such as the carotid artery and other major blood vessels. Therefore, hypopharyngeal squamous cell carcinoma is one of the worst prognosis malignant tumors. As the special nature of hypopharyngeal carcinoma, surgical treatment may result in language, breathing and swallowing organ dysfunction, which seriously impact on the patients’ life quality. While there are inadequate treatments, complications and pain during therapy for patients about radiotherapy and chemotherapy, therefore, new diagnostic methods based on the molecular biology and simple and effective treatments with less toxic side effects has become an urgent issue to be resolved. Biofilm is a major based structure of cell activity, which is closely related with cell malignant transformation and tumor evolution. In1950s, Yamada discovered and named caveolae under the electron microscope, which is the cell plasma membrane vesicles. In1972, Singer and Nicolson proposed "double-liquid lipid mosaic model". In1990s, researchers found that the double lipid membrane structure was heterogeneous, and caveolin was proved to be related to micro-structural changes of some cholesterol and sphingolipid-rich areas. These areas are known as lipid rafts, which are flat-shaped when there is no structural proteins. Lipid rafts turn to caveolae which is flask-shaped membrane structure after combinding with caveolin.Caveolin-1(CAV1) is an essential structural component of caveolae located at7q31.1, the molecular weight of which is about22kd. Currently, the relationship of CAV1and growth of tumors has become a research hotspot. CAV1has been observed as a candidate tumor suppressor in breast cancer, lung cancer, cervical cancer, ovarian cancer and sarcomas. Expression of CAV1was not found in follicular thyroid carcinoma. Over-expression of CAV1is associated with development of prostate, pancreatic cancers and esophageal squamous cell carcinoma, these evidences indicate that CAV1can act as a cancer-promoting gene. Therefore, the role of CAV1may vary depending on the tissue involved.In the present study, we successfully constructed the caveolin-1-RNAi-lentivirus (CAV1-RNAi-lentivirus) and received transfected FaDu cells. And the effects of CAV1on the growth of FaDu cells in vitro were examined. Then the effects of CAV1in vivo were investigated by injecting CAV1-RNAi-lentivirus construct into tumors created with FaDu cells in the HSCCs mouse model and proceeding xenografts experiments.ObjectiveTo construct the CAV1-RNAi-lentivirus and investigate the effects of CAV1on the growth of hypopharyngeal squamous cell carcinoma (HSCC) FaDu cells in vitro and in vivo. Methods1. A pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting the Caveolin-1gene were designed, synthesized, annealed and inserted into linearized pGCSIL-GFP-lentivirus vector. The recombinant plasmid was identified by double restriction digestion with Age Ⅰ and EcoR Ⅰ and DNA sequencing. The recombinant plasmid and a lentivirus packaging mix were co-transfected into293T cells to obtain packaged lentivirus particles. Viral titer was then determined.2. The CAV1-RNAi-lentivirus construct was transfected into FaDu cells. And FaDu cells with stable CAV1expression were selected using G418. RT-PCR and western blotting analysis were used to test the expression of CAV1. Transferase-medisated dUTP nick-end labeling (TUNEL) assay was used to analyze cell apoptosis.3. The rCAV1-RNAi-lentivirus construct was injected in tumors created with FaDu cells in the HSCC mouse model, with the empty-vector lentivirus as a control, to investigate tumor inhibition effect. After cultured for32days, the mice were sacrificed and the tumor specimens were collected. Tumor growth curves and the inhibition rate were analysed. RT-PCR and immunehistochemistry were used to test CAV1expression in xenografts.Results1. RT-PCR and western blot analysis demonstrated successful construction of the CAV1-RNAi-lentivirus construct producing small hairpin RNA. The titer was1×108.2. After30days of transfection, we obtained FaDu cells with stable CAV1expression using G418selection. The decreased expression of CAV1was proved by results of RT-PCR and western blotting analysis(P<0.05). Downregulation of CAV1increased cell apoptosis in TUNUL(P<0.05).3. The average tumor weight and volume in mice treated with CAV1-RNAi-lentivirus was lower than that with control treatment (P<0.05). RT-PCR revealed weak positive expression of CAV1in CAV1-construct-treated xenografts (P<0.05). Immunohistochemistry revealed CAV1expression lower in CAV1-construct-treated mice than in controls, but the difference was not significant (P>0.05).ConclusionThe growth of HSCCs FaDu cells could be inhibited by recombinant CAV1-RNAi-lentivirus in vitro and in vivo. CAV1was considered to be a cancer-promoting gene in the growth of HSCCs FaDu cells.更多还原