【作者】 陈伟；

【导师】 刘宝林； 孙沫逸；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 涎腺腺样囊性癌(adenoid cystic carcinoma, ACC)具有嗜神经侵袭(perineural invasion, PNI)的特性,常侵袭神经并沿神经生长向远处转移。已有一些研究报道了某些基因在ACC发生PNI过程中可能的作用,但大多研究手段比较局限,未能较全面地了解ACC发生PNI的分子机理。而ACC侵袭神经是涉及多基因、多环节、多途径的复杂过程,因此,研究ACC嗜神经侵袭现象时,明确其嗜神经侵袭相关基因表达谱对ACC基础理论的丰富及指导临床治疗均具有重要意义。研究目的:1、利用激光俘获显微切割技术获得侵袭神经的纯一ACC肿瘤细胞群,进而通过基因表达谱芯片技术构建出ACC嗜神经侵袭相关基因表达谱,以辨别潜在的ACC嗜神经侵袭相关标记物。2、从获得的基因表达谱中,选定靶基因CD146(MCAM),利用RNAi技术高效特异地沉默其表达,分析其对ACC-M细胞的生物学影响及其在抑制ACC-M细胞体外嗜神经侵袭能力中的具体作用。研究方法:1、结合连续冰冻切片H&E染色及免疫组织化学(IHC)方法,从原发ACC肿瘤标本中,判定存在明确PNI现象的ACC标本。利用激光俘获显微切割技术获得侵袭神经的纯一ACC肿瘤细胞群,进而结合RNA线性扩增和基因表达谱芯片技术构建出ACC嗜神经侵袭相关基因表达谱。2、采用实时定量PCR(QRT-PCR)及IHC方法对部分差异表达基因进行验证,以确定基因表达谱芯片筛选结果的可靠性。采用生物信息学方法对获得的ACC嗜神经侵袭相关基因表达谱进行分析,选定靶基因CD146用于下一步的RNA干涉研究。3、利用DNA重组技术将针对CD146及GAPDH基因的不同部位所设计的4对dsDNA序列(包括2对CD146的特异性干扰序列、1对CD146的阴性对照干扰序列和1对GAPDH的阳性对照干扰序列)克隆到真核表达质粒pGenesil-1中,限制性酶切和基因测序鉴定,构建CD146的shRNA真核表达载体系统。转染ACC-M细胞,结合有限稀释法及G418压力筛选,构建CD146 shRNA真核表达载体稳定转染细胞株,检测RNA干涉后CD146在mRNA及蛋白水平的表达情况。4、检测CD146基因沉默对ACC-M细胞其生物学特性的影响,包括MTT法测定细胞生长曲线;流式细胞仪检测细胞周期的变化。体外实验应用改良Transwell细胞侵袭实验,以了解CD146基因沉默后ACC-M细胞体外嗜神经侵袭能力的变化。主要实验结果:1、15例原发ACC标本中,6例可判定为存在明确的PNI现象。从6例判定为PNI的肿瘤组织中,激光俘获紧贴神经束且侵袭至神经内膜或神经束膜间的纯一肿瘤细胞群作为嗜神经侵袭组(PNI组);从该6例肿瘤组织中,同样激光俘获未侵袭神经的纯一肿瘤细胞群作为非嗜神经侵袭组(non-PNI组),从而获得侵袭神经的纯一ACC肿瘤细胞群。RNA线性扩增结合人类全基因组芯片Agilent human 1A cDNA microarray,检测差异表达基因。在18,716个基因和1,457个ESTs中,所有的6例芯片共同差异表达的基因有53个,这53个基因能够区分出PNI组及non-PNI组肿瘤细胞,其中38个基因表达上调,15个基因表达下调,从而构建出ACC嗜神经侵袭相关基因表达谱。2、53个差异表达基因中,我们使用QRT-PCR及IHC方法验证了其中7个及1个基因,其表达情况均与芯片筛选结果一致。聚类分析显示53个差异表达基因可聚类为5簇,这5簇聚类可详细描述ACC嗜神经侵袭相关基因表达谱。基因功能分析显示在PNI肿瘤细胞组中特异性高表达的基因,其功能与发育、信号转导、神经发生、癌基因、炎性应答、免疫反应、DNA结合、细胞黏附和细胞外基质相关。部分前期ACC芯片研究中表达增高的基因被我们的实验结果所证实,同时我们也获得了一些之前未被证实的在ACC嗜神经侵袭病理过程中可能起重要作用的新基因。3、以CD146为靶基因,成功构建了CD146 shRNA真核表达载体系统,包括特异性干扰载体pGe-CD146-shRNA1, 2 ;阴性干扰载体pGe-negative-shRNA以及阳性干扰载体pGe-GAPDH-shRNA。结合有限稀释法及G418压力筛选,我们构建了CD146 shRNA真核表达载体稳定转染细胞株,证实稳定转染细胞中,CD146在mRNA及其蛋白的表达水平被显著抑制。4、MTT及流式细胞仪研究结果表明,CD146基因沉默可能通过阻碍ACC-M细胞从G0/G1期进入S期,抑制DNA合成,从而抑制细胞增殖。改良Transwell细胞侵袭实验证实CD146基因沉默可抑制ACC-M细胞体外嗜神经侵袭能力;根据不同位点设计的CD146 shRNA可能导致抑制ACC-M细胞体外嗜神经侵袭能力的差异。结论:1、结合激光俘获显微切割和基因表达谱芯片技术成功构建出ACC嗜神经侵袭相关基因表达谱。聚类分析及基因功能分析可以较全面的分析基因表达谱结果。某些基因在其PNI的病理过程中的作用值得进一步研究。2、CD146 shRNA真核表达载体及其稳定转染细胞株可高效、特异性阻断CD146基因表达,是研究其功能的良好平台。3、CD146基因沉默能够显著抑制ACC-M细胞的恶性表型,尤其可显著抑制其体外嗜神经侵袭能力,提示CD146基因有可能成为ACC嗜神经侵袭相关标志物和治疗的靶基因。更多还原

【Abstract】 Salivary adenoid cystic carcinoma (ACC) has a characteristic of perineural invasion (PNI), it often invades nerves and distantly metastasizes by growing along with nerves. A few of studies have reported the potential effects of some certain genes in the process of PNI in ACC, but most of the studies are limited, so they cannot elucidate the molecule mechanism of PNI in ACC thoroughly. The process of PNI in ACC was complicated which related to multiple genes, component elements, and pathways. So, there is important significance to clarify gene expression profile associated with PNI which can enrich basic theory and guide clinical treatment in ACC.Objectives:1. To investigate the range of gene expression differences between PNI and non-PNI ACC cell groups which procured by laser capture microdissection (LCM) and to correlate gene expression profile associated with PNI to identify potentially new biomarkers of ACC with PNI.2. CD146(MCAM), one of the up-regulated genes from gene expression profile, was chose to be the target for RNAi. To analyze its biological effects in reducing in vitro perineural invasion on ACC-M cell by specifically knock-down the transcription of CD146.Methods:1. Combined use of H&E staining and immunohistochemistry (IHC) method in consecutive frozen sections from primary ACC specimens, obvious PNI was assessed in the cancerous tissues. Pure cancer cells adjacent to the nerve tracts from cancerous tissues judged as PNI were procured by LCM. Then we constructed gene expression profile associated with PNI by combining use of RNA linear amplification and high-throughput cDNA microarray technique.2. Patterns of gene expression were verified by quantitative real-time PCR (QRT-PCR) and IHC. Gene expression profile associated with PNI was analyzed by bioinformatics methods. Target gene CD146 was chose for the following RNAi research.3. According to the CD146 cDNA in the Genebank, we designed two different shRNA targeting the coding sequence of the CD146. The pGe-CD146-shRNA1, 2 were constructed by inserting the designed shRNA in the eukaryotic expression vector pGenesil-1. The vector was identified by restriction endonuclease digestion and partial nucleotide sequencing. ACC-M cells were transfected with different vectors respectively. Monoclones of stable transfectants, were procured by limiting dilution assay, then selected by G418. The expression results of CD146 mRNA and protein were detected after RNAi.4. Effects of CD146 gene silencing on ACC-M cells were detected, including cell proliferation activity analyzed by MTT assay, and cell cycle change detected by flow cytometry (FCM) analysis. The inhibitory effect of RNAi on ACC-M cell’s PNI in vitro was demonstrated in modified Boyden chambers.Results: 1. Obvious PNI was found in 6 out of the 15 primary cancerous tissues. Pure cancer cells adjacent to the nerve tracts from 6 cancerous tissues judged as PNI were laser captured, and pure cancer cells from the same 6 tumors distant from the nerve tracts were also procured. Differential expression genes were detected by Agilent human 1A cDNA microarray. Of 18,716 interrogated genes and 1,457 ESTs, analyzed data showed 53 genes were differentially expressed (fold difference≥2.0 or≤1/2) among all 6 arrays that could distinguish PNI and non-PNI cancer cell groups. Of the 53 genes found differentially expressed, 38 were up-regulated and 15 down-regulated. Thus we constructed gene expression profile associated with PNI.2. Of the 53 differentially expressed genes, we performed QRT-PCR and IHC analysis for a group of selected genes to validate the findings of the microarray analysis. Hierarchical clustering showed the 53 genes could be divided into five clusters discriminating the PNI. Functional gene classes highly represented in PNI cell group included those involved in development, signal transducer activity, neurogenesis, oncogene, inflammatory response, immune response, binding, cell adhesion, and extracellular matrix functions. Several genes previously identified as overexpressed in ACC were confirmed by our study in addition to identifying several novel genes with potential roles in the pathobiology of ACC associated with PNI.3. We successfully constructed CD146 shRNA eukaryotic expression vector system based on target gene CD146, including pGe-CD146-shRNA1, 2; a negative control vector pGe-negative-shRNA and a positive control vector pGe-GAPDH-shRNA. Combined use of limiting dilution assay and G418 pressure selection, we successfully constructed monoclones of CD146 shRNA stable transfectants. It has been proved that the mRNA and protein expression levels of CD146 were significantly inhibited in the stable transfectants.4. Findings based on MTT and FCM analysis indicated that the knockdown of CD146 expression could inhibit proliferation of ACC-M cells by modulating G0/G1 and S cell cycle regulators. Modified Boyden chambers experiment demonstrated that the knockdown of CD146 expression could inhibit PNI activity of ACC-M cells, and that different shRNA transfectants showed striking unequal efficiency in inhibiting PNI activity.Conclusions:1. Combined use of LCM and high-throughput cDNA microarray techniques, we successfully constructed in vivo gene expression profile of salivary ACC and to correlate the profile with PNI. Hierarchical clustering and functional gene classes can analyze the gene expression profile all around. Several novel genes with potential roles in the pathobiology of ACC associated with PNI deserved further study.2. CD146 shRNA eukaryotic expression vectors and their stable transfectants can specifically and effectively inhibit the expression of CD146, suggesting that it could be a good platform applied for functional research.3. The knockdown of CD146 expression by RNAi can successfully inhibit the malignant behaviors of ACC-M cells, especially PNI ability in vitro, implicating that CD146 may be a new candidate target gene for treatment of ACC.更多还原