【作者】 王毅；

【导师】 肖利华；

【作者基本信息】 青岛大学 ， 眼科学， 2009， 博士

【摘要】 目的本研究拟:(1)检测泪腺腺样囊性癌(lacrimal gland adenoid cystic carcinoma,LGACC)与正常泪腺的差异基因表达谱,筛选与肿瘤发生相关的基因。(2)对层粘连蛋白(laminin,LN)及Ⅳ型胶原在LGACC和正常泪腺中的表达进行mRNA和蛋白质水平的验证,确定其与LGACC病理类型的关系。(3)观察LGACC与正常泪腺的超微结构,明确肿瘤基底膜(basement membrance,BM)改变与病理类型的关系。(4)运用RNA干扰技术研究LAMB1基因对ACC-2细胞系的生物学作用。材料与方法(1)收集LGACC和正常泪腺标本各5例,提取总RNA,体外转录为aRNA,与全基因组基因芯片杂交,生物信息学分析二者的全基因表达谱,获得显著表达差异的候选基因。(2)采用实时定量PCR(real-time quantitative PCR,RT-qPCR)方法检测ACC和正常泪腺组织LAMB1和COL4A1基因的表达水平;免疫组化方法观察上述基因的蛋白质产物——LN和Ⅳ型胶原在ACC不同病理分型中的分布形式。(3)收集6例LGACC和2例正常泪腺标本行透射电镜检查,观察不同病理分型ACC超微结构的特点和BM形态的改变。(4) PCR检测SGACC-2细胞系上述基因的表达,合成沉默LAMB1基因的siRNA,脂质体转染ACC-2细胞,MTT实验观察RNAi后增殖活性的改变。结果(1)经基因芯片检测,ACC有1103个基因显著上调表达;1001个基因显著下调表达,差异基因主要涉及细胞代谢、通讯、定位、细胞周期,及细胞粘附和增殖等方面。(2) RT-qPCR证实:ACC与正常泪腺相比,LAMB1和COL4A1基因分别上调表达10.06和12.91倍,而PIGR基因下调至0.27倍,与基因芯片结果相符。免疫组化证实ACC较正常泪腺LN及Ⅳ型胶原表达升高,不但瘤细胞胞浆可见染色,在筛状和管状型ACC中,假囊腔的内层、瘤细胞团外周的间质和BM结构呈连续或不连续的条索状染色;实性型染色位于瘤细胞团外周,呈不连续、不规则的条索状。(3)超微结构研究显示,筛状和管状型ACC的BM明显增厚,呈多层,互相交织成网状,肿瘤细胞可伸出伪足侵入BM,使其中断溶解;实性型ACC细胞间界限不清,细胞外堆积大量溶解的细胞外基质,完整BM结构少见。(4)涎腺ACC-2细胞系有LAMB1基因表达,转染沉默该基因的siRNA,可使基因表达降至9%。MTT实验证实转染72小时后,细胞增殖活性明显下降。结论(1) LGACC和正常泪腺的基因表达谱显著不同,基因芯片是筛选肿瘤相关基因的重要工具。肿瘤细胞可明显上调LAMB1和COL4A1的基因表达及LN和Ⅳ型胶原的蛋白质合成,沉默LAMB1基因可致ACC-2细胞增殖活性下降,提示ACC合成细胞外基质有促进肿瘤增殖的作用,可能成为肿瘤治疗的新靶点。(2) ACC的基底膜增厚,结构紊乱,其溶解和中断可能是肿瘤侵袭能力的体现,深入研究可能揭示ACC侵袭和转移的机理。更多还原

【Abstract】 ObjectivesThe present study aims to(1) use large-scale microarray analysis to characterize the expression profiles of lacrimal gland adenoid cystic carcinoma(LGACC) and normal lacrimal gland,and to specifically identify those genes differentially expressed between them;(2) validate laminin(LN) and typeⅣcollogen at mRNA and protein levels,and to identify the relationship between these markers and histological subtypes;(3) examine the ultrastructure of ACC and normal lacrimal gland,and determine the appearance of basement membrance(BM) of different histological substypes;(4) study the cellular biological effect of LAMB1 by RNA interference(RNAi) in ACC-2 cell line.Methods(1) Total RNAs were isolated from LGACC tissues and normal lacrimal glands from respective 5 cases.The aRNA prepared from in vitro transcription were hybridized with the Phalanx Human Onearray and general gene expression profiles were obtained.Genes with significant differential expression were identified by bioinformatics analytic tools.(2) Real-time quantitative PCR(RT-qPCR) was used to detect expression of LAMB1 and COL4A1 in LGACC and normal larcrimal gland at mRNA level.Immunohistocheical analysis was used to determine the expression of LN and typeⅣcollagen,which were the proteins of targets genes,and to explore the correlation between the distribution of these proteins and histological subtypes.(3) Materials for electron microscopy were obtaind from 6 cases with LGACC and 2 cases with normal lacrimal gland.The ultrastructural features and changes of BM in different histologic patterns were focused. (4) PCR examination was used to validate the expression of targets genes in ACC-2 cells. RNAi-mediated RNA silencing was used to downregulate LAMB1 expression in ACC-2 by transfection with cationic lipid complexes.MTT colorimetric assay was used to detect the changes of cell proliferation after RNAi.Results Camparing with normal tissues,the gene expression profile of LGACC showed that expression of 1103 genes was significantly increased,while expression of 1001 genes was significantly decreased.The functions of these genes were involved in cellular metabolism,communication,localization,cell cycle,cell adhesion and proliferation,etc.RT-qPCR showed there were a 10.61-fold increase of LAMB1 transcript,a 12.91-fold increase of COL4A1 transcript and a 0.27-fold descrease of PIGR transcript in LGACC, as compared to normal lacrimal gland,which were consistent with the results of genechips.Both LN and type IV collagen were more intensively expressed in LGACC than in normal tissue.Besides in cytoplasm of tumor cells,immunostaining was in the inner border of pseudocysts,the surrounding areas of tumor nests and BM of cribriform and tubular types,which showed continuous or noncontinuous streak.The solid type showed noncontinuous,irregular immunopositivity in the surrounding areas of massive tumors.(3) In ultrastructure,the BM of tubular and cribirform types showed obvious thickening,multi-layered and reticular structure.Pseudopodia of tumor cells were seen extending into BM and making it discontinuation and dissolution.The massive dissoluted extracellular matrix was revealed in the intercellular space of solid pattern.The intact BM was unusual.(4) The ACC-2 cell line,derived from salivary glands,was proved expression of LAMB 1 by PCR test LAMB1 expression was downregulated to 9%after transfection of siRNA.MTT test showed cell growth was obviously reduced 72 hours after transfection.Conclusions(1) Microarray,an important biological technique,can win the aim to screen out the genes related to tumorigenesis.Through it,we confirmed the gene expression profile of LGACC was significant different from that of normal lacrimal gland.There were obvious overexpression of LAMB 1 and COL4A1 and oversynthesis of LN and type IV collagen in LGACC.Cell growth was decreased by knockdown of LAMBl,which suggested extracellular matrix(ECM) synthesized by LGACC promote tumor proliferation.Further study maybe lead to new therapeutic targets for this carcinoma.(2) The thickening and structural disorder of BM were important features of LGACC,dissolution and discontinuation of which could be the evidence of tumor invasive growth.More findings might serve as one of the explanations on invasion and metastasis mechanism of LGACC.更多还原