【作者】 黄安乐；

【导师】 蔡建春；

【作者基本信息】 福建医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 恶性肿瘤的主要特征是浸润和转移,细胞外基质(ECM)和基底膜的降解为关键环节。基质金属蛋白酶-9(MMP-9)作为蛋白水解酶的一种,能降解基底膜和细胞外基质成分,从而促进肿瘤的浸润、转移及间质血管新生。胃癌组织中MMP-9检测结果与临床病理资料对比的研究表明,该酶不仅与胃癌的分化程度、分期、病理类型、转移和预后有关,然而MMP-9基因在转录和翻译水平的抑制作用尤其是RNA干扰对胃癌的影响尚未明了。RNA干扰是由dsRNA介导的同源mRNA降解,从而引起相应基因沉默的一种广泛存在于酵母、真菌、植物、动物体内古老的生物防御机制。1998年后,许多研究小组对RNAi的机制和应用进行了大量的研究。在应用方面主要是利用RNAi技术对线虫、果蝇、植物、动物及病毒的某些基因进行干扰,从而达到基因敲除或抑制病毒复制的目的。本实验目的是应用质粒载体将基质金属蛋白酶-9(MMP-9)特异性siRNA通过脂质体转染胃癌细胞,构建了低分化胃腺癌细胞BGC823的MMP-9基因沉默细胞模型;应用免疫荧光、western blot、明胶酶谱分析等方法验证RNA干扰所得到的基因沉默胃癌细胞株。本实验应用分子克隆技术结合RNA干扰技术在转录和翻译水平对特定基因MMP-9进行抑制,从而对该基因在胃癌浸润、转移中的作用及机制进行探讨。实验关键在于获得较高干扰效果并筛选获得成功转染质粒的细胞克隆。这将为后续的体内外胃癌模型建立提供基础。该RNA干扰模型建立,从分子水平探讨胃癌细胞浸润与基质金属蛋白酶9的关系,在胃癌基因水平研究做了初步探索,结果可能对胃癌的早期诊断、预后判断和基因治疗提供参考。更多还原

【Abstract】 The major characteristics of malignant tumour cells are invasion and metastasis, of which proteolytic degradation of the extra cellular matrix, including basement membrane by matrixmetalloproteinases (MMPs) is one of the essential events. As a kind of matrixmetallo-proteinases, matrixmetalloproteinase-9(MMP-9) can degradate extra-cellular matrix and basement membrane, which would facilitate the invasion, metastasis and interstitial angiogenesis. MMP-9 is expressed predominantly by tumour cells in various cancers. Immunohistochemical studies have shown that the expression of MMP-9 correlates with tumor invasion,metastasis and prognosis in both colorectal adenocarcinoma and esophageal squamous cell carcinoma. But it is not certain what will happen when MMP-9 is inhibitted with RNAi.Over the last years, RNA interference(RNAi) has been recognized as a major mechanism of post-transcriptional gene silence in the nematode Caenorhabditis elegans and the fruit fly Drosophila, as well as in plants. This phenomenon is based on double-stranded RNA(dsRNA) that triggers the silence of gene expression in a sequence-specific manner. Now, there are many study on the mechanism, while has become a routine tool for transient knockdown of gene expression in a wide range of organisms. In our experiment, we constructed a modal with MMP-9 geneknockout by RNAi in poorly differentiated gastric adenocarcinoma cell BGC823. In the process of construction, we cloned a stable RNAi system with liposome transfection. Then applied immunofluorescence technique , gelatin zymography and western blot to detect the expression of MMP-9 in BGC823.The experiment applies molecular cloning and RNA infection techniques to inhibit MMP-9 gene in level of transcription and translation, for researching the role and mechanism of the invasion and metastasis in gastric cancer cell. Experimental key is to obtain a higher interference effects gastric cancer cell line that transfected plasmid successfully. The experiment is the basis of the experimental model in vitro. The resulds would show the relationship between gastric cancer cell invasion and MMP-9, provide evidences for early diagnosis, gene therapy, and judging the prognosis in gastric cancer.更多还原