【作者】 付彦超；

【导师】 张庆瑜； 康春生； 张洁；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 硕士

【摘要】 目的构建针对肿瘤相关基因AKT1、COX-2和PI3Kp85基因的shRNA重组腺病毒质粒表达载体,检测其在胃腺癌细胞株SGC-7901中对AKT1、COX-2和PI3Kp85基因表达的抑制作用,以及敲低靶基因的表达后,观察MMP-2、MMP-9、TIMP-2和VEGF的表达变化。在体外实验中检测肿瘤细胞侵袭和迁移活性的变化,并进一步探讨COX-2和PI3K/AKT信号通路在胃腺癌侵袭迁移过程中的作用机制。方法本课题分两部分进行：第一部分设计可形成小干扰RNA的AKT1、COX-2和PI3Kp85 shRNA对应模板DNA序列,克隆至线性化质粒PEGFP6-1、PGENESIL-2、PEGFP6-4上,构建一个编码AKT1、COX-2和PI3Kp85三条shRNA的质粒表达载体。将此质粒亚克隆至腺病毒表达载体上,经酶切、测序鉴定正确后包装扩增并鉴定其病毒滴度,然后转染至SGC7901细胞中检测其转染率。第二部分在体外应用RNA干涉技术,以构建的重组腺病毒介导靶向AKT1、COX-2和PI3Kp85的shRNA转染人胃腺癌细胞系SGC-7901后,应用Realtime PCR和Western blot法检测目的基因的敲低情况,并用Western blot法检测RNAi敲低靶基因的表达后,COX-2及EGFR通路下游侵袭相关因子MMP-2、MMP-9、TIMP-2和VEGF的蛋白表达变化。应用ELISA检测转染前后分泌到细胞外的MM9-2和MMP-9的浓度变化。应用划痕实验、Transwell实验、2-D和3-D的Matrigel基质生长实验评价肿瘤细胞转染前后侵袭迁移能力的变化。结果1.通过应用定向克隆、同源重组等技术,获得一种同时包含靶向COX-2、PI3Kp85和AKT1三条RNA干扰序列的真核表达质粒,经酶切分析,测序鉴定表明构建成功后,扩增并亚克隆至重组腺病毒载体上,获得重组腺病毒质粒表达载体rAd5-C-A-P。由于重组腺病毒质粒表达载体上带有荧光蛋白表达框,所以通过转染SGC-7901后观察荧光蛋白表达情况,计算转染率>90%。2. Realtime PCR和Western blot结果提示,转染AKT1、COX-2和PI3Kp85 shRNA后可明显敲低靶基因mRNA和蛋白的表达。在靶基因被下调后通过Western blot检测发现COX-2及PI3K/AKT信号通路下游侵袭相关因子MMP-2、MMP-9和VEGF的表达水平均明显降低,而TIMP-2表达上调。应用ELASA检测发现rAd5-C-A-P转染组细胞培养液中MMP-2和MMP-9明显减低,划痕实验显示与对照组和无义序列组相比,rAd5-C-A-P转染组细胞迁移运动能力明显减弱,Transwell体外侵袭实验结果显示对照组和无义序列组穿过细胞数分别为(105±4)、(102.5±6.4),而rAd5-C-A-P转染组(26.4±4.6) (p<0.001).2-D的Matrigel基质生长实验显示对照组和无义序列组细胞呈正常形态贴壁生长并融合成片,而rAd5-C-A-P转染组细胞贴壁能力与迁移能力均明显减低,3-D的Matrigel基质生长实验显示与对照组和无义序列组比较,rAd5-C-A-P转染组细胞形成的细胞团块较小(p<0.001)。结论1.成功构建靶向AKT1、COX-2和PI3Kp85的重组腺病毒质粒表达载体,其能在体外高效转染肿瘤细胞。2.靶向AKT1、COX-2和PI3Kp85的RNA干扰技术可以序列特异性地抑制SGC7901细胞的AKT1、COX-2和：PI3Kp85表达,在体外实验中对胃腺癌的侵袭迁移起到了明显的抑制作用。因此靶向AKT1、COX-2和PI3Kp85的联合基因治疗有望成为胃癌基因治疗的新措施。更多还原

【Abstract】 AIM The aim of this study was to construct a recombinant adenovirus vector that expresses small hairpin RNA against the COX-2, AKT1 and PI3Kp85 gene and to detect the suppression effect on the expression of COX-2, AKT1 and PI3Kp85 in gastric adenocarcinoma cell SGC-7901. And after COX-2, AKT1 and PI3Kp85 were down-regulated, we examined the effects of COX-2, AKT1 and PI3Kp85 shRNA on the invasion and metastasis of gastric adenocarcinoma, and the effects of the down-regulation of COX-2, AKT1 and PI3Kp85 on expression of MMP-2, MMP-9, TIMP-2 and VEGF. The mechanism of COX-2 and PI3K/AKT signal pathway in the invasion and metastasis of gastric adenocarcinoma is further investigated.METHODS The study was divided into 2 parts.1. AKT1+COX-2+PI3Kp85shRNA template DNA sequence, capable of forming a small hairpin structure was designed. After renaturation, cloned into the linearisation plasmid PEGFP6-1, PGENESIL-2, PEGFP6-4, then construct a plasmid expression vector, which coding three strip of shRNA. Subcloned the AKT1+COX-2+PI3Kp85 shRNA expression frame into adenovirus expression vector. After verification, enclose、amplify and titered, then transfected the SGC7901 cell to detect the transfection efficiency.2. The recombinant adenovirus vector plasmid expression vector which contained short hairpin RNA (shRNA) expression construct targeting COX-2, AKT1 and PI3Kp85 was transfected into human gastric adenocarcinoma SGC7901 cells. Realtime PCR and Western blot analysis were used to detecte COX-2, AKT1 and PI3Kp85 expression. And the expression of MMP-2, MMP-9, TIMP-2 and VEGF were also detected by Western blot when COX-2, AKT1 and PI3Kp85 were downregulated. ELESA was used to detecte the ectocytic density change of MMP-2 and MMP-9.The invasion and metastasis ability of the tumor cells were measured by Scarification Transwell、2-dementinal and 3-dementional matrigel matrix.RESULTS 1. By using directional cloning, homologous recombination techniques, to obtain a plasmid which includes three shRNA sequence targeting COX-2, PI3Kp85 and AKT1. Restriction digest patterns and squencing analysis showed that rAd5-C-A-P adenovirus expression vector was constructed successfully. And the expression vector contained fluorescin expression frame, so we detected the transfection efficiency by observing the expression of fluorescin after transfection. And the transfection efficiency was over 90%.2. Recombinant adenovirus vector rAd5-C-A-P mediated shRNA targeting COX-2, AKT1 and PI3Kp85 dramatically down-regulated their expression in SGC7901 cells. And MMP-2, MMP-9 and VEGF were also downregulated, while TIMP-2 was upregulated. The ectocytic density of MMP-2, and MMP-9 decreased. Scarification indicates that compared with control group and nonsense sequence group the ability of invision was decreased obviously. Transwell showed that the number of cells invading through the matrigel was control group (105±4), nonsense sequence group (102.5±6.4). rAd5-C-A-P transfection group (26.4±4.6) (p<0.001). Cell growth on matrigel matrix showed normal cell appearances and mix together in control group and nonsense sequence group cells, while the cells of rAd5-C-A-P transfection group were detached from the matrix or grew in a scattered clustering patterns, signifying poor cell metastasis activities in 2-D matrigel. The rAd5-C-A-P transfection group cells formed only small aggregates as compared with the control group and nonsense sequence group in 3-D matrigel matrix growth(p<0.001).CONCLUSION1. Recombinant adenovirus vector expression vector targeting COX-2, AKT1 and PI3KP85 was constructed successfully, and it may transfect the tumor cell with high efficiency.2. ShRNA targeting COX-2, AKT1 and PI3KP85 downregulates significantly their expression in a sequence-specific manner, exerted invasion and metastasis inhibition effect on SGC7901 cells in vitro. Consequently, combined gene therapy by targeting COX-2, AKT1 and PI3KP85 would be a new strategy in targeting gene therapy of gastric adenocarcinoma.更多还原