摘要
目的构建针对肿瘤相关基因蛋白激酶B1（protein kinase B1，AKT1）、环氧合酶2（cyclooxygenase 2，COX-2）、磷脂酰肌醇-3-激酶调节亚基1（phosphatidylinositol kinase 3 regulatory subunit1，PIK3R1）的小发卡RNA（small hairpin，shRNA）重组腺病毒表达载体，体内外研究其对人胃腺癌SGC7901细胞增殖的影响，为胃腺癌的联合基因治疗提供参考。方法构建一个编码COX2，AKT1及PIK3R1三条shRNA的重组腺病毒质粒表达载体，包装扩增并鉴定其病毒滴度。在体内外转染SGC7901细胞，检测肿瘤细胞生长情况，蛋白表达和细胞周期变化。结果经酶切分析，测序鉴定表明成功构建并扩增rAd5-C—A—P腺病毒表达载体。转染SGC7901后COX2，AKT1及PIK3R1的mRNA和蛋白的表达水平均明显降低，甲基氮唑蓝（methyl thiazolyltetrazolium，MTT）法显示转染后第6d细胞存活率为27．6％，流式细胞周期显示G0／G1期阻滞，2-D的Matrigel基质生长实验显示rAd5-C—A-P转染组细胞贴壁生长能力明显减低，体内实验中肿瘤生长也明显减慢（P％0．01）。结论靶向COX-2，AKT1和PIK3R1的shRNA技术可以序列特异性地抑制SGC7901细胞的COX-2，AKT1和PIK3R1表达，在体内外对SGC7901细胞增殖产生明显抑制作用。
Objective To construct a recombinant adenovirus vector that expresses small hairpin RNAs（shRNA） against COX-2, AKT1 and PIK3R1 gene and to evaluate its potential for suppressing the cell proliferation of human gastric adenocarcinoma SGC701 cell in vitro and in vivo, which will enable the development of a gene therapy protocol for the treatment of human gastric adenocarcinoma. Methods Three strips of shRNA targeting AKT1, COX-2 and PIK3R1, was subcloned into adenovirus expression vector. After verification, it was amplified and titered. The recombinant adenovirus expression vector was infected into human gastric adenocarcinoma SGC7901 cells in vitro and the infected cells were injected in nude mice. The mRNA and protein expression levels of AKT1, COX-2 and PIK3R1 were determined by real time PCR and Western blot respectively. Cell proliferation in vitro was determined by methyl thiazolyltetrazolium（MTT） assay and flow cytometry, tumor growth in vivo was measured by volume of tumor in nude mice. Results Restriction digestion and sequencing analysis showed that the rAdS-C-A-P adenovirus expression vector was constructed successfully. It significantly inhibited the expression of AKTI, COX-2 and PIK3R1, and cell growth was inhibited over 70% as indicated by MTT assay and accompanied with Go/G1 phase arrest. Cell growth on matrigel matrix showed that the rAd5 C-A-P transfected cells were detached from the matrix or grew in a scattered clustering pattern, indicating poor cell growth activities in 2-D matrigel. Tumor growth in nude mice in the C ＋ A ＋ P group was inhibited （P〈 0. 01）. Conclusion shRNA targeting COX-2, AKT1 and PIK3R1 down regulated significantly the expression of the three genes in a sequence-specific manner, exerted proliferation inhibition effect on SGC7901 cells in vitro and in vivo.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2009年第4期383-387,共5页
Chinese Journal of Medical Genetics
基金
天津市自然科学基金（07JCZDJC07700）
关键词
重组腺病毒
RNA干扰
联合基因治疗
胃腺癌
recombinant adenovirus
RNA interference
combined gene therapy
gastric adenocarcinoma
