摘要
目的：应用基因芯片技术,检测野生型K-ras2基因在结肠癌Caco2细胞中表达诱发的基因谱改变,进一步阐明野生型K-ras2基因可能的分子生物学功能.方法：抽取正常人的静脉血,提取RNA,反转录成cDNA,以此为模板,采用PCR方法克隆K-ras2野生型全编码cDNA序列.以常规的分子生物学技术将获得的K-ras2 cDNA克隆到T Easy载体中进行核苷酸序列的测定,构建真核表达载体pCI-neo-K-ras2,以脂质体转染结肠癌细胞系Caco2,提取mRNA逆转录为cDNA与转染空白表达载体pCI-neo的Caco2细胞进行cDNA芯片分析.结果：构建的表达载体经过限制性内切酶分析和DNA序列测定证实准确无误,提取高质量的RNA逆转录为cDNA进行DNA芯片技术分析.在135个差异表达基因中发现有24个基因表达水平显著上调,121个基因表达水平显著下调.差异表达基因与细胞增殖、分化、凋亡和信号传导等有关.结论：应用基因表达谱芯片技术成功筛选了野生型K-ras2转染结肠癌细胞后差异表达基因,反映出野生型K-ras2对细胞增殖、代谢、转录调控等过程的负性调控状态,为进一步阐明野生型K-ras2可能的生物学功能提供了新的线索.
AIM： To investigate the genes differentially expressed in human colon carcinoma cell line Caco2 transfected with wild type K-ras2- expressing plasmid and further elucidate the potential molecular biological function of wild type K-ras2.
METHODS： Sequence specific primers were designed and synthesized, and the wild type K-ras2 DNA fragment was amplified with polymerase chain reaction （PCR） technique. The expressive vector of pCI-neo-K-ras2 was constructed by routine molecular biological methods. cDNA microarray technique was employed to detect the mRNA expression in Caco2 cells transfected with pCI-neo-K-ras2 and pCI-neo, respectively, using lipofectarnine.
RESULTS： The expressive vector was constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis. High quality RNA and cDNA were prepared and successful microarray screening was conducted. The scanning results indicated that among 8568 genes which were obtained from gene expression profile analysis, there were 135 different ones of which 121 were down-regulated and 24 were up-regulated in the wild type K-ras2-expressing Caco2 cells. These genes differentially regulated by wild type K-ras2 included human genes encoding proteins involved in cell signal transduction, cell apoptosis, cell proliferation and differentiation.
CONCLUSION： cDNA microarray is successfully used to screen the genes differentially expressed in wild type K-ras2-expressing Caco2 cells, and the alteration of expression profile induced by the wide-type K-ras2 suggested the negatively regulatory function of wild type K-ras2 for cell signal transduction, apoptosis, cell proliferation and differentiation.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第20期1970-1976,共7页
World Chinese Journal of Digestology
基金
国家自然科学基金青年基金项目
No 302000326
