目的：验证细胞周期聚合酶链反应（PCR）芯片筛选出来的差异表达基因。方法胃泌素刺激的SW480细胞作为实验组，无胃泌素刺激的SW480细胞作为对照组。用蛋白免疫印迹（Western blot）法对胃泌素刺激前后的钙周期素结合蛋白（CacyBP/SIP）表达定位进行检测；用实时荧光定量PCR（qRT-PCR）技术对细胞周期PCR芯片筛选出的差异表达基因周期素依赖性蛋白激酶8（CDK8）和细胞周期蛋白依赖性激酶亚基2（CKS2）进行验证。结果胃泌素刺激前CacyBP/SIP主要表达于细胞质，刺激后可同时表达于细胞质和细胞核；同细胞周期PCR芯片结果一致，基因CDK8和CKS2在实验组的表达量较对照组明显上调（P<0.05）。结论胃泌素刺激可使CacyBP/SIP发生核转位；基因芯片筛选出的差异基因大体可靠，对筛选出的差异基因可以进行进一步研究。%Objective To verify the genes screened by the polymerase chain reaction (PCR) chip of cell cycle. Methods The colon cancer cells SW480 were randomized into two groups, the test group (with gastrin stimulation) and con-trol group (without gastrin stimulation). The method of Western blot was used to detect the expression of calcylin binding pro-tein/Siah-1 interacting protein (Cacybp/SIP) before and after gastrin stimulation. The differential expression genes, cyclin de-pendent kinase 8 (CDK8) and cyclin dependent kinase subunit (CKS2), were verified by using real-time quantitative PCR (qRT-PCR). Results It was found that before the stimulation, CacyBP/SIP was located and expressed in cytoplasm, and then in both cytoplasm and nucleus after gastrin stimulation. The qRT-PCR results of CDK8 and CKS2 genes were consis-tent with those of microarray detection. The expressions of CDK8 and CKS2 were up-regulated (P < 0.05). Conclusion The stimulation of human gastrin can lead to the nuclear translocation of CacyBP/SIP. The results of microarray are reliable, and the differentially expressed genes screened through gene chip deserve further study.
展开▼
