应用基因芯片研究康莱特对人胰腺癌Patu-8988细胞的凋亡诱导作用

Study of Kanglaite-induced apoptosis on human pancreatic cancer cells by cDNA microarray

目的 探讨康莱特注射液诱导人胰腺癌细胞 (Patu 8988)凋亡过程中相关基因表达的变化。方法 通过流式细胞仪AnnexinV/PI双染法研究康莱特诱导Patu 8988细胞凋亡的过程 ,应用基因芯片技术分析加药前后凋亡相关基因的表达差异 ,并以Western印迹对部分基因蛋白产物表达进行验证。结果 康莱特对Patu 8988细胞的凋亡诱导作用呈时间依赖性。康莱特作用 2 4h后 ,在 96条有关凋亡的目的基因中共有 17条基因表达发生大于 3倍的显著变化 ,其中表达上调基因 12条 ,下调基因5条。Western印迹表明P5 3、Bcl 2、Bax、Caspase 3等蛋白表达改变与基因芯片结果一致 ,同时Caspase 3底物多聚ADP核糖多聚酶被降解。结论 康莱特可使Patu 8988细胞中多个凋亡相关基因的表达发生改变 ,进一步揭示了其诱导胰腺癌细胞凋亡的作用机制。

Objective To assess the expression patterns of apoptosis-related genes in human pancreatic cancer cells (Patu-8988)induced by Kanglaite (KLT), a novel anti-cancer botanical product. Methods The apoptosis of Patu-8988 cells was determined by flow cytometry with Annexin V-FITC and propidium iodide staining. A human apoptosis microarray containing 96 cDNA fragments was used to detect gene expressions, and followed by Western blot analysis to confirm changes in expression of selected gene products. Results KLT induced apoptosis of Patu-8988 cells in a time-dependent manner. cDNA microarray analysis identified 5 down-regulated genes and 12 up-regulated genes (more than three fold) in the first 24 h as a consequence of treatment. Western blot performed for P53, Bcl-2, Bax and Caspase-3 were consistent with the microarray results. The enhanced activity of Caspases-3 was evidently verified by the cleavage of the 89×103 form of poly ADP-ribose polymerase. Conclusion KLT could alter the expression profile of apoptosis- related genes in human pancreatic Patu-8988 cancer cells, which provides valuable insight into the tentative signaling pathways mediating the outcome of KLT-induced apoptosis.