摘要
背景与目的:实验证明bcl-xL 在鼻咽癌细胞株CNE-2Z 中存在高表达,它可能在鼻咽癌的发生发展、转移及治疗过程中产生耐药等方面发挥重要作用。本研究拟探讨bcl-xL 短发夹状RNA (shorthairpin RNA ,shRNA )诱导CNE-2Z 细胞凋亡的作用。方法:把表达bcl-xL shRNA 的重组质粒pm U6-RNAi转染到CNE-2Z细胞中,用荧光染色和流式细胞术等方法检测细胞凋亡;用逆转录聚合酶链反应(reverse transcription-polym erase chain reaction,RT-PCR )检测bcl-xL、bcl-2、survivin和caspase-3的m RNA 表达水平;用W estern blot检测Bcl-xL、Caspase-3和P53的蛋白表达水平。结果:流式细胞术检测发现,pm U6-RNAi重组质粒作用24h 后,细胞出现明显的凋亡峰;H oechst33258单染在荧光显微镜下可见细胞缩小、染色质固缩和核断裂;RT-PCR 分析pm U6-RNAi重组质粒作用细胞后明显下调了bcl-xL、bcl-2和caspase-3的m RNA 表达水平, 对survivin 的m RNA 表达水平无影响;W estern blot分析pm U6-RNAi质粒作用细胞后明显下调了Bcl-xL、Caspase-3的蛋白表达水平,而大幅度上调了P53的蛋白表达水平。结论:bcl-xL shRNA 可诱导CNE-2Z 细胞凋亡;CNE-2Z 细胞的凋亡可能与bcl-2、caspase-3和p53有着密切的关系;质粒表达的bcl-xL shRNA
BACKGROUND & OBJECTIVE: Recent studies showed overexpression of bcl-xL in human nasopharyngeal carcinoma (NPC) cell line CNE-2Z; it may play a pivotal role in tumorigenesis, metastasis, and drug resistance of NPC. This study was to explore inducing effect of bcl-xL short hairpin RNA (shRNA) on apoptosis of CNE-2Z cells. METHODS: After transfection of recombinant plasmid pmU6-RNAi expressing bcl-xL shRNA, apoptotic CNE-2Z cells were detected by fluorescent staining and flow cytometry (FCM). mRNA levels of bcl-xL, bcl-2, survivin, and caspase-3 was detected by reverse transcription-polymerase chain reaction (RT-PCR); while protein levels of Bcl-xL, Caspase-3, and P53 were detected by Western blot. RESULTS: When treated with pmU6-RNAi for 24 h, an obvious apoptotic peak of CNE-2Z cells appeared; cell shrinkage, chromatin condensation, and nuclear fragmentation were observed in most cells under fluorescent microscope. RT-PCR analysis showed that pmU6-RNAi down-regulated mRNA levels of bcl-xL, bcl-2, and caspase-3, but had little or no effect on mRNA level of survivin; Western blot analysis showed an obvious reduction in protein levels of Bcl-xL and Caspase-3, and a great increase in protein level of P53. CONCLUSIONS: bcl-xL shRNA can induce apoptosis of CNE-2Z cells, which may be closely related to down-regulation of bcl-2, caspase-3 and p53. bcl-xL shRNA may be helpful for developing gene therapy for NPC.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2005年第6期646-652,共7页
Chinese Journal of Cancer
基金
广东省自然科学基金(No.31962)
广东省重大攻关项目(No.ZKM 04205S)
广东省重点学科经费资助项目 (No.9906)~~
