【摘要】 背景与目的:FLICE抑制蛋白基因在凋亡信号传递中发挥重要作用,研究靶向cFLIP的反义基因治疗药物可能为临床治疗胃癌提供新的方法和思路。本研究拟探讨cFLIP反义寡核苷酸对人胃癌细胞株BGC823凋亡的影响。方法:以脂质体Lipofectame2000为载体,介导cFLIP反义寡核苷酸片段导入胃癌细胞株BGC823。RT-PCR和Westernblot方法检测cFLIPL/SmRNA和蛋白在HeLa、BGC823细胞中的表达;以cFLIP基因翻译起始区为靶点,人工合成20个碱基全硫代修饰的反义寡核苷酸,脂质体介导转染BGC823细胞后,MTT法检测细胞抑制率;原位末端标记、流式细胞术检测细胞凋亡;Westernblot检测蛋白表达变化。结果:cFLIPmRNA的2种拼接体和表达蛋白在HeLa、BGC823细胞中呈阳性表达。MTT显示脂质体介导反义寡核苷酸可显著抑制BGC823细胞增殖,其抑制作用随浓度增加而增强;TUNEL检测可见阳性染色;流式细胞仪分析发现在G1峰前出现凋亡峰;Westernblot检测可见cFLIPL和cFLIPS蛋白表达显著下降。结论:cFLIPL/SmRNA和蛋白在HeLa、BGC823细胞中表达。脂质体介导反义寡核苷酸转染BGC823细胞后抑制cFLIPL/S蛋白表达,促进凋亡。更多还原

【Abstract】 BACKGROUND &OBJECTIVE: Cellular FLICE inhibitory protein (cFLIP) p lays an important role in cell apoptosis,researches of antisense oligonucleotide s (ASODN) of cFLIP gene may provide a new method or protocol for treatment of hu man gastric cancer. This study was to explore effect of cFLIP ASODN on apoptosis of human gastric cancer cell line BGC823. METHODS: Human cervical cancer cell l ine HeLa was used as control,cFLIP ASODN was introduced into BGC823 cells and He La cells,reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detect cFLIPL/S(cellular FLIPShort and cellular FLIPlong)mRN A and protein. The 5′FAM-conjugated ASODN was created complementary to a seque nce that included the start site of FLIP open reading frame. After introducing,M TT was used to detect cell inhibition rate,TUNEL and flow cytometry (FCM) were u sed to detect cell apoptosis,and Western blot was used to detect protein level o f cFLIP. RESULTS: The encoding mRNA and protein of cFLIPL and cFLIPS can be dete cted in both HeLa and BGC823 cells.MTT revealed that cFLIP ASODN significantly i nhibited proliferation of BGC823 cells (P< 0.05)in a concentration-dependent manner. TUNEL staining detected positive FLIP expression,specific apoptotic peak can be detected before G1 peak by FCM, and Western blot revealed that protein l evel of cFLIPL and cFLIPS decreased significantly (P< 0.05). CONCLUSION: The c FLIPL/S mRNA and encoded proteins expressed in both HeLa and BGC823 cells. ASODN may down-regulate cFLIPL/S protein level,and initiate apoptosis of BGC823 cell s.更多还原