脂质体介导反义C-FLIP对BGC823细胞凋亡的影响  

Liposome-mediated Antisense Oligonucleotides of Cellular FLICE Inhibitory Protein Induce Human Gastric Cell Line BGC823 to Apoptosis

作　　者：张国强[1] 庞达[1] 蔡莉[2] 张岂凡[1] 赵家宏[1] 

机构地区：[1]哈尔滨医科大学附属肿瘤医院腹部外科,哈尔滨市150040 [2]哈尔滨医科大学附属肿瘤医院内四科,哈尔滨市150040

出　　处：《中国肿瘤临床》2005年第19期1100-1103,共4页Chinese Journal of Clinical Oncology

基　　金：黑龙江省教育厅科研基金资助(编号:10541130)

摘　　要：目的:探讨脂质体介导下c-FLIP(Cellular-FLICEInhibitoryProtein)反义寡核苷酸对胃癌细胞系BGC823的作用。方法:RT-PCR和WesternBlot方法检测c-FLIPL、c-FLIPS的mRNA和蛋白的表达;MTT法检测细胞抑制率;原位末端标记法、流式细胞术检测细胞凋亡;WesternBlot检测蛋白表达变化。结果:c-FLIP基因编码的2种mRNA和蛋白在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;反义寡核苷酸作用细胞36h后,TUNEL检测可见明显阳性染色,流式细胞分析发现在G1峰前出现凋亡峰;WesternBlot可见c-FLIPL和c-FLIPS蛋白表达显著下降。结论:c-FLIPL、c-FLIPSmRNA和蛋白在Hela、BGC823细胞中表达。脂质体介导c-FLIP反义寡核苷酸转染BGC823细胞后抑制目的蛋白表达,抑制细胞生长、促进凋亡,其作用呈剂量依赖性。研究靶向c-FLIP的反义基因治疗药物可能为胃癌的临床治疗提供新的方法和思路。Objective： To study the important role of c-FLIP, which structurally simulates the apoptotic effector caspase-8, in cell apoptosis and to provide a new methodology or protocol of antisense oligonucleotides （ASODN） of the gene for treatment of human gastric cancer. Methods： Reverse transcriptase polymerase chain reaction and Western Blot were used to detect c-FLIPI/s （cellular FLIP.short and cellular FLIPlong） mRNA and protein in both Hela and BGC823 cell lines. Whether c-Flip is functionally significant for the cell line or not was determined and 5＂ FAM-conjugated antisense oligonucleotides （ASODN） were created complementary to a sequence that included the start site of the c-Flip open reading frame. C-FLIP ASODN was introduced into cell line BGC823 with lipofectamine 2000 as the vector, and its effect and mechanism were investigated by MTT, TUNEL, FACs and Western blot assay. Results： The encoding mRNA and protein of c-FLIPL and c-FLIPs can be detected in both cell lines. MTT assay revealed that liposome mediated antisense oligonucleotides （Lipo-AS） can significantly inhibit BGC823 cell proliferation, with the effect apparently related with the concentration; TUNEL staining was positive, specific apoptotic peak can be detected before G1 peak by FACs, and Western Blot analysis revealed that the protein expression of c-FLIPL and c-FLIPs decreased significantly after the cell was treated with ascending concentration ASODN. Conclusions： The c-FLIPI/s mRNAs and proteins express in Hela and BGC823 cell line. Liposome-mediated ASODN down-regu-lates the c-FLIPI/s protein expression and initiates the apoptosis. Researches on antisense drugs targeting on c-FLIP gene wound provide the new methods and protocols for treatment of gastric carcinoma.

关 键 词：反义寡核苷酸 c—FLIP BGC823细胞 凋亡 

分 类 号：R735.2[医药卫生—肿瘤]