Zhang Guo-Qiang, Pang Da, Wang Kuan, Xue Ying-Wei, Zhang Qi-Fan, Zhao Jia-Hong
Department of Abdominal Surgery, Affiliated Tumor Hospital, Harbin Medical University, Harbin, Heilongjiang, 150040 P.R.China.
Ai Zheng. 2004 Nov;23(11 Suppl):1390-5.
BACKGROUND & OBJECTIVE: Cellular FLICE inhibitory protein (cFLIP) plays an important role in cell apoptosis, researches of antisense oligonucleotides (ASODN) of cFLIP gene may provide a new method or protocol for treatment of human gastric cancer. This study was to explore effect of cFLIP ASODN on apoptosis of human gastric cancer cell line BGC823.
Human cervical cancer cell line HeLa was used as control, cFLIP ASODN was introduced into BGC823 cells and HeLa cells, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detect cFLIP(L/S) (cellular FLIP(Short) and cellular FLIP(long)) mRNA and protein. The 5'FAM-conjugated ASODN was created complementary to a sequence that included the start site of FLIP open reading frame. After introducing, MTT was used to detect cell inhibition rate,TUNEL and flow cytometry (FCM) were used to detect cell apoptosis, and Western blot was used to detect protein level of cFLIP.
The encoding mRNA and protein of cFLIP(L) and cFLIP(S) can be detected in both HeLa and BGC823 cells. MTT revealed that cFLIP ASODN significantly inhibited 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 level of cFLIP(L) and cFLIP(S) decreased significantly (P< 0.05).
The cFLIP(L/S) mRNA and encoded proteins expressed in both HeLa and BGC823 cells. ASODN may down-regulate cFLIP(L/S) protein level, and initiate apoptosis of BGC823 cells.
细胞型FLICE抑制蛋白（cFLIP）在细胞凋亡中起重要作用，对cFLIP基因反义寡核苷酸（ASODN）的研究可能为人类胃癌的治疗提供新方法。本研究旨在探讨cFLIP ASODN对人胃癌细胞系BGC823凋亡的影响。
以人宫颈癌细胞系HeLa为对照，将cFLIP ASODN导入BGC823细胞和HeLa细胞，采用逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测cFLIP（L/S）（细胞型FLIP（短型）和细胞型FLIP（长型））mRNA和蛋白。构建与FLIP开放阅读框起始位点序列互补的5'FAM偶联ASODN。导入后，采用MTT法检测细胞抑制率，TUNEL法和流式细胞术（FCM）检测细胞凋亡，蛋白质印迹法检测cFLIP蛋白水平。
HeLa细胞和BGC823细胞中均可检测到cFLIP（L）和cFLIP（S）的编码mRNA和蛋白。MTT结果显示，cFLIP ASODN可显著抑制BGC823细胞增殖（P<0.05），且呈浓度依赖性。TUNEL染色检测到FLIP阳性表达，FCM检测到G1峰前出现特异性凋亡峰，蛋白质印迹法显示cFLIP（L）和cFLIP（S）蛋白水平显著降低（P<0.05）。
HeLa细胞和BGC823细胞中均表达cFLIP（L/S）mRNA及编码蛋白。ASODN可能下调cFLIP（L/S）蛋白水平，诱导BGC823细胞凋亡。
