TFPI-2基因在胆囊癌中的表达及其抑癌作用的实验研究

摘要

目的 研究组织因子途径抑制物2(TFPI-2)基因在胆囊癌组织中的表达情况及其在胆囊癌细胞株和荷瘤组织中的凋亡诱导作用.方法 通过免疫组化、RT-PCR和Western blot法检测胆囊正常组织、息肉组织及胆囊癌组织中TFPI-2基因和蛋白的表达情况.构建携带TFPI-2基因的重组腺病毒(Ad5-TFPI-2),检测Ad5-TFPI-2抑制胆囊癌荷瘤的生长作用及其诱导细胞凋亡的作用.通过Western blot法检测TFPI-2蛋白对胆囊癌细胞内凋亡相关蛋白表达的影响.结果 RT-PCR结果显示TFPI-2 mRNA在正常组织、胆囊息肉和胆囊癌组织中的阳性表达率分别为87.0％、52.2％、23.3％(x2 =21.104,P=0.000).Western blot结果显示TFPI-2蛋白在正常组织、胆囊息肉和胆囊癌组织中的阳性表达率分别为78.3％、47.8％、26.7％(x2=13.882,P =0.001).TFPI-2基因体内转染显著抑制胆囊癌细胞的生长.TFPI-2蛋白表达增加可以通过诱导Bax蛋白表达,降低Bcl-2,进而激活caspase-3蛋白介导内源性凋亡途径,诱导胆囊癌细胞发生凋亡.结论 TFPI-2基因表达降低可能参与胆囊病变从良性向恶性转变的过程.通过腺病毒基因转染的方法恢复TFPI-2基因的表达能够抑制胆囊癌的生长.%Objective To examine the expression of tissue factor pathway inhibitor-2 (TFPI-2) in gallbladder cancer (GBC) and to investigate the anti-cancer activities of TFPI-2 against the growth of GBC.Methods TFPI-2 expression in gallbladder normal tissues,gallbladder polyp (GBP) tissues and GBC tissues were examined by reverse transcriptase polymerase chain reaction (RT-PCR),Western blot and immunohistochemical staining.Adenovirus carrying human TFPI-2 gene (AdS-TFPI-2) were constructed and its anti-cancer effects were investigated in xenograft tumors.Xenograft tumors were constructed by injection of GBC-SD and SGC-996 cells into the flank of nude mice and the volume of xenograft tumors was measured every 3 days until the sacrifice of mice.The apoptosis index of xenograft tumors was examined by TUNEL assay.The status of Bax,Bcl-2 and caspase-3 was examined by Western blot assay.Results TFPI-2 expression was profoundly lower in GBC tissues (87.0％) when compared to normal tissues (23.3％) and GBP tissues (52.2％ ; x2 =21.104,P =0.000).Ad-TFPI-2 significantly inhibited the growth of xenograft tumors in nude mice.Ad-TFPI-2 inhibited GBC-SD cell growth through the induction of apoptosis.The means of total apoptotic cells per field were much higher in Ad5-TFPI-2 group than those in PBS and Ad5-GFP groups.Ad5-TFPI-2 elevated the expression of Bax and cleaved caspase-3,while it decreased the expression of Bcl-2.Conclusions TFPI-2 gene and protein was down-regulated in GBC and the downregulation of TFPI-2 may play a role in the tumorigenesis of GBC.Adenovirus-mediated TFPI-2 can inhibit GBC growth through the induction of apoptosis.