靶向抑制survivin基因对肝细胞癌血管生成的影响

The effect of survivin targeting inhibition on angiogenesis of hepatocellular carcinoma

目的研究靶向抑制survivin基因对肝细胞癌血管生成的影响，探讨survivin在血管生成中的作用机制。方法采用脂质体介导survivin ASODN体外转染人肝癌细胞株HepG2，Western blot检测细胞中survivin蛋白的表达；RT-PCR检测细胞中VEGF mRNA的表达；FCM检测细胞的凋亡率；利用皮下注射法建立人肝癌裸鼠皮下移植瘤模型；采用脂质体包裹survivin ASODN直接瘤内注射，观察其对移植瘤生长情况的影响；SABC法检测移植瘤组织MVD。结果400nmol／Lsurvivin ASODN转染可明显下调HepG2细胞survivin蛋白表达，但不影响VEGFmRNA表达；ASODN组HepG2细胞凋亡率显著高于空白对照组和SODN组（F=428．19，P〈0．01）；ASODN组瘤体生长速度较空白对照组和SODN组明显减慢（F=12．63，P〈0．01）：ASODN组瘤体重量较空白对照组和SODN组明显减轻（F=5．90，P〈0．05）；ASODN组MVD较空白对照组和SODN组明显降低（F=34．29，P〈0．01）。结论survivin ASODN可以抑制人肝癌裸鼠移植瘤生长，其机理可能是诱导HepG2细胞自发性凋亡和抑制肿瘤血管生成。

Objective To investigate the effect of survivin targeting inhibition on angiogenesis of hepatocellular carcinoma and the role of survivin gene during tumor angiogenesis. Methods Human HCC cell line HepG2 was transfected in vitro with survivin antisense oligonucleotide （ASODN） mediated by lipofectamine TM2000. The expression of survivin protein was detected by Western blot. The expression of VEGF mRNA was determined by RT-PCR. Apoptotic index（AI） was examined by flow cytometry. Human HCC model in nude mice was established by subcutaneous injection of HepG2 cells. Tumor growth was evaluated after intratumoral injections with a mixture of survivin ASODN- lipofectamine ^TM2000 Microvessal density （MVD） of the tumor tissue was detected by immunohistochemical staining （SABC Method）. Results The expression of survivin protein in HepG2 cells was obviously downregulated after transfected with 400 nmol/L survivin ASODN, but the expression of VEGF mRNA was not affected. Apoptosis index （AI） of HepG2 cells in ASODN group was significantly higher than that of the control and SODN group（ F = 428. 19 ,P 〈 0. 01 ） ; The tumor growth in ASODN group was significantly slower than that of the control and SODN group（ F = 12.63, P 〈 0. 01 ） ; Tumor weight in ASODN group was significantly lower than that of the control and SODN group（ F = 5.90, P 〈 0. 05 ） ; MVD of ASODN group was significantly lower than that of the control and SODN group （ F = 34. 29, P 〈 0. 01 ）. Conclusions Survivin ASODN suppresses the growth of human HCC xenograft in nude mice, possibly by inducing the apoptosis of HepG2 cells and suppressing tumor angiogenesis.