缺氧诱导因子-1a反义寡核苷酸对喉鳞癌荷瘤裸鼠化疗的增敏作用

Chemosensitization effect of hypoxia inducible factor-1α anti-sense oligodexynucleotide on the model of human laryngeal squamous cell cacinoma in nude mice

目的探讨应用缺氧诱导因子-1α(HIF-1α)反义寡核苷酸对荷瘤裸鼠化疗(顺铂)的增敏作用及可能的途径。方法将肿瘤最大直径长至8～10mm的裸鼠,选择20只随机分为四组:A组(正义+化疗),B组(反义+化疗),C组(化疗),D组(空白对照),每次注射反义寡核苷酸100μg/只(按脂质体:AS-ODN=1∶1进行包裹加入等量的细胞培养液),0.2ml向肿瘤基底部3点以及中央1点注射,隔日注射1次,共8次,前5次注射反义寡核苷酸24h后给予荷瘤裸鼠顺铂(日化疗剂量3mg/kg,共5次)腹腔注射,在最后一次注射反义寡核苷酸24h后将实验鼠脱颈处死,应用流式细胞仪、免疫组化观察肿瘤细胞的凋亡、周期变化及HIF-1α、P53和VEGF蛋白表达。结果反义组凋亡率最大(44.750±5.710),明显高于空白对照组、化疗对照以及正义对照组(P<0.01);反义组细胞G0/G1期比例明显低于其他3组(P<0.05),其他3组细胞阻滞在G0/G1期,S期和G2/M期细胞减少。免疫组化SP法染色显示HIF-1α、P53及VEGF在反义治疗组表达较其他3组明显较少;流式细胞仪检测结果显示反义治疗组的HIF-1α、P53、VEGF蛋白表FI值(0.905±0.141,0.773±0.128,0.848±0.068)明显低于化疗对照以及正义对照组(P<0.01)。结论缺氧诱导因子(HIF-1α)反义寡核苷酸联合化疗对肿瘤的抑瘤作用最为明显,对化疗有明显增敏作用。

Objective To investigate the sensitization effect and the possible pathway of hypoxia inducible factor-1α（HIF-1α） anti-sense oligodexynucleotide in the chemotherapy of human laryngeal squamous cell cacinoma in nude mice.Methods Twenty-eight nude mice were divided into four groups：group A（HIF-1a S-PS-ODN transfection＋chemotherapy）,group B（HIF-1a AS-PS-ODN transfection＋ chemiotherapy）,group C（chemiotherapy） and group D（blank for constrol）.100 μg DNA was injected into the mice in group A and B（lipofectamine2000∶DNA=1∶1）.Mice of all groups were injected with DDP every 48 h.The duration was 16 days.The nude mice were killed 24 h after the last injection.Flow cytometry（FCM） was used for detecting cell apoptosis.The expression of HIF-1α,P53 and VEGF was detected with immunocytochemical method.Results The apoptosis rate of group B（44.75±5.71%） was significantly higher than that of the other 3 groups（P0.01）.Proportion of G0/G1 in Group B was significantly lower than those in the other 3 groups（P0.05）.Expression of these proteins in Group B was significantly lower than that in the other 3 groups.After transfected by HIF-1α AS-PS-ODN,the expression of HIF-1α,P53,VEGF proteins in Hep-2 cells was restrained.The expression in Hep-2 cells of group B（0.905±0.141,0.773±0.128,0.848±0.068） was significantly lower than that in the other 3 groups（P0.01）.Conclusion Treatment strategy in group B may be the most effective in the inhibition of tumors.Expression of P53 protein and VEGF protein may be the major apoptotic pathway.