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目的:通过HIF-1α反义寡核苷酸转染hep-2细胞株,观察细胞凋亡及对放疗的反应情况。方法:体外培养人喉鳞状细胞癌hep-2细胞株,脂质体介导反义寡核苷酸转染,6 h后4 Gyγ射线照射。低氧培养24 h后RT-PCR检测HIF-1α mRNA表达,流式细胞仪检测HIF-1α蛋白水平及细胞凋亡率,MTT检测细胞存活情况。结果:低氧使人喉鳞状细胞癌hep-2细胞株对放射的敏感性下降;低氧并未影响HIF-1α mRNA的表达,HIF-1α蛋白在低氧6 h时表达已开始升高,低氧36 h时达高峰,之后表达逐渐下降;放射刺激对HIF-1α蛋白表达差异无统计学意义(P>0.05);各浓度的反义寡核苷酸转染组降低HIF-1α的转录及蛋白水平;MTT结果显示随反义核苷酸浓度增高,其增殖抑制效应越强(P<0.05),正义对照核苷酸及脂质体对照没有显著的抑制效应(P>0.05),但转染浓度在400 nmol/L以上时,正义对照也表现出一定的增殖抑制作用(P<0.05);放疗后反义寡核苷酸转染组蛋白表达下降明显,较对照组及单纯放射组细胞凋亡率增加(P<0.01)。结论:低氧环境下人喉鳞状细胞癌hep-2细胞株对放射有抵抗作用,这种抵抗作用与低氧时HIF-1α蛋白表达上调有关,而放射本身并未上调HIF-1α蛋白的表达;HIF-1α反义寡核苷酸转染hep-2细胞株能够降低HIF-1α蛋白表达,提高低氧环境下人喉鳞状细胞癌hep-2细胞株对放疗的敏感性。
OBJECTIVE: To investigate the apoptosis and the response to radiotherapy by transfecting hep-2 cell line with HIF-1α antisense oligonucleotide. Methods: Human laryngeal squamous cell carcinoma hep-2 cell line was cultured in vitro. Lipofectamine was used to transfect antisense oligonucleotide and irradiated with 4 Gy γ-ray 6 h later. The expression of HIF-1αmRNA was detected by RT-PCR, the protein level of HIF-1α and the apoptosis rate were detected by flow cytometry, and the cell viability was detected by MTT assay. Hypoxia did not affect the expression of HIF-1α mRNA and the expression of HIF-1α protein began to increase at 6 h of hypoxia , Peaked at 36 h after hypoxia, and then gradually decreased. The expression of HIF-1α protein was not significantly different between the two groups (P> 0.05). The concentrations of antisense oligonucleotide transfection group decreased HIF-1α (P <0.05). The results of MTT assay showed that the inhibitory effect of antisense nucleotide and liposome were not significant (P> 0.05) However, the transfection concentration of 400 nmol / L or more, the positive control also showed a certain degree of inhibition of proliferation (P <0.05); after radiotherapy antisense oligonucleotide transfection group protein decreased significantly compared with the control group and simple radiotherapy Group apoptosis rate increased (P <0.01). CONCLUSIONS: Human laryngeal squamous cell carcinoma hep-2 cell line is resistant to radiation in hypoxia environment. This resistance is related to the up-regulation of HIF-1α protein expression in hypoxia, while radiation itself does not up-regulate HIF-1α protein HIF-1α antisense oligonucleotide transfected hep-2 cell line can reduce the expression of HIF-1αprotein and enhance sensitivity of radiotherapy to human laryngeal squamous cell carcinoma hep-2 cell line under hypoxia.