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目的体外研究C-MYC反义寡核苷酸(ASODN)对人宫颈癌HELA细胞凋亡的影响,寻求提高肿瘤细胞放射敏感性的方法。方法免疫组化、流式细胞术、半定量RT-PCR法鉴定ASODN的有效性;GIEMSA染色、流式细胞术检测凋亡指数(AI)、DNA LADDER检测细胞凋亡。结果转染后,免疫组化结果显示C-MYC蛋白表达降低;半定量RT-PCR结果显示C-MYC MRNA表达较空白对照组及阴性对照组明显减弱(P<0.05)。GIEMSA染色可见凋亡小体;流式细胞术结果显示转染后凋亡指数为(10.29±0.66)%,转染ASODN(C-MYC)联合放射的凋亡指数为(16.83±0.57)%,均明显高于空白对照组(1.79±0.19)%(P<0.05);DNA LADDER呈现具有凋亡特征的梯带。结论本实验所设计的ASODN能有效地抑制C-MYC基因的表达;转染ASODN(C-MYC)能够显著增加HELA细胞凋亡,从而提高肿瘤细胞的放射敏感性。
Objective To investigate the effect of C-MYC antisense oligonucleotide (ASODN) on the apoptosis of human cervical cancer HELA cells in vitro and to find a method to improve the radiosensitivity of tumor cells. Methods Immunohistochemistry, flow cytometry and semi-quantitative RT-PCR were used to identify the effectiveness of ASODN. Apoptotic index (AI) was detected by GIEMSA staining and apoptosis by DNA LADDER. Results The results of immunohistochemistry showed that the expression of C-MYC protein was decreased after transfection. Semi-quantitative RT-PCR results showed that the expression of C-MYC MRNA was significantly decreased compared with the blank control group and the negative control group (P <0.05). The apoptotic index of the transfected ASODN (C-MYC) combined with radiation was (16.83 ± 0.57)%, (1.79 ± 0.19)%, respectively (P <0.05). DNA LADDER showed a ladder with apoptotic features. CONCLUSIONS: ASODN designed in this study can effectively inhibit the expression of C-MYC gene. Transfection of ASODN (C-MYC) can significantly increase the apoptosis of HELA cells and enhance the radiosensitivity of tumor cells.