RNA干扰信号转导和转录活化因子3基因提高喉癌放疗疗效的实验研究

Silencing of signal transducer and activator of transcription 3 gene expression using RNAi enhances the efficacy of radiotherapy for laryngeal carcinoma in vivo

目的探讨RNA干扰信号转导和转录活化因子3（signal transducer and activator of transcription 3，STAT3）基因联合放射治疗对喉癌Hep-2裸鼠移植瘤生长的抑制作用。方法建立人喉癌Hep-2裸鼠移植瘤动物模型28只，采用随机数字表法将动物随机分为4组：阴性质粒对照组、干扰组、放疗组和干扰＋放疗组，定期测量肿瘤体积。放射治疗结束后15d处死动物，称量瘤重，计算抑瘤率，绘制肿瘤生长曲线。免疫组化SP法结合图像分析技术对各组移植瘤磷酸化STAT3、B淋巴细胞2（bcl-2）、p53、血管内皮生长因子（VEGF）蛋白的表达情况进行定量分析，检测移植瘤的微血管密度，流式细胞术检测肿瘤细胞凋亡率。结果干扰组、放疗组和干扰＋放疗组的抑瘤率分别为19．68％、34．76％和67．70％。干扰＋放疗组磷酸化STAT3蛋白表达较其他组显著降低（P〈0．01），肿瘤微血管密度明显低于阴性对照组和放疗组（P〈0．01），肿瘤细胞凋亡率显著增加（P〈0．01）。干扰＋放疗组磷酸化STAT3表达与p53、bcl-2、VEGF及微血管密度呈正相关趋势（r值分别为0．738、0．727、0．735、0．691，P值均〈0．01），与肿瘤细胞凋亡率呈负相关趋势（r=-0．765，P〈0．01），p53和VEGF蛋白表达分别与微血管密度呈正相关趋势（r值分别为0．784、0．641，P值均〈0．01）；bcl-2蛋白表达与凋亡率呈负相关趋势（r=-0．883，P〈0．01）。结论RNA干扰STAT3基因联合放射治疗能显著抑制喉癌裸鼠移植瘤的生长。

Objective To study the inhibitory effect of signal transducer and activator of transcription 3 （STAT3） shRNA generated by vector pSilence in conjunction with radiotherapy on laryngeal squamous cell carcinoma of nude mice xenograft tumor. Methods The animal models of xenotransplanted human laryngeal carcinoma cell line Hep-2 were set up in 28 nude mice which were divided into 4 groups at random： the negative plasmid control group, the group that received pshSTAT3 （pGPU6/GFP/NeoshSTAT3） transfecion, the radiation group, and the group of pshSTAT3 transfection combined with irradiation. Tumor volume was determined regularly. On the fifteenth day after termination of radiation treatment, the mice were sacrificed, the tumor weight was measured in all the groups, the inhibition rate for tumor growth was calculated and tumor growth curve was plotted. Meanwhile, the expressions of p-STAT3, B cell lymphoma 2 （bcl-2）, p53, vascular endothelial growth factor（VEGF） protein and intratumor microvessel density （MVD） were observed by immunohistochemistry. Computer-assisted image analysis was used to obtain the results. Flow cytometry was used to detect the cell apoptosis rate. Results There was a significant difference in tumor volume among the groups （P 〈 0. 01 ）. The rate of tumor inhibition in the pshSTAT3 group, radiation group and pshSTAT3 plus radiation group was 19. 68%, 34. 76% and 67.70%, respectively. The p-STAT3 protein expression decreased significantly in the group of pshSTAT3 plus simple radiotherapy （P 〈 0. 01 ）. The intratumoral MVD in the group of pshSTAT3 plus simple radiotherapy was significantly lower compared to the negative plasmid control group and the radiotherapy group （ P 〈 0. 01 ）, while the apoptosis rate was much higher （ P 〈 0.01 ） . There was a positive correlation between the expressing of p-STAT3 and bcl-2, p53, VEGF and MVD（r value was 0. 738,0. 727,0. 735,0. 691, all P 〈 0. 01 ）, and there was a negative correlation concerning cell apoptosis rate. Moreover, a statistically positive association was present between MVD and p53, VEGF protein expression, respectively（r value was 0. 784, 0. 641 ,all P 〈 0. 01 ） ; and the correlation was negative between expression of bcl-2 and apoptosis rate （ r = -0. 883,P 〈 0. 01 ）. Conclusions Using pshSTAT3 in conjunction with radiotherapy can significantly inhibit the growth of laryngeal carcinoma.