摘要
目的探讨Stat3反义寡核苷酸(antisense oligodnucleotides,ASODN)联合放疗对喉癌Hep-2细胞的作用及其机制。方法 Hep-2细胞株传代培养,分别进行以下处理:无处理、60Co照射5Gy、不同浓度Stat3ASODN转染Hep-2细胞、不同浓度Stat3ASODN转染联合放疗,倒置荧光显微镜下观察细胞形态学改变。结晶紫法检测各组作用24h后的细胞增殖抑制率。流式细胞学检测不同浓度Stat3ASODN转染联合放疗后细胞周期的变化及不同浓度Stat3ASODN转染组、200nmol/LStat3ASODN转染组联合放疗前后对Stat3、p-Stat3、cyclin D1、p21和cdk4蛋白表达的影响。结果荧光显微镜下Stat3ASODN转染联合放疗组细胞形态改变显著,出现明显的病变。对细胞增殖抑制的作用差异显著,与单纯放疗和单纯ASODN转染组相比差异具有统计学意义(P<0.01)。流式细胞学检测显示:随着Stat3ASODN转染浓度的增加,Hep-2细胞G0/G1期比例上升,S期比例下降;Stat3、p-Stat3及cyclin D1、cdk4蛋白荧光指数呈一致性降低趋势,而p21蛋白荧光指数呈升高趋势,其中Stat3、p-Stat3、p21蛋白表达组间差别具有统计学意义(P<0.05)。Pearson相关性分析结果显示:Stat3与p-Stat3呈显著正相关(r=0.986,P<0.01)、与cdk4呈显著正相关(r=0.910,P<0.05),与p21呈负相关(r=-0.981,P<0.05);p-Stat3与cdk4呈正相关(r=0.949,P<0.05),与p21呈负相关(r=-0.917,P<0.05);p21与cdk4之间呈现一定的负相关性(r=-0.973,P<0.01);细胞抑制率的变化与Stat3、p-Stat3蛋白荧光指数的变化呈负相关(r=-0.989、r=-0.971,P<0.01)。结论 Stat3ASODN转染喉癌Hep-2细胞,通过下调Stat3蛋白的表达而对细胞周期蛋白进行调控,从而引起细胞周期分布的改变,达到增强放疗敏感性的作用。
Objective To study the synergistic effect and mechanism of transfecting Stat3 antisense oligonucleotides（ASODN）and irradiation on laryngeal squamous carcinoma cell line Hep-2.Methods Hep-2 cells which were in the logarithmic growth phase were divided into untreated group,transfection with Stat3 ASODN group,radiotherapy group and transfection combined with radiotherapy group.Cell morphological changes were observed by inverse-phase microscope.Growth inhibition rates was detected by MTT assay.Cell cycle and the expressions of stat3,p-stat3,cyclin D1,p21 and cdk4 protein were detected by flow cytometry（FCM）.Results The combined treatment group had significantly morphological changes and growth inhibition rate（P0.01）.FCM results showed that the proportion of cells in G0/G1 phase increased and cells in S phase continuously reduced with augmentation of ASODN concentration.The protein expressions of stat3,p-stat3,cyclin D1 and cdk4 decreased,while the expression of p21 protein increased in combined treatment group.The protein expressions of stat3,p-stat3 and p21 were statistically different among the groups（P0.05）.Pearson＇s correlation analysis showed that stat3 had positive correlation with p-stat3（r=0.986,P0.01）and Cdk4（r=0.910,P0.05）,while had negative correlation with p21（r=-0.981,P0.05）;The p-stat3 protein expression were positively correlated with Cdk4（r=0.949,P0.05）and negatively correlated with p21（r=-0.917,P0.05）;The expressions of p21 protein and Cdk4 were negatively correlated（r=-0.973,P0.01）.Cell inhibition rate were negatively correlated with stat3,p-stat3 protein fluorescent index（r=-0.989 and r=-0.971 respectively,P0.01）.Conclusions Hep-2 cells transfected with Stat3 ASODN can change the distribution of cell cycle and then increase the radiosensitivity of cells,by reducing the expression of stat3 which regulates cell cycle proteins.
出处
《中国癌症防治杂志》
CAS
2010年第4期261-265,共5页
CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT
基金
全军医药卫生十一五科技攻关项目(200606M076)
关键词
STAT3
喉癌
反义寡核苷酸
放疗
细胞周期
Stat3
Laryngeal squamous cell carcinoma
Antisense oligonucleotide
Radiotherapy
Cell cycle
作者简介
通讯作者：李晓明。E—mail：xmlmo@126．com
