^131I对分化型甲状腺癌细胞核因子κB表达和功能的影响

Changes of NF-kB expression and function in differentiated thyroid cancer cells by^131I therapy

目的探讨^131I对DTC细胞核因子KB（NF-KB）表达和功能的影响，以及^131I和NF-KB抑制剂Bay11-7082联合治疗的可行性。方法用不同放射性浓度^131I和不同浓度Bay11-7082处理DTC细胞，加入四氮唑盐显色，测定450nm的吸光度（A）值，用公式（A药物处理组-A空白）／（A对照组-A空白）×100％计算癌细胞存活率（A空白为空白孔的吸光度，A对照组为单纯细胞孔的吸光度）；选择癌细胞存活率约60％左右的^131I和Bay11-7082浓度进行联合实验。将^131I和Bay11-7082作用于DTC细胞6、24和48h后，提取核蛋白，分别与NF-KB结合序列探针相结合，测定450nm的A值，NF-KB的DNA结合率=（A药物处理组-A空白）／（A对照组-A空白）×100％。用Westernblot鉴定单用^131I、Bay11-7082和联合用药6h后NF-KB核蛋白相对表达水平的变化，并以B-actin作为内参对照进行半定量分析。用配对t检验、F检验和q检验进行统计分析。结果细胞存活分析发现不同放射性浓度^131I和不同浓度Bay11-7082处理后癌细胞的存活率不同（F=281．07和173．84，P均〈0．01）；单用^131I组、单用Bay11-7082组与联合用药组癌细胞的存活率分别为（67．33±5．65）％、（61．83±6．68）％和（36．67±5．35）％，差异有统计学意义（F=45．79，P〈0．01），其中前2组分别与联合处理组相比q=8．20和8．35，P均〈0．01。DNA结合实验证实”。I可以诱导癌细胞内NF-KB结合率增高，24h为（255．33±29．86）％（最高）；而联合使用Bay11-7082后，在6、24和48h时间点能够抑制到相应刺激状态下NF-KB功能的22．10％、39．75％和43．18％，联合处理组与单用^131I组比较，3个时间点差异均有统计学意义（t：24．58、26．29和8．20，P均〈0．01）；6h时NF-KBp65功能受抑程度最明显，DNA结合率仅为（35．33±8．21）％，与对照组相比差异有统计学意义（t=28．98，P〈0．01）。半定量分析：^131I作用6h后p65和pSO相对表达水平均升高，Bay11-7082联合^131I作用6h后两者的表达水平均受到明显抑制；不同给药方式作用后p65和p50的相对表达水平差异均有统计学意义（F=100．93和193．55，P均〈0．01），^131I组和对照组两两比较，q=4．75和8．22，P均〈0．05，联合处理组与对照组两两比较，q=30．80和22．83，P均〈0．01。结论^131I会导致DTC细胞NF-KB表达增加、功能增强，联合使用NF-（B抑制剂可以抑制这种改变，获得协同疗效。

Objective To study the effects of ^131I on the expression and function of nuclear factor- kappa B （NF-KB） in a DTC cell line and to investigate the possibility of combined therapy of ^131I and a NF- KB inhibitor （Bay 11-7082）. Methods By cell survival assay, effects of monotherapy and the combination of ^131I and Bay 11-7082 were assessed. The absorbent values （As） were read at 450 nm. Cell survival rate was calculated using the following formula： （Atherapy -- Ablank ）/（A 1 - Ablank ） ~ 100%. By DNA binding assay, nuclear proteins extracted at 6, 24 and 48 h after treatment were added to the NF-KB DNA binding probes. The A was read at 450 nm. DNA binding rate was obtained according to the formula： （Atherapy -Ablank ）/（Acontrol --Ablank ）×100%. The changes of NF-KB protein expression after different therapies were de- tected by Western blot, and 13-actin was used as control for semi-quantitative analysis. Paired t test, F test and q test were performed for statistical analysis. Results Significant differences of cancer cell survival rates were shown after monotherapy of different^131I activities and different concentrations of Bay 11-7082 （F = 281.07 and 173.84, both P 〈 0. 01 ）. The cancer survival rates after 131I treatment alone, Bay 11-7082 treatment alone and combined therapy were （67.33 ± 5.65 ） %, （61. 83 ± 6.68） % and （36. 67 ± 5.35 ） % respectively （F =45.79, P 〈 0. 01 ）. Also, significant reduction of cell survival rates were shown betweenthe combined therapy and monotherapy of ^131I or Bay 11-7082 （ q = 8.20 and 8.35, both P 〈 0.01 ）. DNA binding assay showed^131I could greatly induce NF-KB binding rate, which reached a peak of （255.33 ± 29. 86）% at 24 h. Combined therapy suppressed NF-KB binding rates to 22.10% , 39.75% and 43.18% at 6, 24 and 48 h respectively, which were significantly different compared with those of 131I monotherapy （t ： 24.58, 26. 29, 8.20, all P 〈 0.01 ）×The most suppressed NF-KB p65 binding rate （35.33 ± 8.21 ） % was observed at 6 h, which was significantly different from that of control group （ t = 28.98, P 〈 0. 01 ）. The relative protein expression levels of 1365 and p50 increased 6 h after 131I therapy. However, the com- bined treatment could effectively decrease the expression levels of p65 and 1350. Both p65 and p50 were sig- nificantly different after different treatments （ F = 100.93 and 193.55, both P 〈 0.01 ）. Statistical differ- ence was found with p65 and p50 expression levels when compared ^131I and combined therapy with the control group （ q = 4.75 and 8.22, both P 〈 0.05 for ^131 I ; q = 30.80 and 22.83, both P 〈 O. O1 for combined thera- py）. Conclusions^131I could induce the expression and function activation of NF-KB in DTC cells. However, NF-KB inhibitor could effectively suppress the changes and enhance therapeutic effects synergistically.