目的：探讨双特异性酪氨酸磷酸化调节激酶1b（Dyrk1b）蛋白在不同程度子宫颈病变组织及子宫颈癌细胞中的表达及意义。方法（1）组织标本检测：选取2011年1月至2013年12月间就诊于大连医科大学附属第一医院，经病理检查证实的子宫颈鳞癌75例（其中Ⅰ期60例，Ⅱ期15例）、低级别鳞状上皮内病变（LSIL）12例、高级别鳞状上皮内病变（HSIL）40例患者的石蜡组织标本；另取同期经病理检查确诊的28例慢性子宫颈炎患者的子宫颈组织为对照。采用免疫组化SP法检测不同程度子宫颈病变组织中Dyrk1b蛋白的表达情况，并分析子宫颈癌组织中Dyrk1b蛋白的表达与其不同临床病理指标的关系。（2）细胞实验：采用子宫颈癌细胞系HeLa和SiHa细胞，实验分为两组，实验组细胞中加入Dyrk1b抑制剂——AZ191，对照组细胞中不加AZ191。采用蛋白印迹法检测不同浓度（5、10μmol/L）的AZ191作用后两组细胞中Dyrk1b蛋白的表达；四甲基偶氮唑蓝（MTT）比色法检测不同浓度（2.5、5、10、25、50、100μmol/L）的AZ191作用后两组细胞的增殖情况；流式细胞仪检测不同浓度（5、10μmol/L）的AZ191作用后两组细胞的凋亡情况。结果（1）免疫组化SP法检测显示，慢性子宫颈炎、LSIL、HSIL和子宫颈鳞癌组织中Dyrk1b蛋白阳性表达率分别为11%（3/28）、1/12、42%（17/40）、71%（53/75），子宫颈鳞癌明显高于慢性子宫颈炎、LSIL、HSIL（P<0.01）；HSIL明显高于慢性子宫颈炎、LSIL（P<0.05）；而LSIL与慢性子宫颈炎比较，差异无统计学意义（P>0.05）。子宫颈鳞癌组织中Dyrk1b蛋白的表达与子宫颈肌层浸润深度显著相关（P<0.01），而与年龄、临床分期、淋巴结有无转移以及血清鳞状上皮细胞癌抗原（SCC-Ag）水平均无显著相关性（P>0.05）。（2）蛋白印迹法检测显示，不同浓度（5、10μmol/L）的AZ191作用后，实验组HeLa和SiHa细胞中Dyrk1b蛋白的表达强度均随着AZ191浓度的增加而减弱，且均明显弱于对照组。MTT比色法检测显示，不同浓度（2.5、5、10、25、50、100μmol/L）的AZ191作用后，实验组HeLa和SiHa细胞增殖的抑制率随着AZ191浓度的增加均逐渐增高，呈明显的浓度依赖性（P<0.01）；且均明显高于对照组（P<0.05）。但实验组中同一浓度A2191作用后HeLa细胞与SiHa细胞增殖的抑制率分别比较，差异均无统计学意义（P>0.05）。流式细胞仪检测显示，5μmol/L的AZ191作用后，实验组HeLa和SiHa细胞的凋亡率[分别为（12.8±0.7）%、（3.8±1.8）%]分别与对照组[分别为（9.1±0.9）%、（2.2±1.1）%]比较，差异均无统计学意义（P>0.05）；而10μmol/L的AZ191作用后，实验组HeLa和SiHa细胞的凋亡率[分别为（15.5±1.1）%、（8.4±3.3）%]均明显高于对照组（P<0.05）。但实验组中同一浓度AZ191作用后HeLa细胞与SiHa细胞的凋亡率分别比较，差异均无统计学意义（P>0.05）。结论随着子宫颈病变程度的进展，Dyrk1b蛋白的表达逐渐增高，子宫颈癌组织和细胞中Dyrk1b蛋白均呈高表达；Dyrk1b抑制剂AZ191下调Dyrk1b蛋白表达后，可抑制子宫颈癌细胞的增殖，诱导细胞发生凋亡。%Objective To detect and explore the expression and clinical significance of dual specificity tyrosine phosphorylation regulated kinase1b (Dyrk1b) in the specimens and cells of cervical lesions. Methods (1)All the data were collected from 75 patients with cervical cancer and 52 cases with squamous intraepithelial lesion(SIL)admitted in the First Affiliated Hospital of Dalian Medical College during Jan. 2011 to Dec. 2013 and confirmed by pathological examination, included 60 cases of stageⅠand 15 cases of stageⅡ, 12 cases with low-grade squamous intraepithelial lesion(LSIL)and 40 cases with high-grade squamous intraepithelial lesion(HSIL). While, 28 cases with chronic cervicitis were chosen as the control group. The protein expression of Dyrk1b was detected by immunohistochemistry among the four groups.(2)The expression of Dyrk1b in HeLa and SiHa cells were detected by western blot method and the expression of Dyrk1b protein were also detected after treatment of AZ191 (5, 10 μmol/L) for 48 hours in HeLa and SiHa cells.(3)The cellular survival and proliferation of HeLa and SiHa cells treated by different concentrations of AZ191(2.5, 5, 10, 25, 50, 100 μmol/L)for 48 hours were detected by methyl thiazolyl tetrazolium (MTT) assay.(4)The rate of apoptosis of HeLa and SiHa cells was detected by flowcytometry after treatment of AZ191 (5, 10μmol/L) for 48 hours. Results (1)The positive rates of Dyrk1b protein in chronic cervicitis, LSIL, HSIL and cervical squamous cancer by immunohistochemistry were 11%(3/28), 1/12, 42%(17/40)and 71%(53/75), respectively. The expression of Dyrk1b in cervical squamous cancer and HISL were higher than those in LSIL and chronic cervicitis (P<0.01), there were significant difference between cervical squamous cancer and HSIL, or between HSIL and LSIL(all P<0.05), while there were not significant difference between LSIL and chronic cervicitis(P>0.05). Expression of Dyrk1b was correlated with stromal invasion depth of cervical cancer (P<0.05), but not with age, clinical stage, lymph node metastasis, and serum squamous cell carcinom antigen(SCC-Ag)levels (all P>0.05). (2) Dyrk1b protein was expressed in different levels in HeLa and SiHa cells, and the expression of Dyrk1b was decreased gradually as the increased of the concentration of AZ191 in both HeLa and SiHa cells by treatment of AZ191 for 48 hours. (3) Different concentration of AZ191 treated on cervical cancer cells could inhibit the cellular proliferation and induce cell apoptosis in a concentration-dependent manner(P<0.01), concomitant to the decreased cell survival rate. The apoptosis rate of HeLa and SiHa were increased significantly after 10μmol/L AZ191-treatment for 48 hours, but no any difference induced by 5 μmol/L AZ191-treatment compared to control group. Also,there was no any difference between Hela and SiHa cells in either inhibitory effect or apoptosis rate induced by AZ191. Conclusions Dyrk1b is over-expressed in either specimens or cells of cervical cancer. The expression of Dyrk1b protein in cervical lesions is increased as the progression of disease. Dyrk1b inhibitor AZ191 could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner in cervical cancer cells.
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