CAJ | 학술논문

目的研究Notch信号通路对小细胞肺癌(SCLC)细胞增殖及神经内分泌标志物表达的调控作用.方法采用包含Notch1胞内段(NIC)的重组质粒转染NCI-H446细胞,并建立稳定转染株.实验细胞分为3组,即NIC转染组、空质粒转染组和未转染组.应用二苯基溴化四氮唑蓝(MTT)法,连续6 d测定各组细胞的增殖活性.应用细胞爬片、免疫细胞化学染色法以及Western blot技术,对神经元特异性烯醇化酶(NSE)和嗜铬素蛋白A(CgA)的表达量进行检测.结果 MTT法检测结果显示,NIC转染组细胞的增殖活性明显降低,其连续6 d测得的吸光度(A)值均小于空质粒转染组和未转染组(均P<0.05).免疫细胞化学染色结果显示,NIC转染组、空质粒转染组和未转染组cgA染色的阳性单位(Pu)值分别为8.81±0.77、38.10±1.55和38.97±0.80;NSE染色的PU值分别为7.21 ±0.59、28.25±1.46和30.57±1.31;NIC转染组CgA和NSE的Pu值均显著小于空质粒转染组和未转染组(均P<0.01).Westem blot检测结果显示,NIC转染组、空质粒转染组和未转染组CgA条带的灰度值分别为0.54±0.03、0.99±0.05和1.00;NSE条带的灰度值分别为0.43±0.02、1.07±0.09和1.00;NIC转染组CgA和NSE条带的灰度值均显著小于空质粒转染组和未转染组(均P<0.01).结论 Notch1在SCLC中可能是一种抑癌基因,其介导的Notch信号通路的上调能抑制SCLC细胞的增殖和神经内分泌标志物的表达.
목적 연구Notch신호통로대소세포폐암(SCLC)세포증식급신경내분비표지물표체적조공작용.방법 채용포함Notch1포내단(NIC)적중조질립전염NCI-H446세포,병건립은정전염주.실험세포분위3조,즉NIC전염조、공질립전염조화미전염조.응용이분기추화사담서람(MTT)법,련속6 d측정각조세포적증식활성.응용세포파편、면역세포화학염색법이급Western blot기술,대신경원특이성희순화매(NSE)화기락소단백A(CgA)적표체량진행검측.결과 MTT법검측결과 현시,NIC전염조세포적증식활성명현강저,기련속6 d측득적흡광도(A)치균소우공질립전염조화미전염조(균P<0.05).면역세포화학염색결과 현시,NIC전염조、공질립전염조화미전염조cgA염색적양성단위(Pu)치분별위8.81±0.77、38.10±1.55화38.97±0.80;NSE염색적PU치분별위7.21 ±0.59、28.25±1.46화30.57±1.31;NIC전염조CgA화NSE적Pu치균현저소우공질립전염조화미전염조(균P<0.01).Westem blot검측결과 현시,NIC전염조、공질립전염조화미전염조CgA조대적회도치분별위0.54±0.03、0.99±0.05화1.00;NSE조대적회도치분별위0.43±0.02、1.07±0.09화1.00;NIC전염조CgA화NSE조대적회도치균현저소우공질립전염조화미전염조(균P<0.01).결론 Notch1재SCLC중가능시일충억암기인,기개도적Notch신호통로적상조능억제SCLC세포적증식화신경내분비표지물적표체.
Objective To investigate the effects of Notchl signal activation on proliferation and neuroendocrine marker expression in small cell lung cancer cells. Methods The active form of Notch1 (NIC) was over-expressed in NCI-H446 cells by constitutive transfection and a stable transfected cell line was established. Proliferation of NCI-H446 cells was analysed by MTT assay on 6 successive days. Expression of neuroendocrine markers (CgA, NSE) was observed by immunohistochemistry and Western blot analysis. Statistical analysis was conducted to compare the results in ceils with NIC transfected and those in control groups. Results MTT assay showed that absorbance (A) of cells overexpressing Notchl was significantly depressed compared with that of the control cells (P < 0. 05). Immunohistochemistry of CgA showed that Pus in the NIC transfected group, sham group and negative control group were 8.81 ±0.77, 38.10 ± 1.55, 38.97±0.80, respectively, the former one was significantly smaller than that of the latter two (P <0.01). Lmmunohistochemistry of NSE showed that Pus in the NIC transfected group, sham group and negative control group were 7.21±0.59, 28.25 ± 1.46, 30.57 ± 1.31, respectively, the former one was significantly smaller than that in the latter two (P <0.01). Western blot analysis showed that the gray scales of CgA in the NIC transfected group and sham group were 0. 54 ± 0. 03 and 0. 99 ± 0. 05, respectively, (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P < 0.01). The gray scales of NSE in the NIC transfected group and sham group were 0.43 ± 0.02 and 1.07 ± 0.09, respectively (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P < 0. 01). Conclusion Notch1 may behave as a tumor suppressor in small cell lung cancer. Notchl signal activation can inhibit the proliferation and neuroendocrine marker expression in small cell lung cancer cells, suggesting that Notchl gene could be a new target for small cell lung cancer treatment and probable relief of paraneoplastic syndrome.