摘要
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Notchl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6d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M1Tr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6d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)。Western 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Notchl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。
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 Notehl (NIC) was over-expressed in NCI-H446 cells by constitutive transfection and a stable transfected cell line was established. Proliferation of NCI-H446 ceils 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 cells 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 ). Immunohistochemistry 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 transfeeted 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 Notchl may behave as a tumor suppressor in small cell lung cancer. Notchl signal activation can inhibit the proliferation and neuroendoerine ma
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2009年第5期335-339,共5页
Chinese Journal of Oncology
基金
浙江省科技厅计划基金(2006C33060)