摘要
目的探讨Notch信号转导对小细胞肺癌的调控作用及可能机制。方法应用重组质粒转染的方法，在小细胞肺癌细胞株NCI—H446中表达组成性活化的Notch1（NIC转染组），同时设立转染空质粒组和未转染组作为对照组，待筛选出稳定细胞株后，以MTT法检测细胞活力，应用半定量RT—PCR技术测定Notch1及其下游基因（HES1和hASH1）表达，并应用免疫细胞化学标记和Westernblot技术对神经内分泌标志物嗜铬粒素A（CgA）、神经元特异性烯醇化酶（NSE）的表达行半定量[阳性单位（PU）值]分析。结果未转染组和转染空质粒组Notch1及其下游基因HES1表达不明显，而hASH1表达显著，转染组细胞Notch1及HES1表达升高，同时伴有hASH1表达明显降低。与两对照组比较，转染组细胞增殖速度显著降低，连续6d测得的吸光度（4）值均小于未转染组和转染空质粒组（均P〈0．05）。免疫细胞化学染色显示，NIC转染组、转染空质粒组、未转染组的CgA染色的PU值分别为8．81±0．77、38．10±1．55、38．97±0．80，NSE染色的PU值分别为7．21±0．59、28．25±1．46、30．57±1．31，NIC转染组CgA和NSE的PU值均小于转染空质粒组和未转染组（均P〈0．01）。Westernblot检测结果中，将未转染组的条带灰度值设为1．00，NIC转染组、转染空质粒组的CgA条带灰度值分别为0．54±0．03、0．99±0．05，NSE条带灰度值分别为0．43±0．02、1．07±0．09，NIC转染组CgA和NSE条带的灰度值均小于转染空质粒组和未转染组（均P〈0．01）。结论Notch信号途径对小细胞肺癌的调控可能是通过靶基因HES1对分化效应基因hASH1的转录抑制来实现的；Notch信号途径可以抑制小细胞肺癌细胞的增殖，降低其神经内分泌标志物的表达。
Objective To investigate the status of Notch signaling pathway in small cell lung cancer （SCLC）. Methods Expression plasmids of pEFBOS-NIC-MYC and pEFBOS-neo were transfected into NCI-H446 cells. Stably transfected cell lines were selected and their growth rates were examined by MTT method. Expression of downstream genes along the Notch signaling pathway were studied by RT-PCR. Protein expression of euroendocrine markers of CgA and NSE were detected by Western blot analysis and immunocytocbemistry. Results The expression of HES1 was increased in the pEFBOS-NIC-MYC group, but the expression of hASH in the pEFBOS-NIC-MYC group was decreased significantly. The transfected cells with pEFBOS-NIC-MYC plasmid showed a significantly slower growth rate compared with that of two control groups（P 〈 0. 05, Student＇s t-test）. Immunocytoehemistry 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 smaller than the values of the latter two significantly （ P 〈 0. 01 ）. Western blot analysis showed the grave scales of CgA in NIC transfected group and sham group to be 0. 54 ± 0. 03 and 0. 99 ± 0. 05 respectively （ grave scales of the negative control was set as 1.00）, the former one significantly smaller than that of the other two groups （ P 〈 0.01 ）. The grave scales of NSE in the NIC transfected group and sham group were 0. 43 ± 0. 02 and 1.07±0. 09 respectively （ grave scales of the negative control was set as 1.00） and the former one was significantly smaller than the other two groups （ P 〈 0. 01 ）. Conclusion Notch signaling pathway regulates SCLC cells through its inhibitory effect on hASH1 transcription via HES1 along with an expression inhibition of neuroendocrine markers in SCLC.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2010年第2期95-99,共5页
Chinese Journal of Pathology
