中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2010年
2期
95-99
,共5页
张秀明%王洁心%雷小光%程慧%王玲玲%姚根有
張秀明%王潔心%雷小光%程慧%王玲玲%姚根有
장수명%왕길심%뢰소광%정혜%왕령령%요근유
肺肿瘤%癌%小细胞%受体%Notch%肿瘤细胞%培养的
肺腫瘤%癌%小細胞%受體%Notch%腫瘤細胞%培養的
폐종류%암%소세포%수체%Notch%종류세포%배양적
Lung neoplasms%Carcinoma%small cell%Receptor%Notch%Tumor cells%cultured
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6 d测得的吸光度(A)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).Western blot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.
目的 探討Notch信號轉導對小細胞肺癌的調控作用及可能機製.方法 應用重組質粒轉染的方法,在小細胞肺癌細胞株NCI-H446中錶達組成性活化的Notch1(NIC轉染組),同時設立轉染空質粒組和未轉染組作為對照組,待篩選齣穩定細胞株後,以MTT法檢測細胞活力,應用半定量RT-PCR技術測定Notch1及其下遊基因(HES1和hASH1)錶達,併應用免疫細胞化學標記和Westernblot技術對神經內分泌標誌物嗜鉻粒素A(CgA)、神經元特異性烯醇化酶(NSE)的錶達行半定量[暘性單位(PU)值]分析.結果 未轉染組和轉染空質粒組Notch1及其下遊基因HES1錶達不明顯,而hASH1錶達顯著,轉染組細胞Notch1及HES1錶達升高,同時伴有hASH1錶達明顯降低.與兩對照組比較,轉染組細胞增殖速度顯著降低,連續6 d測得的吸光度(A)值均小于未轉染組和轉染空質粒組(均P<0.05).免疫細胞化學染色顯示,NIC轉染組、轉染空質粒組、未轉染組的CgA染色的PU值分彆為8.81±0.77、38.10±1.55、38.97±0.80,NSE染色的PU值分彆為7.21±0.59、28.25±1.46、30.57±1.31,NIC轉染組CgA和NSE的PU值均小于轉染空質粒組和未轉染組(均P<0.01).Western blot檢測結果中,將未轉染組的條帶灰度值設為1.00,NIC轉染組、轉染空質粒組的CgA條帶灰度值分彆為0.54±0.03、0.99±0.05,NSE條帶灰度值分彆為0.43±0.02、1.07±0.09,NIC轉染組cgA和NSE條帶的灰度值均小于轉染空質粒組和未轉染組(均P<0.01).結論 Notch信號途徑對小細胞肺癌的調控可能是通過靶基因HES1對分化效應基因hASH1的轉錄抑製來實現的;Notch信號途徑可以抑製小細胞肺癌細胞的增殖,降低其神經內分泌標誌物的錶達.
목적 탐토Notch신호전도대소세포폐암적조공작용급가능궤제.방법 응용중조질립전염적방법,재소세포폐암세포주NCI-H446중표체조성성활화적Notch1(NIC전염조),동시설립전염공질립조화미전염조작위대조조,대사선출은정세포주후,이MTT법검측세포활력,응용반정량RT-PCR기술측정Notch1급기하유기인(HES1화hASH1)표체,병응용면역세포화학표기화Westernblot기술대신경내분비표지물기락립소A(CgA)、신경원특이성희순화매(NSE)적표체행반정량[양성단위(PU)치]분석.결과 미전염조화전염공질립조Notch1급기하유기인HES1표체불명현,이hASH1표체현저,전염조세포Notch1급HES1표체승고,동시반유hASH1표체명현강저.여량대조조비교,전염조세포증식속도현저강저,련속6 d측득적흡광도(A)치균소우미전염조화전염공질립조(균P<0.05).면역세포화학염색현시,NIC전염조、전염공질립조、미전염조적CgA염색적PU치분별위8.81±0.77、38.10±1.55、38.97±0.80,NSE염색적PU치분별위7.21±0.59、28.25±1.46、30.57±1.31,NIC전염조CgA화NSE적PU치균소우전염공질립조화미전염조(균P<0.01).Western blot검측결과중,장미전염조적조대회도치설위1.00,NIC전염조、전염공질립조적CgA조대회도치분별위0.54±0.03、0.99±0.05,NSE조대회도치분별위0.43±0.02、1.07±0.09,NIC전염조cgA화NSE조대적회도치균소우전염공질립조화미전염조(균P<0.01).결론 Notch신호도경대소세포폐암적조공가능시통과파기인HES1대분화효응기인hASH1적전록억제래실현적;Notch신호도경가이억제소세포폐암세포적증식,강저기신경내분비표지물적표체.
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 immanocytochemistry. 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). Immunocytochemistry 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.
