中华皮肤科杂志

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Bcl-2对皮肤鳞状细胞癌细胞系SCL-1细胞Fas凋亡通路调控的研究

金鑫, 宋智琦, 王雅坤, 肖汀, 陈光, 姜奕, 陈洪铎, 何春涤   

  1. 中国医科大学附属第一医院皮肤科, 卫生部免疫皮肤病学重点实验室(中国医科大学), 沈阳, 110001
  • 收稿日期:2006-08-24 出版日期:2007-07-15 发布日期:2007-07-15
  • 通讯作者: 何春涤,email:chundihe@hotmail.com E-mail:chundihe@hotmail.com
  • 基金资助:
    国家自然科学基金(30271198);教育部留学回国人员科研启动基金(2002247)

Bcl-2 regulates the Fas signaling pathway in the apoptosis of skin squarnous cell carcinoma cell line SCL-1

JIN Xin, SONG Zhi-qi, WANG Ya-kun, XIAO Ting, CHEN Guang, JIANG Yi, CHEN Hong-duo, HE Chun-di   

  1. Department of Dermatology of No.1 Hospital, Key Laboratory of Immunodermatology of Ministry of Health, China Medical University, Shenyang 110001, China
  • Received:2006-08-24 Online:2007-07-15 Published:2007-07-15

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摘要： 目的 建立稳定转染bcl-2的鳞状细胞癌(鳞癌)细胞系,研究人皮肤鳞癌细胞凋亡的相关通路.方法 应用脂质体介导的基因转染法.将含有bcl-2 cDNA的逆转录病毒表达载体pIRESneo质粒导入皮肤鳞癌细胞系SCL-1细胞中.并以空质粒作为对照.经过G-418筛选后获得转入目的基因的细胞克隆,细胞扩大培养后用细胞免疫荧光染色法、RT-PCR和免疫蛋白印迹检测bcl-2的表达情况.然后用C2-ceramide和抗Fas单克隆抗体诱导细胞株凋亡,利用ELISA法检测可溶性DNA-组蛋白复合物,分析其凋亡的差异.结果 转染了pIRESneo-bcl-2的SCL-1细胞bcl-2蛋白和mRNA表达阳性,而转染了pIRESneo空载体的SCL-1细胞为阴性.不同浓度的C2-ceramide对SCL-1/bcl-2凋亡的诱导差异均无统计学意义,而对SCL-1/vector诱导的凋亡差异均有统计学意义.抗Fas单克隆抗体对SCL-1/bcl-2和SCL-1/vector诱导的凋亡.差异均有统计学意义,P<0.01.结论 用基因转染法成功地建立了稳定高表达bcl-2蛋白的SCL-1细胞系,C2-ceramide诱导的SCL-1凋亡基本可以被bcl-2阻滞,而Fas所诱导的调亡仅部分被阻滞,即SCL-1细胞系在被阻断了线粒体途径后,还可以经由死亡受体通路进行凋亡.

关键词: 细胞凋亡, 癌, 鳞状细胞, 抗原, CD95, 原癌基因蛋白质c-bcl-2, Ceramide

Abstract: Objective To establish a bcl-2 transfected squamous cell carcinoma cell line SCL-1 which stably expresses bcl-2 protein,and to investigate the Fas signaling pathway in the apoptosis of SCL-1 cells.Methods SCL-1 cells were transfected by lipofection with bcl-2 cDNA-containing retrovirus expression vector pIRESneo(SCL-1/bcl-2)or plRESneo vector plasmid(SCL-1/vector)as control.The transfected cells were selected with G-418,and passaged.The protein and mRNA expression of bcl-2 was detected by immunofluorescence,Western blot and RT-PCR,respectively.Then,varying concentrations(0～120μmol)of C2-ceramide,CH-11(a monoclonal antibody to Fas protein)were applied to induce the apoptosis of SCL-1/bcl-2 and SCL-1/vector cells.The apoptosis rate of these cells was measured by detection of soluble DNA-histone complexes with ELISA.Results Bcl-2 protein was found to be expressed in the SCL-1/bcl-2 cells,but not in the SCL-1/vector cells.The apoptosis rate of SCL-1/bcl-2 cells induced by C2-ceramide was of no significant difference from the control cells without any stimulation,while statistical difference was observed in that of SCL-1/vector cells between the C2-ceramide-stimulating group and the control without any stimulation.There was a significant difference in the apoptosis rate of both SCL-1/bcl-2 and SCL-1/vector cells induced by CH-11 from that of the control free of any stimulation.Conclusion s The SCL-1 cell clone expressing bcl-2 protein highly and stably,is established successfully by gene transfection technique.Bcl-2 could generally antagonize the apoptosis in SCL-1 cells induced by C2-ceramide,but only partly inhibit that induced by CH-11,which may suggest that the apoptosis of SCL-1 cells could occur via death receptor pathway after the blockage of mitochondrial apoptosis pathway.

Key words: Carcinoma,squamous cell, Apoptosis, Antigens,CD95, Proto-oncogene proteins c-bcl-2, Ceramide