跨膜接头蛋白CBP对皮肤鳞癌细胞A431生长增殖的负调控作用研究

发布时间：2018-06-17 05:30

  本文选题：皮肤鳞癌 + 跨膜接头蛋白　； 参考：《中国癌症杂志》2017年07期

【摘要】：背景与目的:跨膜接头蛋白(Csk-binding protein,CBP)是新发现的Src家族成员,与多种肿瘤的发生有关。该研究旨在观察CBP基因过表达对皮肤鳞癌细胞系A431增殖及细胞凋亡的影响,探讨其相关的分子机制。方法:构建CBP-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合蛋白的慢病毒过表达载体,采用反转录病毒转染的方法建立CBP过表达的A431细胞株。实验分为亲本细胞组(未进行基因转染的A431细胞)、对照组(A431细胞转染仅含EGFP阴性对照病毒)和实验组(A431细胞转染CBP-EGFP病毒)。在激光共聚焦显微镜下观察细胞转染率,验证转染成功与否;CCK-8法检测CBP过表达对A431细胞增殖能力的影响,并采用流式细胞术(flow cytometry,FCM)检测对细胞凋亡的影响;实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测Lck、Csk和Fyn三种上游信号转导分子分别在m RNA和蛋白中的表达水平变化。结果:建立了稳定过表达CBP的A431细胞株;CCK-8法结果提示,CBP过表达明显抑制细胞生长,第2~6天的组间差异有统计学意义(P0.05);FCM检测显示,实验组细胞凋亡率显著增加,与亲本细胞组及对照组相比差异有统计学意义(P0.001);RTFQ-PCR结果显示,实验组A431细胞Lck m RNA的相对表达水平显著下调(P0.001),实验组细胞Csk和Fyn m RNA的表达分别约为亲本细胞组的1.6倍和3.8倍,表达显著上调(P0.001);Western blot结果表明,实验组Lck蛋白的相对表达水平明显下降(P0.001),实验组细胞Csk和Fyn蛋白与亲本细胞组和对照组相比表达明显增加(P0.001)。结论:CBP过表达可抑制皮肤鳞癌细胞增殖,诱导细胞凋亡及坏死。CBP通过调节上游信号转导通路中的蛋白酪氨酸激酶Lck、Csk和Fyn来调控细胞的增殖活性。
[Abstract]:Background & AIM: transmembrane junction protein Csk-binding protein (CBP) is a newly discovered member of Src family, which is involved in the development of many kinds of tumors. The aim of this study was to investigate the effects of CBP gene overexpression on the proliferation and apoptosis of cutaneous squamous cell carcinoma cell line A431, and to explore its molecular mechanism. Methods: the lentivirus overexpression vector of CBP- enhanced green fluorescent protein (EGFP) fusion protein was constructed, and a CBP overexpression A431 cell line was established by retrovirus transfection. The experiment was divided into parent cell group (A431 cells without gene transfection and control group with EGFP negative control virus only) and experimental group with CBP-EGFP virus transfection. The transfection rate of A431 cells was observed under confocal laser microscope, and the effect of CBP overexpression on the proliferation of A431 cells was detected by CCK-8 method, and the apoptosis was detected by flow cytometry (FCM). Real-time fluorescence quantitative polymerase chain reaction- PCR (RTFQ-PCR) and Western blot were used to detect the expression levels of LckCsk and Fyn in mRNA and protein, respectively. Results: CCK-8 assay was established for stable overexpression of CBP in A431 cell line. The results showed that the overexpression of CBP significantly inhibited the growth of A431 cells. The results of FCM analysis showed that the apoptosis rate of the experimental group was significantly higher than that of the control group. The results of RTFQ-PCR showed that the relative expression level of Lck mRNA in A431 cells of experimental group was significantly down-regulated, and the expression of Csk and Fyn mRNA in experimental group was about 1.6 times and 3.8 times as much as that in parent cell group, respectively. The results of Western blot showed that the relative expression level of Lck protein in the experimental group was significantly lower than that in the control group. The expression of Csk and Fyn protein in the experimental group was significantly higher than that in the parent cell group and the control group, and the expression of Csk and Fyn protein in the experimental group was significantly higher than that in the parent cell group and the control group. Conclusion the overexpression of CBP can inhibit the proliferation of skin squamous cell carcinoma cells, induce apoptosis and necrosis. CBP regulates the proliferation activity of the cells by regulating the protein tyrosine kinase LckCsk and Fyn in the upstream signal transduction pathway.
【作者单位】： 青岛大学附属医院烧伤整形外科;青岛大学医学院免疫教研室;
【分类号】：R739.5
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本文编号：2029911