Notch信号报告系统在前列腺肿瘤细胞系中的建立

发布时间：2018-01-29 16:16

本文关键词： 前列腺肿瘤 LNCa P细胞人胚肾T细胞 Notch基因信号报告系统　出处：《山东医药》2016年22期 　论文类型：期刊论文

【摘要】：目的在前列腺肿瘤细胞系LNCa P中建立Notch信号报告系统。方法采用In-Fusion酶系统将巨细胞病毒(CMV)启动子和Notch蛋白胞内结构域(ICD)细胞核内DNA结合蛋白CSL的8拷贝串联DNA序列克隆入p LVX-ac GFP-N1慢病毒载体,构建Notch信号报告重组慢病毒表达质粒(p LVX-8XCSL-ac GFP)。在人胚肾293T细胞中转染p LVX-8XCSL-ac GFP质粒或对照质粒p LVX-ac GFP,6 h后再转染Notch受体的细胞内结构域(NICD)的表达质粒p ETP-NICD(过表达NICD)或对照质粒p ETP。转染48 h后用荧光显微镜观察GFP的表达,用流式细胞仪进行荧光定量分析。然后将p LVX-8XCSL-ac GFP质粒及对照质粒包装成慢病毒转导前列腺癌细胞系LNCa P,72 h后荧光显微镜观察绿色荧光,用流式细胞仪分选出GFP荧光强度最强5%及最弱5%的细胞。提取这两部分细胞的RNA,采用qRT-PCR方法检测细胞中Notch1、Notch2以及Notch信号通路下游靶基因Hey1的表达。结果在转染p LVX-8XCSL-ac GFP的293T细胞中,转染p ETP-NICD的细胞荧光强度较转染p ETP对照质粒的细胞增加了约10倍;而在转染p LVX-ac GFP的293T细胞中,转染p ETP-NICD的细胞与转染p ETP对照质粒的细胞荧光强度无明显差异。根据p LVX-8XCSL-ac GFP荧光强度分选出的LNCa P细胞,荧光强度高的细胞Notch1、Notch2、Hey1表达高于荧光强度弱的细胞(P均0.05)。结论在LNCa P细胞中p LVX-8XCSL-ac GFP可以作为报告Notch信号水平的有效工具。
[Abstract]:Objective to establish a Notch signal reporting system in prostate cancer cell line LNCa P. methods the cytomegalovirus (CMV) was detected by In-Fusion enzyme system. The 8-copy tandem DNA sequence of DNA binding protein CSL in the nucleus of promoter and Notch protein was cloned into the lentivirus vector of p LVX-ac GFP-N1. Construction of Notch signal reporting Recombinant Lentivirus expression plasmid, p LVX-8XCSL-ac GFP. P LVX-8XCSL-ac GFP plasmid or control plasmid p LVX-ac GFP were transfected into human embryonic kidney 293T cells. Expression plasmid pETP-NICD (overexpression of NICD) in cells transfected with Notch receptor after 6 h. The expression of GFP was observed by fluorescence microscope after transfection of pETP. or control plasmid for 48 h. Fluorescence quantitative analysis was performed by flow cytometry. Then the plasmids of p LVX-8XCSL-ac GFP and control plasmids were packaged into lentivirus transducted prostate cancer cell line LNCa P. After 72 hours, green fluorescence was observed by fluorescence microscope. The cells with the strongest GFP fluorescence intensity of 5% and the weakest 5% were selected by flow cytometry. The RNA of these two parts of cells were extracted. Notch1 in cells was detected by qRT-PCR method. The expression of Notch2 and downstream target gene Hey1 in Notch signaling pathway was detected in 293T cells transfected with p LVX-8XCSL-ac GFP. The fluorescence intensity of the cells transfected with p ETP-NICD was about 10 times higher than that of the control plasmids transfected with p ETP. But in 293T cells transfected with p LVX-ac GFP. There was no significant difference in fluorescence intensity between the cells transfected with p ETP-NICD and the control plasmids transfected with p ETP. LNCa P cells isolated by GFP fluorescence intensity. The cells with high fluorescence intensity were Notch1 and Notch2. The expression of Hey1 was higher than that of cells with weak fluorescence intensity (P < 0.05). Conclusion p LVX-8XCSL-ac GFP can be used as an effective tool to report the level of Notch signal in LNCa P cells.
【作者单位】：上海交通大学医学院附属仁济医院;
【基金】：国家自然科学基金资助项目(81371507) 上海交通大学多学科交叉项目培育项目(医工YG2013MS40);上海交通大学医学院科技基金(3XJ10016)
【分类号】：R737.25
【正文快照】： Notch信号通路是一条在生物进化过程中高度保守的信号通路,其通过相邻的两个细胞细胞膜上的受体和配体相互作用,参与调控生物体从胚胎到成体组织的众多发育过程,在肿瘤的发生发展过程中也起重要作用[1,2]。成熟的Notch蛋白为Ⅰ型跨膜蛋白,主要分为胞外结构域(ECD)、跨膜结构域

【相似文献】