Notch信号报告系统在前列腺肿瘤细胞系中的建立

Establishment of Notch signaling pathway reporter system in prostate cancer cell line

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。

Objective To establish a Notch signaling reporter system in prostate cancer cell line LNCa P. Methods We set up a Notch signaling reporter system by first constructing a lentivirus plasmid（ p LVX-8XCSL-ac GFP） which containing a tandem of 8 copy transfactor CSL DNA binding elements driven by a CMV promoter. Then the 293 T cells were transfected with p LVX-8XCSL-ac GFP plasmids for 6 h or with p LVX-ac GFP plasmids as a control group,respectively. Furthermore,293 T cells in each of the above group were re-transfected with p ETP-NICD or p ETP control plasmids. The green fluorescence was observed with fluorescence-microscope at 48 h,and the fluorescence intensity was quantified by flow cytometry. Then,p LVX-8XCSL-ac GFP plasmids were packed to produce p LVX-8XCSL-ac GFP lentivirus to transduce prostate cancer cell line Ln Ca P. The green fluorescence was observed under fluorescence-microscope at 72 h. Furthermore,the cells were divided into two groups with flow cytometry according to the fluorescence intensity： the 5% highest intensity group and the 5% lowest group. Then the expression of Notch1,Notch2 and Hey1 were monitored by Q-PCR in both groups. Results In the 293 T cells transfected with p LVX-8XCSL-ac GFP,the green fluorescence intensity of cells transfected with p ETP-NICD was 10 times higher than that of cells transfected with p ETP control plasmid,while in the 293 T cells transfected with p LVX-ac GFP,no significant difference was found in the fluorescence intensity between cells transfected with p ETP-NICD and cells transfected with p ETP control plasmid. Q-PCR test displayed that the expression of Notch1,Notch2 and Hey1 in the 5% highest fluorescence intensity group were significant higher than that in the 5% lowest intensity group（ all P 0. 05）. Conclusion In the LNCa P cells,p LVX-8XCSL-ac GFP can be used as an effective tool to report Notch signal level.