人类新血小板反应素R-spondin 3的细胞定位及其对肿瘤细胞功能的影响

Cellular localization of a novel human thrombospondin R-spondin 3 and its effects on tumor cells

目的观察人类新血小板反应素R-spondin 3(Rspo 3)的细胞定位,并探讨其在肿瘤发生发展中的可能作用。方法利用荧光显微成像法观察EGFP-Rspo 3在HEK293细胞中的分布。将重组质粒pcDNA-Rspo 3转染结肠癌细胞HT-29、LoVo,采用流式细胞术观察Rspo 3基因过表达对肿瘤细胞周期与凋亡的影响;采用Matrigel、Transwell实验分别检测Rspo 3基因过表达对肿瘤细胞黏附及侵袭能力的影响。结果荧光显微成像法观察发现,EGFP-Rspo 3融合蛋白在细胞核表达,呈弥散点状分布。流式细胞术观察发现,Rspo 3基因过表达对肿瘤细胞生长周期没有明显影响;Rspo 3基因过表达不影响低恶性的HT-29细胞凋亡,但诱导高恶性的LoVo细胞凋亡(P<0.01)。Rspo 3基因过表达增强肿瘤细胞黏附性(P<0.01),降低肿瘤细胞的侵袭力(P<0.01),对肿瘤细胞移行有一定抑制作用。结论 Rspo 3有核定位功能,能诱导部分肿瘤细胞凋亡并抑制其转移扩散。

Objective To study the cellular localization of a novel human thrombospondin R-spondin 3 and its role in the development and progression of tumors.Methods The subcellular localization of R-spondin 3 was investigated in HEK293 cells by fluorescence micrography.Colon carcinoma cell lines HT-29 and LoVo were transfected with pcDNA-Rspo 3 recombinant expression plasmid;and the cell cycle and apoptosis were detected by Flow Cytometry（FCM）.Cell adhesion and invasion ability were determined by Matrigel reagents and Transwell system,respectively.Results Fluorescence micrography showed that EGFP-Rspo 3 fusion protein was mainly localized in nuclei as dispersed particles.Overexpression of R-spondin 3 showed no effect on cell cycle in both colon carcinoma cell lines.Overexpression of R-spondin 3 induced apoptosis of LoVo cells with high malignancy（P〈0.01） although it showed no effect on the apoptosis of HT-29 cells with low malignancy.The overexpression also promoted the adhesion ability（P〈0.01）,restrained invasion ability（P〈0.01） and motility of both colon carcinoma cell lines.Conclusion Our research indicates that R-spondin 3 localize in the nuclei;it can induce apoptosis and restrain metastatic potential of some tumor cells.