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目的:探讨基质细胞衍生因子1(SDF-1)及其受体CXC趋化因子受体4(CXCR4)在肾癌细胞转移中的作用机制,观察不同区段CXCR4在肾癌细胞内的定位。方法:应用核定位分析软件结合实验,构建不同长度的CXCR4与绿色荧光蛋白pEGFP-N1重组表达载体,转染肾透明细胞癌A498细胞株后用共聚焦显微镜观察重组表达载体在细胞内的定位。结果:生物信息学分析软件PSORTⅡPrediction发现第146~149位氨基酸残基RPRK可能是CXCR4的核定位序列,我们构建的重组质粒EGFP-CXCR4(1~510bp)、EGFP-CXCR4(1~765bp)、野生型全长EGFP-CXCR4分别加SDF-1刺激因子后其表达产物主要呈细胞核分布,而加入SDF-1刺激因子后重组质粒EGFP-CXCR4(1~267bp)的表达产物呈细胞质分布,未经SDF-1刺激野生型全长EGFP-CXCR4其表达产物呈细胞质分布,与生物信息学分析软件预测结果初步吻合。结论:CXCR4的第90~170位氨基酸残基含有核定位序列,为进一步精确定位CXCR4在肾癌细胞内的核定位序列以及寻找抑制肾癌转移的可能靶标奠定了实验和理论基础。
OBJECTIVE: To investigate the mechanism of CXCR4 and SDF-1 in the metastasis of renal cell carcinoma, and to investigate the location of different regions of CXCR4 in renal cell carcinoma. Methods: The nuclear localization analysis software was used in combination with experiments to construct CXCR4 and EGFP expression vector pEGFP-N1 with different length. The transfected cells were transfected with A498 cell line and the localization of the recombinant expression vector was observed by confocal microscopy. Results: Bioinformatics analysis software PSORTⅡPrediction found that the amino acid residues RPRK at positions 146-149 may be the nuclear localization sequence of CXCR4. The recombinant plasmids EGFP-CXCR4 (1 ~ 510bp), EGFP-CXCR4 (1-765bp) The expression products of full-length EGFP-CXCR4 with SDF-1 stimulating factor were mainly nuclear distribution, and the expression product of EGFP-CXCR4 (1 ~ 267bp) after addition of SDF-1 stimulating factor showed cytoplasmic distribution without SDF -1 stimulated the full-length wild-type EGFP-CXCR4 expression in cytoplasm distribution, preliminary agreement with the results of bioinformatics analysis software. CONCLUSION: The amino acid residues 90-170 of CXCR4 contain nuclear localization sequences, which provide the experimental and theoretical basis for further pinpointing the nuclear localization sequence of CXCR4 in renal cell carcinoma and finding the possible target of inhibiting the metastasis of renal cell carcinoma.