Expression and Subcellular Localization of Recombinant SDF-1 and Its Mutant Intrakine in Transfected COS-7 Cells

Objective:This paper is to explore a method of transferring human SDF-1 and its mutant SDF-154 intrakine gene into COS-7 cells for determining their expression and subcelluar localization of the fusion protein.This could offer feasibility for inhibiting the metastasis of malignant tumors by phonotypic knockout for blocking functional expression of receptor on the cell-surface.Methods:Amplify the target gene with PCR from the constructed plasimid SDF-WT-Gly×4-DecPET-30a(+) with a C-terminal retention signal fragment KDEL. After the pcDNA3.1 SDF-1KDEL,pcDNA3.1 SDF-154KDEL,pEGFPSDF-1KDEL and pEGFPSDF-154KDEL eukaryotic expression vectors were constructed and the DNA sequence was accurate,they were transferred into COS-7 cells with liposome . The exogenous expressions were observed, fusion protein SDF-1His and SDF-154His were confirmed by Western blot,and the SDF-1EGFP and SDF-154EGFP were determined by Laser Scanning Confocal Microscopy. Results:Four expression vectors were constructed successfully, the fusion protein SDF-1 KDELHis and SDF-1 54 KDELHis expressed in COS-7 cells.Subcelluar localization analysis showed that SDF-1KDELEGFP and SDF-154KDELEGFP were located mainly in endoplasmic reticulum.Conclusion:Four expression vectors pcDNA3.1 SDF-1KDEL, pcDNA3.1SDF-154KDEL, pEGFPSDF-1KDEL and pEGFPSDF-154KDEL were constructed successfully, which could express in eukaryotic cell and locate mainly in the endoplasmic reticulum .

Preparation and Targeting Evaluation of Recombined Intrakine Plasmid pEGFP/SDF-1/KDE/Folate-Liposome Complexes

Objective:To prepare folate liposome complexes of recombinant plasmid pEGFP/SDF-1/KDEL wich contained intrakine SDF-1/KDEL gene and to observe its targeting for breast cancer cells.Methods:Reverse phase evaporation method was employed for preparing folate-liposome complexeses of pEGFP/SDF-1/KDEL,so that preparation condition was optimized;and fluorometric method was taken for testing encapsulation efficiency.Particle diameter of liposome was tested by transmission electron microscopy.Complexeses encapsulated with 0.05-0.25 mg/ml calcein were got and incubated with breast cancer cell line MDA-MB-231 cells for 2 to 12 h,then dissolved them with dimethyl sulfoxide,then detected absorbance with microplate reader.Results:The optimized encapsulation condition are as follows:molecular ratio of lecithin and cholesterol was 3:1,rotary speed was 150 r/min under temperature of 43℃,and encapsulation efficiency reached 81%in this experiment.Average liposome particle diameter was 210 nm;8 h after incubation, the intake of MDA-MB-231 cells to 0.25 mg/ml compounds achieved saturation. Conclusion:The pEGFP/SDF-1/KDEL folic acid liposome complexes prepared has a high encapsulating rate,liposome particle diameters are homogeneous,which is available for targeting for breast cancer cells in vitro.

CXCR4在不同骨转移潜能小细胞肺癌细胞系中的差异表达及意义

目的:应用intrakine技术构建CXCR4(chemokine receptor 4趋化因子受体4)敲减表型的肺癌细胞,体外观察其增殖、凋亡、侵袭、转移能力的变化。方法:经克隆PCR、测序鉴定质粒后,给高表达CXCR4、高骨转移细胞系SBC-5中转染pCMV-S-K,经流式及细胞免疫化学检测成功构建了CXCR4稳定敲减表型的肺癌细胞SBC-5/S-K,检测CXCR4敲减后细胞增殖、凋亡、侵袭及转移能力的变化。结果:与对照SBC-5/neo相比,SBC-5/S-K在增殖、克隆形成、凋亡及细胞进程方面没有变化,而侵袭、转移能力明显减弱。结论:体外实验证实CXCR4是肺癌转移基因,为后续体内功能研究打下基础。