| Selectin is a animal lectin family of three numbers which are E-, P-and L-selectin. They are expressed as adhesion molecular on cell surface ofactivated endothelium, platelets and white blood cells, respectively. Amongthem, L-selectin is continually expressed in the greater number of whiteblood cells , and it is the "homing receptor" of lymph cells. The gene of L-selectin locates in 1 chromosome, exhibiting highhomogeneity among different species. L-selectin belongs to I-typetransmembrane glycoprotein and consists of 324 amino acids. Its molecularweight is 74-95 kDa. L-selectin is made up of five domains, from N-end toC-end, which are calium dependent lectin domain also called carbohydraterecognition domain, epidermal growth factor-like domain, 2 shortconsensus repeats also called complement regulatory protein domain,transmembrane domain and short cytoplasmic tail domain. Among them,the lectin domain and the epidermal growth factor-like domain exhibitstrong relationship with L-selectin function. L-selectin functions not merelyin blood system such as facilitating leukocyte rolling along the vessel wall,and also required for efficient recirculation of lymphocytes into lymphnodes. Recently, Olga D. Genbacev's studies revealed that L-selectin andits ligand expressed on the surface of embryonic saccan and endometrium,respectively, in the implantation phase which contribute to the embryoimplantation. Because the process of tumor cell metastasis to lymph node is similarto that of lymph cells'homing, the relationship between L-selectin andtumor cell lymphogenous metastasis is more and more attractive. Manystudy revealed that: T cell hybrid tumor cells which specifically metastasizeto lymph node highly express L-selectin; Non-Hodgkin lymphoma cellsinvading the lymph nodes is considered to play the key role in themetastasis, while those propagated in lymph node frequently expressL-selectin and those prefer to propagate in peripheral tissues rarely expressL-selectin. Fawn Qian group used transgenic mouse to demonstrate thatL-selectin can facilitate tumor metastasis to lymph nodes. All these factssupport that L-selectin plays a role in the process of tumor cells metastasisto lymph nodes. Previous work in our laboratory showed that L-selectin expression inmouse Hepatocacinoma cell line Hca-F and Hca-P related positively tolymph node metastasis potential. So, it is necessary to research themechanism of L-selectin function in tumor cell lymphogenous metastasisusing L-selectin protein. Object: To express, purify L-selectin/IgG chimeric protein and toinvestigate its effect on the adhesion between P388d1 cells and frozensection of DBA mouse lymph node. Methods: After the expression vector of pCDM8-L-selectin/IgGplasmid being amplified through E.coli. HB10/P3, the plasmid wastransfected into COS-7 cells with lipid reagent Lipofect AMINE 2000TM.The expression of chimera protein in the supernatant of the cell culturemedia was detected by ELISA and the transfection condition was optimized.The expressed L-selectin/IgG protein in 48 hours cell culture media waspurified through protein A affinity chromatography column. The proteinwas visualized by SDS-PAGE, Dot blot and Western blot analysis. Finally,the effect of L-selectin/IgG protein on P388d1 adhesion to frozen section ofDBA mouse lymph node was detected in vitro. Results:The plasmid was amplified and purified as mainly superhelixstructure which visualized by 1% agarose gel. The 500bp fragment ofL-selectin was amplified from the purified plasmid by PCR, as expected.The chimeric protein was detected by ELISA in the cell culture media of 48hours after the transfection of pCDM8 L-selectin/IgG expression vector toCOS-7 cells. After purification through protein A affinity columnchromatography, the purified protein showed a band with molecular weightof about 94.41 kDa by SDS-PAGE. The analysis of Dot-blot andWestern-blot showed that this protein not only specifically bound with goatanti L-selectin polyclonal antibody but also bound with anti-human IgGpolyclonal antibody. The ad... |
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