Gene Cloning,Expression Of The Human Chemokine SLC And The Study On Immuogenicity Of MCF-7

Secondary Lymphoid-Tissue Chemokine(SLC) is a special chemeokine for T cell, NK and mature DC cell. T cell,NK cell and DC, are three important effectors in the anti-tumor immunity, and T cell play the critical role in anti-tumor immunity. Studies have revealed that lack of mouse-SLC in mouse can lead to the mistake of localization of DCs and homing of T cells, as well as delayed activation of T cells and disturbance of the structure of secondary lymphoid tissues. However, overexpression of SLC always results in progression of many kinds of autoimmue disorders. Recent research revealed that the receptor of SLC--CCR7 is expressed on naive T cells, B cells, dendritic cells and some cancer cells, for example, Melanoma and breast cancer cell, and Present researches demonstrated that the combination of lymphatic SLC and CCR7 induces cells'migration to secondary lymphoid tissue. Therefore, SLC is closely related with lymphocytes homing and lymphatic metastasis. Therefore, we may developed a new strategies based on its bio-activities to treat transplant rejection and relevant diseases. Our lab has discovered that a certain concentration of SLC can inhabit tumor grow, and improve the proliferation of PBMC.【Objective】Based on previous theories, The functional recombinant human SLC protein was successfully produced, transfecting into MCF-7,then detected its expressing, affection on MCF-7 and PBMC,.This research makes further studies on novel therapeutic strategies possible.1.Construction and expression of human CCL21 plasmid(1)Construction of human CCL21The SLC gene was amplified by reverse transcription PCR ,then cloned into green fluorescent protein eukaryotic expression vector pgenesil-8-GFP,BglII和EcoR I restriction sites. The construction of recombinant plasmid was identified with digestion and sequencing.(2)Transfection and expression of SLC vector Recombinant plasmid was transfected into MCF-7. 24 hours later, the intracellular expression of green fluorescence was observed with inverted fluorescence microscope. The supernatant was harvested and detected with western blot. The results indicated that SLC-GFP was secreted into the supernatantan and its concentration peaked at 72 hours after transfection.2.Effects of SLC on MCF-7 apoptosisGroups:1)Untransfected MCF-7 control:MCF-72)Vector-transfected MCF-7 control:MCF-7-vector3)SLC-transfected MCF-7:MCF-7-SLCThe result of MCF-7 cell apoptosis analyzed by FCM suggested that Compared with the controls, more MCF-7-SLC underwent apoptosis.3.Effects of SLC on PBMC proliferationGroups1)Blank: DC stimulated with saline2)PBMC /MCF-7:PBMC stimulated with MCF-73)PBMC /MCF-7-vector:PBMC stimulated with MCF-7-vector4)PBMC /MCF-7-CCL21:PBMC stimulated with MCF-7-SLCMethods and results1) PBMC preparation:Human peripheral mononuclear cells were isolated by Ficoll-Hypaque and then was isolated.2)2×105MCF-7,MCF-7-vector and MCF-7-SLC were all treated with mitomycin (30ug/ml) for 20 mins, and cocultured with 106PBMC for 5 days.3)5 days later the proliferation of PBMC was analysis with Midifest softwell.Compared with the controls, MCF-7-SLC could enhance the proliferation of PBMC.【Conclusion】The functional recombinant human SLC protein was successfully produced, and more MCF-7-SLC underwent apoptosis, enhancing the proliferation of PBMC.